**Meet the editor**

Ibrokhim Y. Abdurakhmonov received his B.S. Degree (1997) *in biotechnology* from the National University of Uzbekistan, M.S. degree i*n plant breeding* (2001) from Texas A&M University of USA, Ph.D. degree (2002) *in molecular genetics*, Doctor of Science degree (2009) *in genetics*, and full professorship (2011) i*n molecular genetics and molecular biotechnology* from the Institute of Genetics

and Plant Experimental Biology, Academy of Sciences of Uzbekistan. He founded (2012) and is currently leading the Center of Genomics and Bioinformatics of Uzbekistan. He serves as an associate editor/editorial board member of several international and national journals on plant sciences. He received Government award, 2010 chest badge "Sign of Uzbekistan," 2010 TWAS prize, and "ICAC Cotton Researcher of the Year 2013" for his outstanding contribution to cotton genomics and biotechnology. He was elected as the World Academy of Sciences (TWAS) Fellow (2014) on *Agricultural Science* and as a co-chair/chair of "Comparative Genomics and Bioinformatics" workgroup (2015) of International Cotton Genome Initiative (ICGI).

## Contents

#### **Preface XIII**



#### **X** Contents



#### **Section 6 RNA Interference in Pest and Pathogen Control 339**

Chapter 7 **ER-targeted Intrabodies Mediating Specific In Vivo Knockdown of Transitory Proteins in**

Chapter 8 **RNAi Therapeutic Potentials and Prospects in CNS Disease 165**

Emine Şalva, Ceyda Ekentok, Suna Özbaş Turan and Jülide Akbuğa

Tamara Martínez, Maria Victoria González, Beatriz Vargas, Ana

Chapter 9 **RNAi-based Gene Therapy for Blood Genetic Diseases 191** Mengyu Hu, Qiankun Ni, Yuxia Yang and Jianyuan Luo

Chapter 11 **siRNA-Induced RNAi Therapy in Acute Kidney Injury 223**

Chapter 12 **Preclinical Development of RNAi-Inducing Oligonucleotide**

**Section 5 RNA Interference for Immune and Infectious Diseases 273**

Chapter 14 **Perspectives on RNA Interference in Immunopharmacology**

Zhaohua Hou, Qiuju Han, Cai Zhang and Jian Zhang

Chapter 15 **RNA Interference as a Tool to Reduce the Risk of Rejection in**

Chapter 16 **Utility of Potent Anti-viral MicroRNAs in Emerging Infectious**

Zhabiz Golkar, Donald G. Pace and Omar Bagasra

Constanca Figueiredo and Rainer Blasczyk

Oliver Backhaus and Thomas Böldicke

Kyoung Joo Cho and Gyung Whan Kim

Chapter 10 **Non-viral siRNA and shRNA Delivery Systems in**

**Therapeutics for Eye Diseases 245**

Isabel Jiménez and Covadonga Pañeda

**Cancer Therapy 201**

Cheng Yang and Bin Yang

Chapter 13 **RNAi-Induced Immunity 275** Wenyi Gu

**Diseases 325**

**and Immunotherapy 285**

**Cell-Based Therapies 311**

**Section 4 RNA Interference for Disease Therapy 163**

**Comparison to RNAi 137**

**VI** Contents


## Preface

RNA interference (RNAi), being a revolutionary discovery of all biological sciences of twenty-first cen‐ tury and historically known as *co-suppression, quelling*, and/or *post-transcriptional gene silencing* (PTGS), is an evolutionarily conserved, double-stranded RNA-dependent, universal eukaryotic process to clean "foreign-like" mRNA transcripts in a sequence-specific manner before they generate harmful proteins or invade the genome. Opportunity to induce and trigger RNAi for any desired sequence signature and its systematic spreading property from cell-to cell, tissue to tissue, or in whole body system made it an efficient approach to study the function of any genes and/or sequence signatures of an organism.

Increasing body of our knowledge on a sequence/gene of interest, improved design of RNAi inducers with its various modifications, development of efficient and specific delivery systems, and optimized targeting strategies as well as the use of organism's own gene or gene fragment further made RNAi advantageous over existing gene manipulation "transgenic" technologies. Hence, RNAi holds a great potential for agricultural biotechnology and crop improvement, food security, industrial biotechnology and biofuel production, molecular pharmacology, and treatment of various inflammatory, infectious, and hereditary diseases such as complex immune and cancer therapy. Addressing all of these, RNAi research and application significantly advanced in past decade period.

The book *RNA interference*, a collection of 19 chapters from distinguished laboratories and eminent sci‐ entists of the world conducting state-of-the-art RNAi research, aims to provide readers "up-to-date" knowledge and progress on basics; types of inducers, triggers, and delivery systems; design and optimi‐ zation requirements; and performance of RNAi in various cell models and organisms. Further, chapters of this book discuss a wide variety of potential "bench-to-clinic" applications of RNAi and lessons learned in crop improvement and protection, veterinary and animal protection, molecular pharmacolo‐ gy, and medicine, including its current and future therapeutic potential on inflammatory, blood, central nervous system, eye, liver, immune and cancer disease therapies with some aspects of limitations, alter‐ native tools, safety, and risk assessment.

Although we missed the latest developments on RNAi repositories, screens, and databases as well as indepth coverage of crop RNAi applications and commercialization efforts, I trust that various topics on advancements of RNAi research and its application, compiled in this single book, should add informa‐ tion to currently available literature sources and be useful for university students as well as private and public life science researchers, enhancing the reader's knowledge.

I would like to express my sincere appreciation to all eminent authors of the book chapters for their contributions, hard work, and full cooperation with my editorial requests. I thank the InTech book de‐ partment, for giving me an editorship opportunity of this book, and Ms. Sandra Bakic and Ms. Iva Sim‐ cic, InTech's Publishing Process Managers, for their initiation of this book project, coordination of entire book processing, correspondence with authors, and support and help with this book publication.

#### **Ibrokhim Y. Abdurakhmonov**

Center of Genomics and Bioinformatics, Academy of Sciences of Uzbekistan, Ministry of Agriculture and Water Resource, "Uzcottonindustry" association, Tashkent, Uzbekistan

**Section 1**

## **Introductory**

## **RNA Interference – A Hallmark of Cellular Function and Gene Manipulation**

Ibrokhim Y. Abdurakhmonov

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62038

#### **Abstract**

The discovery of RNA interference (RNAi) and its utilization in downregulation of spe‐ cific target transcripts have revolutionized gene function analysis and elucidation of many key biochemical/genetic pathways. The insights into gene function, combined with a technology that made silencing of gene function possible using the potent, highly spe‐ cific and selective RNAi approaches, provided the solution to longstanding complex ob‐ stacles in targeted crop improvementsfor agriculture, and disease therapies for medicine. In this introductory chapter, I aim briefly to cover the basics and peculiarities of RNAi and the advances made in understanding the mechanisms, components, function, evolu‐ tion, application, safety and risk assessment of RNAi, while at the same time highlighting the related chapters of this book.

**Keywords:** Gene silencing, RNA interference, RNAi inducers and delivery, RNAi-based disease therapy, biosafety

#### **1. Introduction**

The "central dogma" of genetics as first presented by Francis Crick is that genes, packed inside the cell as the deoxyribonucleic acid (DNA) molecule, are transcribed into messenger ribonu‐ cleic acids (mRNA), which are subsequently translated into proteins (or enzymes). These final protein products provide all life functions, and together with DNA and RNA, constitute the molecules of life. Therefore, if there is a disruption (interference) of a gene function, messenger RNA synthesis, or protein translation, normal life processes get altered or even stopped. "No gene-no messenger", or "no messenger-no protein", has been the basis of understanding biological processes. One of the easy-to-access points in cellular processes is messenger RNA due to its cytoplasmic location, "naked" structure, comparatively short half-life, and temporal existence between transcription and translation. Further, mRNA is in between the chain of

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

events from DNA to protein; it has the universal chemical structure, consisting of only four nucleotides, regardless of the encoded message. In contrast, proteins are chemically much more variable, consisting of combinations of 21 different amino acids, with side chains that vary from very hydrophilic to highly hydrophobic. If mRNA is altered or eliminated before translation, there is no functional gene product, which results in changing the cellular process from the native state. This is the entire rationale of RNA interference (RNAi).

RNA interference is a process in eukaryotic cells in which double stranded endogenous or exogenous RNA molecules trigger a cytoplasmic response, which involves sequence specific target identification and destruction. This may include native messenger RNAs (mRNAs) that code vitally important proteins [1]. Any type of double-stranded RNA (dsRNA) molecules can activate RNAi where long dsRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs) and their various forms and modifications are considered the main players/inducers [1]. Let us take a look at RNAi discovery history.

Plant scientists in the 1990s first used targeted gene silencing by introducing an antisense gene into plants. The first example was silencing of a nopaline synthase (NOS) gene, for which the silencing was only visible by loss of a band on a Norther blot and loss of NOS activity [2]. The second antisense gene used in plants targeted the petunia chalcone synthase (CHS) gene, encoding the first step in floral pigment production, and the result was visible in the loss of petal pigmentation [3]. Curiously, attempts to create dark pigmented petunia flowers by overexpression of the same CHS gene resulted in similar colorless petunia petals [4, 5]. It was thought that such a phenotype was "*due to post-transcriptional inhibition of gene expression via an increased rate of mRNA degradation*" [6]. The observed phenomenon was named as "*co-suppres‐ sion*" of gene expression and the molecular mechanism behind "*co-suppression*" remained unknown for many years [7]. Later, a transient gene inactivation of the carotenogenic *albino-3* (*AL-3*) and *albino-1* (*AL-1*) genes was reported after transformation with homologous sequen‐ ces in *Neurospora crassa* [8]. This phenomenon, named as gene "*quelling*", was observed to be severely destructive but spontaneously and progressively reversible and monodirectional, resulting in mutant, intermediate, and wild-type phenotypes [8]. In the years to follow, the cosuppression phenomenon were attributed to inverted repeat T-DNA insertions, which result in RNA transcripts with internal complementary sequences that can fold back on themselves, generating double-stranded RNA and can seed the now well-known Argonaute/dicer silencing system.

Following these seminal discoveries, similar phenomena were discovered in other organisms including the nematode (*Caenorhabditis elegans*) and insects (*Drosophila melanogaster*) from studying the function of a *PAR-1* gene (required for establishing embryo polarity) in the former and alcohol dehydrogenase in the latter (*ADH*) [9, 10]. These studies not only demonstrated a wide range of functionality of "*co-suppression*" phenomenon but also prompted an intense effort to understand the exact mechanism causing this process. In one experiment, injection of dsRNAs associated with muscle protein production into nematodes successfully silenced the targeted gene. The effect on muscle production was not observed using either mRNA or antisense RNA [11]. With this work, for the first time, the agent directly responsible for "*cosuppression*" was identified and formally named as "*RNA interference*" or RNAi. This work was later recognized with the 2006 Nobel Prize.

In plants, the suppression of targeted genes during viral infections was discovered [12] and subsequently developed into a system by which plant gene function may be studied through inhibition by infection with viruses bearing a short sequence targeted against plant mRNAs [13]. This phenomenon was termed as "*virus-induced gene silencing*" (VIGS) and is often used to study gene function in plant species that are recalcitrant to transformation or just take a very long time to regenerate.

events from DNA to protein; it has the universal chemical structure, consisting of only four nucleotides, regardless of the encoded message. In contrast, proteins are chemically much more variable, consisting of combinations of 21 different amino acids, with side chains that vary from very hydrophilic to highly hydrophobic. If mRNA is altered or eliminated before translation, there is no functional gene product, which results in changing the cellular process

RNA interference is a process in eukaryotic cells in which double stranded endogenous or exogenous RNA molecules trigger a cytoplasmic response, which involves sequence specific target identification and destruction. This may include native messenger RNAs (mRNAs) that code vitally important proteins [1]. Any type of double-stranded RNA (dsRNA) molecules can activate RNAi where long dsRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs) and their various forms and modifications are considered the main players/inducers

Plant scientists in the 1990s first used targeted gene silencing by introducing an antisense gene into plants. The first example was silencing of a nopaline synthase (NOS) gene, for which the silencing was only visible by loss of a band on a Norther blot and loss of NOS activity [2]. The second antisense gene used in plants targeted the petunia chalcone synthase (CHS) gene, encoding the first step in floral pigment production, and the result was visible in the loss of petal pigmentation [3]. Curiously, attempts to create dark pigmented petunia flowers by overexpression of the same CHS gene resulted in similar colorless petunia petals [4, 5]. It was thought that such a phenotype was "*due to post-transcriptional inhibition of gene expression via an increased rate of mRNA degradation*" [6]. The observed phenomenon was named as "*co-suppres‐ sion*" of gene expression and the molecular mechanism behind "*co-suppression*" remained unknown for many years [7]. Later, a transient gene inactivation of the carotenogenic *albino-3* (*AL-3*) and *albino-1* (*AL-1*) genes was reported after transformation with homologous sequen‐ ces in *Neurospora crassa* [8]. This phenomenon, named as gene "*quelling*", was observed to be severely destructive but spontaneously and progressively reversible and monodirectional, resulting in mutant, intermediate, and wild-type phenotypes [8]. In the years to follow, the cosuppression phenomenon were attributed to inverted repeat T-DNA insertions, which result in RNA transcripts with internal complementary sequences that can fold back on themselves, generating double-stranded RNA and can seed the now well-known Argonaute/dicer

Following these seminal discoveries, similar phenomena were discovered in other organisms including the nematode (*Caenorhabditis elegans*) and insects (*Drosophila melanogaster*) from studying the function of a *PAR-1* gene (required for establishing embryo polarity) in the former and alcohol dehydrogenase in the latter (*ADH*) [9, 10]. These studies not only demonstrated a wide range of functionality of "*co-suppression*" phenomenon but also prompted an intense effort to understand the exact mechanism causing this process. In one experiment, injection of dsRNAs associated with muscle protein production into nematodes successfully silenced the targeted gene. The effect on muscle production was not observed using either mRNA or antisense RNA [11]. With this work, for the first time, the agent directly responsible for "*cosuppression*" was identified and formally named as "*RNA interference*" or RNAi. This work was

from the native state. This is the entire rationale of RNA interference (RNAi).

[1]. Let us take a look at RNAi discovery history.

silencing system.

4 RNA Interference

later recognized with the 2006 Nobel Prize.

Over the past decade, RNAi has been demonstrated in many eukaryotes including humans as well as some prokaryotic life forms [14] and has been recognized to form an integral part of many gene regulatory networks during development. This revolutionary breakthrough in biological science has become a valuable *in vitro*, *in vivo*, and *ex vivo* manipulation of gene expression, allowing for large-scale studies of gene function. It is now a routine laboratory practice to introduce the desired gene-specific dsRNA inducers into cells and selectively, robustly, and systematically silence the targeted sequence signature revealing its cellular function. In addition, RNAi has become an efficient tool for agricultural biotechnology to improve production [15] and combat disease pests as well as for medicine and molecular pharmacology to cure complex infectious, inflammatory, and hereditary diseases [16].

**Figure 1.** Dynamics of scientific publications devoted to the RNA interference for the past three decades. Source: PubMed [18] data sorted by the year of publications, which were retrieved by the search with the unquoted keyword "*RNA interference*"]

RNAi research has rapidly advanced and expanded over the past decade, evidenced by increasing numbers of publications, research projects, and practical applications in both agriculture and medicine. For example, searching *Google Scholar* [17] with the unquoted keyword "*RNA interference*" retrieved over 1 million (1,110,000) documents. Repeating the same search with "organism-specified RNA interference" in *PubMed* database [18] on the same date returned a total of 50,824 indexed scientific documents with a major pick after 2002 reaching to over 1,000 scientific publications per year (Figure 1). The distribution of specified search results revealed a number of PubMed-indexed, RNAi-related publications for human (32,007), plant (3,701), animal (27,751), insect (4,145), fungal (690), and prokaryotic (119) organisms. Moreover, the therapeutic application of RNAi is also expanding rapidly with 9,953 articles related to this topic and found in *PubMed* searching with "RNA interference therapy" keyword. In this brief introductory chapter, I aim to cover the basic understanding behind RNAi and an update knowledge on its applications, limitations, safety, and risks, highlighting and discussing some of the key points presented in this book.

#### **2. Components, mechanism, and function**

The principle mechanism of RNAi is complex, but very straightforward and easy to under‐ stand. RNAi is induced by the introduction of specific exogenous dsRNA either by virus genome RNAs, injection of synthetic dsRNAs or, in plants, is mediated by Agrobacterium. RNAi is also part of the normal development and dsRNAs are produced by endogenous genes encoding miRNA precursors or other long dsRNA molecules. In either case, the dsRNAs are recognized by the enzyme dicer and cleaved into short, double-stranded fragments of ~19-25 base pair long siRNAs [1]. These siRNAs are separated into two single-stranded RNAs (ssRNAs), which are referred to as the "*passenger*" and the "*guide*" strands. The passenger strand is degraded, while the guide strand is picked up by the RNA-induced silencing complex (RISC) that has enzymatic digestion activity and contains the key components of Argonaute (AGO) and P-element induced wimpy testis (PIWI) proteins [1]. The RISC proteins perform the unwinding of the *guide* and *passenger* strands in ATP-independent manner [19, 20]; however, ATP is required to unwind and remove the cleaved mRNA strand from the RISC complex after catalysis [21]. There are effector proteins such as RDE-4 (nematodes) and R2D2 (insects) that recognize exogenous dsRNAs and stimulate dicer activity. R2D2 also has a differentiating function for siRNA strands by stably binding to 5' end of the *passenger* strand, thus directing the *guide* strand to the RISC [22]. Here, it should be noted that the 5′ end of the *guide* strand is involved in matching and binding the target mRNA while the 3′ end physically arranges target mRNAs into the cleavage-favorable site of the RISC complex [21]. AGO/PIWI proteins localize within the specific P-body regions in the cytoplasm, considered to be a critical site for RNAi [23–25].

It is not clear as yet how the *guide* strand-bound active RISC complex finds mRNA targets within the cell, but it is known that this process is sequence-specific. Once the target mRNA is identified and captured though RNAi machinery, RISC cleaves the target mRNA rendering it untranslatable [1]. In most cases, the entire process is triggered by amplification of the cleavage process through synthesis of additional dsRNAs from primarily digested fragments of mRNA. Upon annealing to the mRNA target, the guide RNA may also be extended by RNA-dependent RNA polymerase (RdRP), resulting in extended "secondary" dsRNAs which in turn may lead to the formation of new siRNAs that enhance and further systematically spread the degrada‐ tion of the target mRNA in cytoplasm [26, 27].

Although the pathways toward RNAi from exogenous and endogenous dsRNA converge at the RISC and use the same downstream RNAi machinery, there are also some clear differences in their processing and handling [1]. Endogenous dsRNAs cleaved by dicer (**1**) produce 20–25 bp fragments with a two-nucleotide overhang at the 3′end of siRNA duplex [1], while the length of exogenous dsRNAs-derived siRNAs, required for specificity, is unknown. Exoge‐ nous dsRNAs are distinctly (**2**) handled by the above-mentioned effector proteins, RDE-4 or R2D2 [26, 27], whereas siRNA derived from endogenous dsRNAs (i.e., miRNA precursors) are handled by double-stranded miRNA precursor-binding DGCR8 and Drosha proteins with RNAse III enzyme activity. Plants do not have Drosha homologs, instead, processing of miRNA to siRNAs is carried out by one of four dicer-like proteins. Endogenous miRNAs (**3**), except some plant miRNAs, typically have several mismatches to the target sequence, while siRNAs derived from exogenous dsRNAs usually are designed to have a perfect match to the target. Most importantly, (**4**) endogenous dsRNA-derived miRNAs are capable of mildly inhibiting the translation of hundreds of mRNAs [28–30], while exogenous dsRNA-derived ones usually silence only single specific target [31]. Depending on organisms, for instance in *C. elegans* and *D. melanogaster*, (**5**) distinct Argonaute proteins and dicer enzymes [32, 33] process miRNAs and exogenous siRNAs. Furthermore, endogenously processed miRNAs prevalently (**6**) interact with miRNA response elements (MREs) located within the 3'-UTRs region of target mRNAs. Upon binding to MREs, miRNAs can decrease the gene expression of various mRNAs by either inhibiting translation (in animals) or directly causing degradation of the transcript (in plants). In contrast, exogenous dsRNA-derived siRNAs may interact with any complementary sequence region of the target mRNA, causing direct cleavage of the transcript [1]. miRNAs may actually regulate translation of target mRNAs in dual ways, as translation regulation by miRNAs oscillates between repression and activation during the cell cycle through a yet unknown mechanism [34].

The main biological function of RNAi is regulation of gene activity of cells at the posttranscriptional level (PTGS) either by the inhibition of translation of mRNA or by direct degradation of the mRNA. In addition to PTGS, RNAi pathway components may contribute to maintenance of genome organization and structure, mediated by RNA-induced histone modification. Histone modification in turn affects heterochromatin formation and may silence gene activity at the pre-transcriptional level [35]. This process is referred to as "RNA-induced gene silencing (RITS) and requires dicer, siRNA and RISC component proteins such as AGO and R2D2 [36]. In addition, RNAi components and inducers (siRNA/dicer/AGO) may also possibly upregulate expression of genes in binding into a promoter region and through histone demethylation, a process dubbed RNA activation [37, 38].

Because of sequence-specific recognition, regulatory properties, and the possibility of systemic spreading of dsRNAs, RNAi is the key "sterilizing agent" of cells and tissues, and it functions as potent immune response against foreign nucleic acids from viruses, transposons, or transformation events which can invade and harm the genome and its stability [39]. The chapters presented in Section 2 of this book have a more detailed coverage of the history of the RNAi discovery, mechanism, and functional components and on the biological role of RNAi including natural small RNAs/microRNAs as well as long noncoding RNAs in gene regulations.

#### **3. Differences among organisms**

organisms. Moreover, the therapeutic application of RNAi is also expanding rapidly with 9,953 articles related to this topic and found in *PubMed* searching with "RNA interference therapy" keyword. In this brief introductory chapter, I aim to cover the basic understanding behind RNAi and an update knowledge on its applications, limitations, safety, and risks, highlighting

The principle mechanism of RNAi is complex, but very straightforward and easy to under‐ stand. RNAi is induced by the introduction of specific exogenous dsRNA either by virus genome RNAs, injection of synthetic dsRNAs or, in plants, is mediated by Agrobacterium. RNAi is also part of the normal development and dsRNAs are produced by endogenous genes encoding miRNA precursors or other long dsRNA molecules. In either case, the dsRNAs are recognized by the enzyme dicer and cleaved into short, double-stranded fragments of ~19-25 base pair long siRNAs [1]. These siRNAs are separated into two single-stranded RNAs (ssRNAs), which are referred to as the "*passenger*" and the "*guide*" strands. The passenger strand is degraded, while the guide strand is picked up by the RNA-induced silencing complex (RISC) that has enzymatic digestion activity and contains the key components of Argonaute (AGO) and P-element induced wimpy testis (PIWI) proteins [1]. The RISC proteins perform the unwinding of the *guide* and *passenger* strands in ATP-independent manner [19, 20]; however, ATP is required to unwind and remove the cleaved mRNA strand from the RISC complex after catalysis [21]. There are effector proteins such as RDE-4 (nematodes) and R2D2 (insects) that recognize exogenous dsRNAs and stimulate dicer activity. R2D2 also has a differentiating function for siRNA strands by stably binding to 5' end of the *passenger* strand, thus directing the *guide* strand to the RISC [22]. Here, it should be noted that the 5′ end of the *guide* strand is involved in matching and binding the target mRNA while the 3′ end physically arranges target mRNAs into the cleavage-favorable site of the RISC complex [21]. AGO/PIWI proteins localize within the specific P-body regions in the cytoplasm, considered to be a critical

It is not clear as yet how the *guide* strand-bound active RISC complex finds mRNA targets within the cell, but it is known that this process is sequence-specific. Once the target mRNA is identified and captured though RNAi machinery, RISC cleaves the target mRNA rendering it untranslatable [1]. In most cases, the entire process is triggered by amplification of the cleavage process through synthesis of additional dsRNAs from primarily digested fragments of mRNA. Upon annealing to the mRNA target, the guide RNA may also be extended by RNA-dependent RNA polymerase (RdRP), resulting in extended "secondary" dsRNAs which in turn may lead to the formation of new siRNAs that enhance and further systematically spread the degrada‐

Although the pathways toward RNAi from exogenous and endogenous dsRNA converge at the RISC and use the same downstream RNAi machinery, there are also some clear differences in their processing and handling [1]. Endogenous dsRNAs cleaved by dicer (**1**) produce 20–25 bp fragments with a two-nucleotide overhang at the 3′end of siRNA duplex [1], while the length of exogenous dsRNAs-derived siRNAs, required for specificity, is unknown. Exoge‐

and discussing some of the key points presented in this book.

**2. Components, mechanism, and function**

site for RNAi [23–25].

6 RNA Interference

tion of the target mRNA in cytoplasm [26, 27].

Although the RNAi pathway is a universal process in eukaryotic cells, and it consists of similar component(s), mechanisms, and functions as described above, there are some variations among organisms in both up-take of exogenous dsRNAs and induction of RNAi. First, RNAi is systemic and heritable in plants and *C. elegans*. The systemic spreading of RNAi in plants occurs because of transfer of siRNAs between cells through plasmodesmata and the phloem [40]. Second, in plants, RNAi induces epigenetic silencing of genes through methylation of promoters of targeted genes which may be passed to the next generation [41], while in Drosophila and mammals this is not the case. Third, plant miRNAs have perfect or nearly perfect complementary to their target genes and directly cleave and degrade targeted mRNA. In contrast, animal miRNAs have one or more mismatches to target sequence and halt the translation process [42].

RNAi is not found in some eukaryotic protozoa (e.g., *Leishmania major* and *Trypanosoma cruzi*) [43, 44]. Some fungi (e.g., *Saccharomyces cerevisiae*) lack specific RNAi component(s) and the reintroduction of these missing components can recover RNAi [45, 46]. Further, prokaryotic organisms have distinctive RNA-dependent gene regulation system controlled by RNA products of translation-inhibiting genes. These regulatory RNAs are not processed by dicer enzymes, differentiating them from eukaryotic RNAi [47]. However, recently, the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) interference system has been characterized in prokaryotes, which is a gene silencing pathway analogous to eukaryotic RNAi systems [14]. The CRISPR interference system has its specific components, advantages, and limitations that are well described in the literature [48, 49], but will not be presented here. The chapter by Dr. Devi Singh and his colleagues in Section 2 of this book has a detailed coverage of RNAi in various organisms. RNAi in various organisms is also discussed in the chapter by Galay et al. presented in Section 6 of this book, highlighting the specifics of RNAi in ticks while Dr. Tayota and his colleagues present an interesting methodological paper on RNAi in the water flea in Section 3.

#### **4. Evolution**

Studies on components, mechanisms, and functions of RNAi have demonstrated variations among organisms, differences in eukaryotes and prokaryotes, and indicate that RNAi is derived from an ancestral immune defense function against transcripts of transposons and viruses [50, 51]. Although some eukaryotes might have lost RNAi components or, even, the entire pathway following the emergence of the Eukaryota, parsimony-based phylogenetic analyses suggest that an ancestral lineage of all eukaryotes possibly had a primitive RNAi capability including relevant components for some key functions such as histone modification [50]. Phylogenetic studies also indicate that miRNAs of plants and animals may have evolved independently, but the conservation of some key proteins involved in RNAi also indicate that the last common ancestor of modern eukaryotes already possessed an siRNA-based gene silencing system. The RNAi-like defense system of prokaryotes is functionally similar, but structurally distinct from the eukaryotic RNAi system [52]. It seems likely that a proto-RNAi system possessed at least some form of dicer-like, AGO, PIWI, and RdRP proteins. These basic components were shared by major eukaryotic lineages and functioned within an RNA degradation exosome complex [53].

Being an important component of an antiviral innate immune defense system in eukaryotes, RNAi components and various interaction/regulatory mechanisms, including the miRNA pathway, evolved later but at faster rates under strong directional selection [54]. This could have been a means of generating an improved response to the evolutionary arms race with viral genes. Correspondingly, some plant viruses have evolved the means to suppress the RNAi response in their host cells [55]. Extensive studies reported that an ancient duplication of RNAi components followed by species-specific gene duplications and losses provided evolutionary diversification, specificity and adaptation of the RNAi system in many organisms [56]. Chapter(s) presented in Section 2 has covered some evolutionary aspects of RNAi.

#### **5. Applications**

among organisms in both up-take of exogenous dsRNAs and induction of RNAi. First, RNAi is systemic and heritable in plants and *C. elegans*. The systemic spreading of RNAi in plants occurs because of transfer of siRNAs between cells through plasmodesmata and the phloem [40]. Second, in plants, RNAi induces epigenetic silencing of genes through methylation of promoters of targeted genes which may be passed to the next generation [41], while in Drosophila and mammals this is not the case. Third, plant miRNAs have perfect or nearly perfect complementary to their target genes and directly cleave and degrade targeted mRNA. In contrast, animal miRNAs have one or more mismatches to target sequence and halt the

RNAi is not found in some eukaryotic protozoa (e.g., *Leishmania major* and *Trypanosoma cruzi*) [43, 44]. Some fungi (e.g., *Saccharomyces cerevisiae*) lack specific RNAi component(s) and the reintroduction of these missing components can recover RNAi [45, 46]. Further, prokaryotic organisms have distinctive RNA-dependent gene regulation system controlled by RNA products of translation-inhibiting genes. These regulatory RNAs are not processed by dicer enzymes, differentiating them from eukaryotic RNAi [47]. However, recently, the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) interference system has been characterized in prokaryotes, which is a gene silencing pathway analogous to eukaryotic RNAi systems [14]. The CRISPR interference system has its specific components, advantages, and limitations that are well described in the literature [48, 49], but will not be presented here. The chapter by Dr. Devi Singh and his colleagues in Section 2 of this book has a detailed coverage of RNAi in various organisms. RNAi in various organisms is also discussed in the chapter by Galay et al. presented in Section 6 of this book, highlighting the specifics of RNAi in ticks while Dr. Tayota and his colleagues present an interesting methodological paper on RNAi in the

Studies on components, mechanisms, and functions of RNAi have demonstrated variations among organisms, differences in eukaryotes and prokaryotes, and indicate that RNAi is derived from an ancestral immune defense function against transcripts of transposons and viruses [50, 51]. Although some eukaryotes might have lost RNAi components or, even, the entire pathway following the emergence of the Eukaryota, parsimony-based phylogenetic analyses suggest that an ancestral lineage of all eukaryotes possibly had a primitive RNAi capability including relevant components for some key functions such as histone modification [50]. Phylogenetic studies also indicate that miRNAs of plants and animals may have evolved independently, but the conservation of some key proteins involved in RNAi also indicate that the last common ancestor of modern eukaryotes already possessed an siRNA-based gene silencing system. The RNAi-like defense system of prokaryotes is functionally similar, but structurally distinct from the eukaryotic RNAi system [52]. It seems likely that a proto-RNAi system possessed at least some form of dicer-like, AGO, PIWI, and RdRP proteins. These basic components were shared by major eukaryotic lineages and functioned within an RNA

translation process [42].

8 RNA Interference

water flea in Section 3.

degradation exosome complex [53].

**4. Evolution**

Since its first discovery as anti-sense gene suppression, *co-suppression* or *quelling* phenomenon, the sequence specificity, efficiency, and systemic spreading (in some organisms) characteristics of RNAi to suppress target gene expression have caught researchers' attention and soon became an attractive and powerful tool for gene function discovery in life sciences [1]. By full or partial suppression of target gene expression using RNAi, the change in cell physiology and/or developmental phenotype helps to reveal the function of the target gene. Therefore, a utilization of RNAi has revolutionized the annotation of cellular functions of many unknown and unique genes, adding to our understanding the complex genetic/biochemical pathways and their interactions. Thanks to its partial silencing effect, RNAi also helped to discover the function of genes when complete knockout would cause lethality [57]. Moreover, by targeting homologous sequences within a gene family, a single RNAi construct can suppress the expression of multiple members of a gene family, and thus reveal phenotypes that would have been missed in a single mutant due to redundancy in gene function.

The results of the functional genomics studies, advances in the understanding of the RNAi mechanism, improved design of trait-specific RNAi inducers (such as miRNAs), selection of target gene sequences combined with the development of proper delivery systems, as well as screens for "off-target" and cross reactivity have brought the practical applications of RNAi far beyond its initial experimental reach.

Agricultural application of RNAi through tissue culture-derived genetic modifications and transgenic research in a wide range of technical, food, and horticulture crops have been particularly successful and have solved many problems. Examples include, but are not limited to, crop yield and quality improvements [15, 58], food/nutrient quality improvements and fortification [59–62], decreasing the harmful precursors and carcinogens [63, 64], and im‐ provement of plant pest and disease resistance [65–66]. Many of these applications are now evaluated for commercialization or are already in commercial production [67]. In this context, targeting far red (FR) photoreceptor gene (*PHYA1*) using RNAi approach [15], our team succeed to develop the world's first RNAi cotton cultivars with improved fiber quality and other key agronomic traits without adversely affecting the yield, which successfully passed multi-environmental large field trials and have been approved for cotton farming in Uzbeki‐ stan.

Therapeutic application of RNAi has also been successful in medicine and molecular phar‐ macology with examples in inflammatory and infectious disease [68-71], cancer [72-75], as well as hereditary and neurodegenerative diseases [76]. Indeed, for many other disorders RNAi may have great potential. To highlight advances made on this field, in Section 4 of this book, we present several relevant chapters on advances of RNAi application in key human diseases of blood, ocular, nervous, kidney, and oncogenic origin. In addition, Section 5 chapters discuss RNAi utilization in various immune and infectious diseases. Section 6 chapters present the latest advances of RNAi application in studies of insects and parasitic pests such as ticks. All of these chapters highlight various aspects of RNAi and add interesting insights to the present RNAi discussions.

#### **6. Safety and risk assessment**

Manipulation of the organisms' own genetic sequence signature(s) (cis-genesis) is usually considered safer compared to "trans-genesis" that utilizes "foreign" genetic material to create genetically modified (GM) crops and its products [77]. However, for RNAi, when broken down to ~21 nucleotides this quickly may lose its meaning, as a trans-RNAi will only work if it has sufficient homology to an endogenous target transcript. Chemically, RNA is "*generally recognized as safe (GRAS)*" or it is "*rarely formally considered in risk assessmen*t" [67]. Despite this and many other examples of successful application of RNAi technology in agriculture and medicine, there may be risks associated with high or repeated dosages of dsRNA, which inadvertently may interfere with unintended target sequences. A growing body of evidences suggests that testing for the safety and assessing possible risks associated with the use of RNAiderived products sound practical, in particular, evidence of the remarkable stability of dsRNAs in the environment, their survival and resistance in the acidic conditions of the digestive tracts of higher organisms, and consequent transmissibility of dsRNA from foods to humans/ animals. Further, production of possibly harmful "secondary" dsRNAs [67] by primary RNAi inducers raised an early warning signal regarding the GRAS signature of any RNA molecule and the possibility of risks for human health and environment.

Safety concerns about RNAi-based drugs are exemplified by the lethality of 23 out of 49 distinct RNAi therapy experiments in mice because of potential "off-target" effects that could shut down non-targeted gene(s) with sequence similarity to therapeutic RNAi inducer [78]. This observed lethality, however, could be due to "oversaturation" of the dsRNA pathway and delivery issues of short hairpin RNAs [79] that needs to be optimized for harmless therapeutic applications. There are several suggested approaches to minimize or eliminate such "offtarget", "oversaturation" or delivery issues, in particular through the use of (1) comprehensive *in silico* target and off-target analyses [80], (2) modified designing of RNAi inducers with improved target selectivity, and (3) efficient delivery systems.

There may also be concerns about the uptake of intact plant miRNA by consumers through plant diet. Plant microRNAs and some long dsRNA molecules, with sequence complimentary and perfect matches to endogenous human genes, were demonstrated to survive the digestive tract of humans and can freely and routinely enter the blood system [67, 81]. *In vitro* human cell culture experiments further showed that such plant siRNA entered into human blood system could silence endogenous human genes due to sequence complementarity. While this may require attention of regulatory systems on one hand, on the other hand, human con‐ sumption of food crops with natural occurring siRNAs is considered safe and so far has not caused any dramatic biohazards or risk [81]. The chapters in Section 3 of this book also present updated information on RNAi delivery methods (e.g. Tayota et al.); synthesis, chemical and structural modifications, and designing for high specificity and selectivity of RNAi inducers (see Gvozdeva and Chernolovskaya), and limitations of RNAi and possible alternative technology such as ER-targeted intrabodies for gene silencing (see chapter by Backhaus and Böldicke).

Risk assessment and available protocols/guidelines are in the early stages of development. Some suggest that dsRNA-derived products must be subject to risk assessment studies [67]. Other findings indirectly support the safety of RNAi [81, 82], provided its use is within specific dosage ranges, the correct delivery system is in place and RNAi inducers without possible offtarget effects, unintended gene silencing and secondary dsRNA production can be designed. However, it is always advisable to admit to possible risks of any novel genomic technology, including RNAi, and consider potential biohazards and evaluate risks for environmental health, before release of a new product [58, 81–85]. To accomplish this, Heinemann et al. [67] proposed the following five-step guidance: (1) to perform detailed *in silico* comparative bioinformatics analyses for targets of designed dsRNA and identify possible "off-targets" in key consumers; (2) to experimentally quantify designed dsRNAs, and the processing of any other unknown sequence signatures or secondary dsRNA as a result of introducing intended RNAi inducer into recipient or its product; (3) to test possible biohazards and risks due to exposure of RNAi product in animal and human cell/tissue culture; (4) to conduct animal feeding experiments for the long-term physiological and toxicological patterns and possible chronic effects; and (5) to perform clinical trials of RNAi-derived products in humans.

#### **7. Conclusions and future perspectives**

Therapeutic application of RNAi has also been successful in medicine and molecular phar‐ macology with examples in inflammatory and infectious disease [68-71], cancer [72-75], as well as hereditary and neurodegenerative diseases [76]. Indeed, for many other disorders RNAi may have great potential. To highlight advances made on this field, in Section 4 of this book, we present several relevant chapters on advances of RNAi application in key human diseases of blood, ocular, nervous, kidney, and oncogenic origin. In addition, Section 5 chapters discuss RNAi utilization in various immune and infectious diseases. Section 6 chapters present the latest advances of RNAi application in studies of insects and parasitic pests such as ticks. All of these chapters highlight various aspects of RNAi and add interesting insights to the present

Manipulation of the organisms' own genetic sequence signature(s) (cis-genesis) is usually considered safer compared to "trans-genesis" that utilizes "foreign" genetic material to create genetically modified (GM) crops and its products [77]. However, for RNAi, when broken down to ~21 nucleotides this quickly may lose its meaning, as a trans-RNAi will only work if it has sufficient homology to an endogenous target transcript. Chemically, RNA is "*generally recognized as safe (GRAS)*" or it is "*rarely formally considered in risk assessmen*t" [67]. Despite this and many other examples of successful application of RNAi technology in agriculture and medicine, there may be risks associated with high or repeated dosages of dsRNA, which inadvertently may interfere with unintended target sequences. A growing body of evidences suggests that testing for the safety and assessing possible risks associated with the use of RNAiderived products sound practical, in particular, evidence of the remarkable stability of dsRNAs in the environment, their survival and resistance in the acidic conditions of the digestive tracts of higher organisms, and consequent transmissibility of dsRNA from foods to humans/ animals. Further, production of possibly harmful "secondary" dsRNAs [67] by primary RNAi inducers raised an early warning signal regarding the GRAS signature of any RNA molecule

Safety concerns about RNAi-based drugs are exemplified by the lethality of 23 out of 49 distinct RNAi therapy experiments in mice because of potential "off-target" effects that could shut down non-targeted gene(s) with sequence similarity to therapeutic RNAi inducer [78]. This observed lethality, however, could be due to "oversaturation" of the dsRNA pathway and delivery issues of short hairpin RNAs [79] that needs to be optimized for harmless therapeutic applications. There are several suggested approaches to minimize or eliminate such "offtarget", "oversaturation" or delivery issues, in particular through the use of (1) comprehensive *in silico* target and off-target analyses [80], (2) modified designing of RNAi inducers with

There may also be concerns about the uptake of intact plant miRNA by consumers through plant diet. Plant microRNAs and some long dsRNA molecules, with sequence complimentary and perfect matches to endogenous human genes, were demonstrated to survive the digestive

RNAi discussions.

10 RNA Interference

**6. Safety and risk assessment**

and the possibility of risks for human health and environment.

improved target selectivity, and (3) efficient delivery systems.

Thus, being a revolutionizing discovery in genome biology to characterize functions of any desired unknown genetic sequences, the discovery of RNAi has significantly widened our knowledge on core cellular processes. This knowledge has created opportunities and solutions to longstanding obstacles in conventional agriculture and medicine, offering a bright future to curing complex human and animal diseases, improve crop production and protection, and a sustained global food security through proper manipulation of key genes with agricultural or medicinal importance. Although key issues on specificity, selectivity, and delivery of RNAi inducing structures still exist, and some safety risks associated with the use of RNAi products have been recognized, the general believe is that RNAi is a safer technology than transgenomics utilizing "foreign" genetic information. Safe applications, however, require proper designing, dosage and delivery of RNAi inducers, and before its delivery for wide consumer market, the safety risks should be assessed. Addressing the advances made over the past three decades in RNAi research and commercialization, in this book, we have compiled and presented a diverse collection of chapters contributed by the science research communities. We all believe that RNAi, in combination with the rapidly expanding genomic information in key organisms and novel genome editing tools, will become even more powerful and efficient, and that we will all enjoy its benefits far into the future.

#### **Acknowledgements**

I thank the Academy of Sciences of Uzbekistan and Committee for Coordination Science and Technology Development of Uzbekistan for basic science FA-F5-T030; and several applied (FA-A6-T081 and FA-A6-T085) and innovation (I-2015-6-15/2 and I5-FQ-0-89-870) research grants. I am particularly grateful and thank the Office of International Research Programs (OIRP) of the United States Department of Agriculture (USDA) – Agricultural Research Service (ARS) and U.S. Civilian Research & Development Foundation (CRDF) for international cooperative grants P121, P121B, and UZB-TA-2292, which are devoted to study, development, application, risk assessment, and commercialization of RNAi cotton cultivars and their products. I sincerely thank Dr. Alexander R. van der Krol, Waginengen University, Nether‐ lands, and Dr. Eric J. Devor, Iowa State University, the USA, for their critical reading and suggestions to this chapter manuscript.

#### **Author details**

Ibrokhim Y. Abdurakhmonov\*

Address all correspondence to: genomics@uzsci.net

Center of Genomics and Bioinformatics, Academy of Science, Ministry of Agriculture and Water Resources, and "UzCottonIndustry" Association of the Republic of Uzbekistan, Tashkent, Uzbekistan

#### **References**


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presented a diverse collection of chapters contributed by the science research communities. We all believe that RNAi, in combination with the rapidly expanding genomic information in key organisms and novel genome editing tools, will become even more powerful and efficient,

I thank the Academy of Sciences of Uzbekistan and Committee for Coordination Science and Technology Development of Uzbekistan for basic science FA-F5-T030; and several applied (FA-A6-T081 and FA-A6-T085) and innovation (I-2015-6-15/2 and I5-FQ-0-89-870) research grants. I am particularly grateful and thank the Office of International Research Programs (OIRP) of the United States Department of Agriculture (USDA) – Agricultural Research Service (ARS) and U.S. Civilian Research & Development Foundation (CRDF) for international cooperative grants P121, P121B, and UZB-TA-2292, which are devoted to study, development, application, risk assessment, and commercialization of RNAi cotton cultivars and their products. I sincerely thank Dr. Alexander R. van der Krol, Waginengen University, Nether‐ lands, and Dr. Eric J. Devor, Iowa State University, the USA, for their critical reading and

Center of Genomics and Bioinformatics, Academy of Science, Ministry of Agriculture and Water Resources, and "UzCottonIndustry" Association of the Republic of Uzbekistan,

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**Acknowledgements**

12 RNA Interference

**Author details**

Tashkent, Uzbekistan

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Address all correspondence to: genomics@uzsci.net

Ibrokhim Y. Abdurakhmonov\*

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18 RNA Interference

## **RNA Interference Technology — Applications and Limitations**

Devi Singh, Sarika Chaudhary, Rajendra Kumar, Preeti Sirohi, Kamiya Mehla, Anil Sirohi, Shashi Kumar, Pooran Chand and Pankaj Kumar Singh

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61760

#### **Abstract**

RNA interference (RNAi), an evolutionarily conserved mechanism triggered by doublestranded RNA (dsRNA), causes gene silencing in a sequence-specific manner. RNAi evolved naturally to mediate protection from both endogenous and exogenous pathogen‐ ic nucleic acids and to modulate gene expression. Multiple technological advancements and precision in gene targeting have allowed a plethora of potential applications, ranging from targeting infections in crop plants to improving health in human patients, which have been reviewed in this chapter.

**Keywords:** RNA interference, miRNA, RNAi mediated gene silencing, RNA-induced si‐ lencing complex

#### **1. Introduction**

Ascribing the structure and function relationship to a gene and modulating its expression to manifest the desired phenotype have been major challenges for scientists. [1] In order to elucidate the phenotype(s) associated with a given gene, various gene-targeting techniques have been tried with mixed success. Gene silencing can be executed at transcriptional gene silencing (TGS) and posttranscriptional gene silencing (PTGS) levels. [2] The TGS involves targeting genes at DNA level by altering promoter and enhancer efficiencies, methylation status of genes, and deleting parts of genes by homologous recombination, transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palin‐ dromic repeats (CRISPR)/Cas9 systems. [3] The PTGS techniques rely upon the breakdown of mRNA by various technologies, including antisense RNA, ribozymes, DNAzymes, micro‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNAs, and RNA interference (RNAi). [4] Among all these techniques, RNAi is the most efficient tool for targeted gene silencing. RNAi is now routinely utilized across multiple biological disciplines to determine gene function. RNAi is also being utilized for therapeutic interventions to downregulate the expression of genes involved in disease pathogenesis. The current review is focused on recent advancements in the biology and applications of RNAi.

### **2. RNAi-mediated gene silencing: A historical perspective across multiple species**

#### **2.1. Discovery of RNAi in plants and fungi**

R. Jorgensen and his colleagues identified a novel mechanism of post-transcriptional gene silencing in *Petunia*. [5] They were attempting to introduce a chalcone synthase gene under a strong promoter to deepen the purple color of *Petunia* flowers; however, instead of getting a stronger purple color flower they observed that most flowers lost their color. Thus, they observed diminished expression of both the homologous endogenous gene and the exoge‐ nously introduced transgenic copy of the gene and termed the phenomenon as co-suppression. [5] Although the exact mechanism of this phenomenon remained undeciphered at that time, the posttranscriptional nature of gene silencing was still appreciated. [5-7]The suppression of endogenous gene expression by transformation of exogenous homologous sequences was later termed as quelling in *Neurospora crassa*. [8, 9]

#### **2.2. RNAi in Ceanorhabdites elegans**

In 1995, Guo and Kempheus attempted to knock down the expression of PAR-1 gene by antisense RNA in *C. elegans*; they observed a similar loss of gene expression with sense RNA controls as well [10]. At that time, they could not explain the mechanistic basis of such an observation. In 1998, Andrew Fire, Craig C. Mello, and their colleagues demonstrated efficient and specific interference of gene expression by introducing double-stranded RNA in the nematode *C. elegans* [11]. The genetic interference was genetically heritable and was stronger than the antisense strategy. This novel phenomenon was termed as RNA interference or RNAi by Fire and colleagues [11].

Subsequently, Lisa Timmons and Andrew Fire demonstrated that *C. elegans*, when fed on bacteria genetically engineered to express dsRNA for unc-22 and fem-1 genes, showed specific and reversible silencing of unc-22 and fem-1 genes in the worm [12, 13]. High-throughput genetic screens have been developed by either feeding the worms on genetically engineered bacteria expressing dsRNA or soaking or injecting the nematode with dsRNA. Functional genomic analysis of chromosomes I and III in *C. elegans* have been performed by Fraser and Gonczy, respectively, utilizing the RNA interference strategy [14, 15].

#### **2.3. RNAi technology in** *Drosophila*

Specific gene silencing has been achieved in the embryo extracts and cultured cells of *Drosophila* flies by utilizing the RNAi tool [16]. Zamore and colleagues utilized *Drosophila melanogaster* embryo lysates to demonstrate the cleavage of long dsRNA strands into short interfering dsRNA fragments (siRNA) of ~22 nucleotides (nt) [16]. Later Elbashir and colleagues demon‐ strated that chemically synthesized 21- or 22-nt-long dsRNA carrying 3′ overhangs could induce efficient RNA cleavage in embryo extracts from *Drosophila* [17].

#### **2.4. RNAi in mammalian systems**

RNAs, and RNA interference (RNAi). [4] Among all these techniques, RNAi is the most efficient tool for targeted gene silencing. RNAi is now routinely utilized across multiple biological disciplines to determine gene function. RNAi is also being utilized for therapeutic interventions to downregulate the expression of genes involved in disease pathogenesis. The current review is focused on recent advancements in the biology and applications of RNAi.

**2. RNAi-mediated gene silencing: A historical perspective across multiple**

R. Jorgensen and his colleagues identified a novel mechanism of post-transcriptional gene silencing in *Petunia*. [5] They were attempting to introduce a chalcone synthase gene under a strong promoter to deepen the purple color of *Petunia* flowers; however, instead of getting a stronger purple color flower they observed that most flowers lost their color. Thus, they observed diminished expression of both the homologous endogenous gene and the exoge‐ nously introduced transgenic copy of the gene and termed the phenomenon as co-suppression. [5] Although the exact mechanism of this phenomenon remained undeciphered at that time, the posttranscriptional nature of gene silencing was still appreciated. [5-7]The suppression of endogenous gene expression by transformation of exogenous homologous sequences was later

In 1995, Guo and Kempheus attempted to knock down the expression of PAR-1 gene by antisense RNA in *C. elegans*; they observed a similar loss of gene expression with sense RNA controls as well [10]. At that time, they could not explain the mechanistic basis of such an observation. In 1998, Andrew Fire, Craig C. Mello, and their colleagues demonstrated efficient and specific interference of gene expression by introducing double-stranded RNA in the nematode *C. elegans* [11]. The genetic interference was genetically heritable and was stronger than the antisense strategy. This novel phenomenon was termed as RNA interference or RNAi

Subsequently, Lisa Timmons and Andrew Fire demonstrated that *C. elegans*, when fed on bacteria genetically engineered to express dsRNA for unc-22 and fem-1 genes, showed specific and reversible silencing of unc-22 and fem-1 genes in the worm [12, 13]. High-throughput genetic screens have been developed by either feeding the worms on genetically engineered bacteria expressing dsRNA or soaking or injecting the nematode with dsRNA. Functional genomic analysis of chromosomes I and III in *C. elegans* have been performed by Fraser and

Specific gene silencing has been achieved in the embryo extracts and cultured cells of *Drosophila* flies by utilizing the RNAi tool [16]. Zamore and colleagues utilized *Drosophila melanogaster*

Gonczy, respectively, utilizing the RNA interference strategy [14, 15].

**species**

22 RNA Interference

**2.1. Discovery of RNAi in plants and fungi**

termed as quelling in *Neurospora crassa*. [8, 9]

**2.2. RNAi in Ceanorhabdites elegans**

by Fire and colleagues [11].

**2.3. RNAi technology in** *Drosophila*

A global nonspecific inhibition of protein synthesis was observed in mammalian cells by exposing them to dsRNAs that were greater than 30 base pairs (bp) in length [18]. RNAdependent protein kinase (PKR), and 2′, 5′ oligoadenylate synthetase (2′, 5′-OAS) were responsible for the nonspecific silencing. PKR phosphorylates eIF-2α, a translation initiation factor,toshutdownglobalproteinsynthesis.Asynthesisproductofenzyme2′,5′-OASactivates RNase L, which induces nonspecific degradation of all mRNAs in a mammalian cell [18]. Long dsRNAs induce interferon response that activates both of these enzymes in mammalian cells [19]. The nonspecific interference pathways represent the mammalian cell response to viral infection or other stress [20]. Tuschl and colleagues demonstrated that RNA interference could be directly mediated by small interference RNA (siRNA) in cultured mammalian cells [21]. However, because siRNA does not integrate into the genome, the RNAi response from siRNA is only transient. In order to induce stable gene suppression in mammalian cells, Hannon and his colleagues utilized RNA PolIII promoter-driven (e.g., U6 or H1) expression of short hairpin RNAs (shRNAs) [22]. Various approaches have since been developed for mammalian cells to obtain successful gene silencing. Some of the successful gene silencing approaches are listed in Table 1.


**Table 1.** Gene silencing approaches

#### **3. The mechanism of silencing**

RNAi-mediated gene silencing is executed by siRNAs. The process of silencing begins with the cleavage of long dsRNAs into 21–25 -nt fragments of siRNAs in cytoplasm [16, 17]. The process is catalyzed by Dicer enzyme [23]. These siRNAs are inserted into multiprotein silencing complex, which is known as RNA-induced silencing complex (RISC). Subsequent unwinding of siRNA duplex, in turn, leads to active confirmation of RISC complex (RISC\*). Next, target mRNA (mRNA to be degraded) is recognized by antisense RNA, which signals RISC complex for the endonucleolytic degradation of the homologous mRNA. Tuschl and his colleagues have defined the directionality of dsRNA processing and the target RNA cleavage sites [17]. According to their results, target mRNA is cleaved in the centre of the region that is recognized by complimentary guide siRNA, which is 10–12 -nt away from the 5′ terminus of siRNA [17]. The RNAi process is completed by the last step of siRNA molecule amplification. It is well established that the next generation of siRNAs is derived from the priming on the target mRNA by RNA-dependent RNA polymerase (RdRp) enzyme by existing siRNAs. The second generation of siRNAs is effective in inducing a secondary RNA interference that is defined as transitive RNAi. The transitive RNAi causes a systemic genetic interference in plants and *C. elegans*. Interestingly, transitive and systemic RNAi is absent in *Drosophila* and mammals owing to the lack of RdRp in both organisms [24]. An illustration of the function of RNAi is demonstrated in Figure 1.

A multitude of studies suggests a possible link between RNAi and chromatin remodeling [24]. The dsRNA works at TGS and PTGS in plants, where both pathways related and assist in gene silencing. Only TGS is heritable and drives methylation of endogenous sequences. Multiple proteins, including Polycomb in *Drosophila* and *C. elegans* [22], and Piwi in *Drosophila* [25], execute silencing at both TGS and PTGS levels. Volpe and his colleagues documented that RNAi complex proteins, including Dicer, Agronaute, and RdRp, assist in centromeric silencing in *Schizosaccharomyces pombe* [26]. This suggests that RNAi contributes to the maintenance of genomic stability [26].

#### **4. Enzymes involved in RNAi**

#### **4.1. Dicer**

Dicer was first characterized and defined in *Drosophila* by Bernstein et al. [27]. Dicer belongs to the RNase III-class and assists in ATP-dependent siRNA generation from long dsRNAs. Importantly, human Dicer does not require ATP for the cleavage of long dsRNAs [28]. Structurally, Dicer is a large (~220-kDa) multi-modular protein that acts as an antiparallel dimer. Dicer has multiple domains, including an N-terminal putative DExH/DEAH box RNA helicase/ATPase domain, an evolutionarily conserved PAZ domain, two neighboring domains that resemble RNase III, and a dsRNA-binding domain. PAZ domain in dicer helps in recognizing the end of dsRNA, whereas RNase III domain helps in the cleavage of dsRNA. Function of other domains is not fully known. Dicer orthologs has been defined in many

**Figure 1.** Mechanism of RNAi-mediated silencing. The model demonstrates double-stranded RNA (dsRNA) can gener‐ ate either from exogenous natural sources, such as a viral infection, exogenous artificial sources such as transfection, or natural synthesis. The dsRNA is then processed by a multimeric Dicer enzyme to generate siRNA that can be further amplified by RNA-dependent RNA polymerase (RdRp). The siRNA subsequently interacts with an array of proteins to form RNA-induced silencing complex (RISC) that is activated in an ATP-dependent manner. The activated RISC (RISC\*) can then induce chromatin remodeling or TGS, or induce target RNA cleavage, or cause miRNA-mediated translational inhibition.

organisms, including *S. pombe*, *Arabidopsis thaliana* (CARPEL FACTORY [CAF]), *Drosophila* (DCR-1 and DCR-2), *C. elegans* (DCR-1), mouse, and humans. In addition to RNAi, Dicer also assists in the generation of microRNAs in multiple organisms [29].

#### **4.2. RNA-Induced Silencing Complex (RISC)**

**3. The mechanism of silencing**

24 RNA Interference

demonstrated in Figure 1.

genomic stability [26].

**4.1. Dicer**

**4. Enzymes involved in RNAi**

RNAi-mediated gene silencing is executed by siRNAs. The process of silencing begins with the cleavage of long dsRNAs into 21–25 -nt fragments of siRNAs in cytoplasm [16, 17]. The process is catalyzed by Dicer enzyme [23]. These siRNAs are inserted into multiprotein silencing complex, which is known as RNA-induced silencing complex (RISC). Subsequent unwinding of siRNA duplex, in turn, leads to active confirmation of RISC complex (RISC\*). Next, target mRNA (mRNA to be degraded) is recognized by antisense RNA, which signals RISC complex for the endonucleolytic degradation of the homologous mRNA. Tuschl and his colleagues have defined the directionality of dsRNA processing and the target RNA cleavage sites [17]. According to their results, target mRNA is cleaved in the centre of the region that is recognized by complimentary guide siRNA, which is 10–12 -nt away from the 5′ terminus of siRNA [17]. The RNAi process is completed by the last step of siRNA molecule amplification. It is well established that the next generation of siRNAs is derived from the priming on the target mRNA by RNA-dependent RNA polymerase (RdRp) enzyme by existing siRNAs. The second generation of siRNAs is effective in inducing a secondary RNA interference that is defined as transitive RNAi. The transitive RNAi causes a systemic genetic interference in plants and *C. elegans*. Interestingly, transitive and systemic RNAi is absent in *Drosophila* and mammals owing to the lack of RdRp in both organisms [24]. An illustration of the function of RNAi is

A multitude of studies suggests a possible link between RNAi and chromatin remodeling [24]. The dsRNA works at TGS and PTGS in plants, where both pathways related and assist in gene silencing. Only TGS is heritable and drives methylation of endogenous sequences. Multiple proteins, including Polycomb in *Drosophila* and *C. elegans* [22], and Piwi in *Drosophila* [25], execute silencing at both TGS and PTGS levels. Volpe and his colleagues documented that RNAi complex proteins, including Dicer, Agronaute, and RdRp, assist in centromeric silencing in *Schizosaccharomyces pombe* [26]. This suggests that RNAi contributes to the maintenance of

Dicer was first characterized and defined in *Drosophila* by Bernstein et al. [27]. Dicer belongs to the RNase III-class and assists in ATP-dependent siRNA generation from long dsRNAs. Importantly, human Dicer does not require ATP for the cleavage of long dsRNAs [28]. Structurally, Dicer is a large (~220-kDa) multi-modular protein that acts as an antiparallel dimer. Dicer has multiple domains, including an N-terminal putative DExH/DEAH box RNA helicase/ATPase domain, an evolutionarily conserved PAZ domain, two neighboring domains that resemble RNase III, and a dsRNA-binding domain. PAZ domain in dicer helps in recognizing the end of dsRNA, whereas RNase III domain helps in the cleavage of dsRNA. Function of other domains is not fully known. Dicer orthologs has been defined in many RISC is a ribonucleoprotein complex that fragments mRNAs through the production of a sequence-specific nuclease. At first, while working on *Drosophila* embryo extracts, Zamore and his colleagues identified ~250 kDa precursor complex, which turns into an activated complex of 100 kDa upon addition of ATP. However, Hannon and his colleagues found a 500-kDa complex from *Drosophila* S2 cells [30, 31]. The siRNA is an important part of RISC and was the first to be identified. It acts as a template and guides RISC to the target mRNA molecule. To date, a number of RISC protein components are known in *Drosophila* and mammalian species. Interestingly, these components are not completely overlapping, which suggests the develop‐ mental stage-specific or evolutionarily non-conserved nature of the components of RISC complex [24].

The first RISC protein component identified was Agronaute-2, a *C. elegans* RDE-1 homologue [32]. Argonaute (AGO) proteins are part of an evolutionarily conserved protein family and they play a central role in RNAi, determination of stem cell developmental regulation, and tumorigenesis. AGOs are ~100 kDa highly basic proteins that contain N-terminal PAZ and mid- and C-terminal PIWI domains [33]. The PAZ domain is an RNA-binding module, which is involved in protein–protein interactions, whereas PIWI is essentially required for target cleavage. Some AGO proteins that are involved in RNAi are listed in the Table 2.


**Table 2.** Argonaute homolog proteins in RNAi

Some RISC components are non-AGO proteins, including dFXR and VIG in *Drosophila,* the fragile X mental retardation 1 (FMR1) homolog in *Drosophila*, and germin3/4 in mammals [34].

#### **4.3. RNA helicase**

RNA helicases cause unwinding of dsRNA. However, Dicer contains its own helicase activity in the N-terminal helicase domain. Hence, the helicase proteins putatively function down‐ stream of the RISC complex. Two major RNA helicase families are involved in RNAi [35]. SDE3 from *A. thaliana* and its homologous proteins in mouse, human, and *Drosophila* constitute the first such helicase family. The second family contains Upf1p from yeast and an Upf1 homo‐ logue (SMG-2) in *C. elegans*. The Upf1/SMG-2 is characterized by cysteine-rich motif conserved across species and multiple C-terminal Ser-Gln (SQ) doublets. MUT-6, a DEAH-box helicase in *C. elegans* is also putatively involved in transposon suppression. Another RNA helicase Germin3 resides in complex with human AGO protein EIF2C2/hAgo2 [36].

#### **4.4. RNA-dependent RNA polymerase (RdRp)**

The first RISC protein component identified was Agronaute-2, a *C. elegans* RDE-1 homologue [32]. Argonaute (AGO) proteins are part of an evolutionarily conserved protein family and they play a central role in RNAi, determination of stem cell developmental regulation, and tumorigenesis. AGOs are ~100 kDa highly basic proteins that contain N-terminal PAZ and mid- and C-terminal PIWI domains [33]. The PAZ domain is an RNA-binding module, which is involved in protein–protein interactions, whereas PIWI is essentially required for target

cleavage. Some AGO proteins that are involved in RNAi are listed in the Table 2.

PTGS

PPW-1 Essential for germline RNAi

dAgro1 Essential in embryos, acts

dAgo2 Required component of RISC dAgo3 Prediction based on DNA sequence PIWI Essential for PTGS and TGS

cleavage

Some RISC components are non-AGO proteins, including dFXR and VIG in *Drosophila,* the fragile X mental retardation 1 (FMR1) homolog in *Drosophila*, and germin3/4 in mammals [34].

RNA helicases cause unwinding of dsRNA. However, Dicer contains its own helicase activity in the N-terminal helicase domain. Hence, the helicase proteins putatively function down‐

generation.

RNA helicase Vasa. Essential for maturation-dependent RNAi

downstream of RNAi generation

catalyzes the miRNA –directed

**Species Argonaute homolog Essentiality for RNAi Citations**

ZWILE Non-essential

ALG-1 Nonessential ALG-2 Nonessential

*Drosophila* Aubergine Localizes with dsRBP Staufen and

Mammals (human) EIF2C2/hAgo2 Part of RISC complex and

**Table 2.** Argonaute homolog proteins in RNAi

**4.3. RNA helicase**

26 RNA Interference

*Arabidopsis* AGO1 Essential for co-suppression and

*Tetrahymena* TWI1 Essential for DNA elimination *Neurospora* QDE2 Required component of RISC *C. elegans* RDE-1 Forms complex with Dicer

RdRp catalyzes the amplification and triggering of RNAi, which is usually in small amounts. RdRp catalyzes the siRNA-primed amplification by polymerase chain reaction to convert mRNA into dsRNAs, a long form that is cleaved to produce new siRNAs [37]. Lipardi and his colleagues demonstrated RdRp-like activity in *Drosophila* embryo extracts, but the enzyme responsible for the RdRp activity in the *Drosophila* or human is not known. Some of the RdRps involved in RNAi have been summarized in Table 3.


**Table 3.** RdRps involved in RNAi

#### **5. Various small RNA isoforms related to RNAi**

#### **5.1. Small interfering RNAs (siRNAs)**

Small interfering RNAs are 21–23-nt-long double-stranded RNA molecules with 2–3-nt overhangs at the 3′ termini. siRNAs are normally generated, as mentioned in the above sections, by the cleavage of long double-stranded RNAs by RNase III (Dicer) [16]. siRNAs must be phosphorylated at the 5′ termini by endogenous kinases to enter into the RISC complex [31]. It is thought that the hydroxylated 3′ termini are essential for the siRNA-primed amplification step catalyzed by RdRps. However, Zamore et al. showed that non-priming alterations in the 3′ hydroxyl group did not adversely affect RNAi-mediated silencing [38]. They went on to explain that siRNAs operate as guide RNAs for gene repression but not as primers in the human and *Drosophila* RNAi pathways [38]. Conversely, Hamada et al. showed in mammalian cells that modifying the 3′ end of the antisense strand of siRNA abolished the RNAi effect, while modifying the 3′ end of the sense strand did not affect the RNAi silencing [39]. These findings support the model that each strand of siRNA has different functions in the RNAi process, and the 3′ hydroxylated end of the antisense strand may prime the amplification. Ambros et al. discovered endogenous siRNA in more than 500 genes in wild-type *C. elegans* [39]. This suggests that siRNA may be a globally conserved and common molecule among species.

#### **5.2. Micro RNAs (miRNAs)**

miRNAs are 19–25-nt small RNA species produced by Dicer-mediated cleavage of endogenous ~70-nt noncoding stem-loop precursors. The miRNAs, while allowing mismatches, can either repress the target mRNA translation (mostly in mammals) or facilitate mRNA destruction (mostly in plants) [40]. miRNAs *lin*-4 and *let-*7 were the first ones to be identified in *C. elegans* [40]. So far, about 2000 different miRNAs have been identified in plants, animals, and lower species. While some miRNAs are evolutionarily conserved, others are specific for some developmental stages or are species-specific. Different terminologies are referred to in literature. According to one terminology, the miRNAs with well-characterized functions (e.g., *lin-4 and let-7*) are referred to as small temporal RNAs (stRNAs), while other similar small RNAs of unknown functions are called miRNAs [40]. Multiple miRNAs have been character‐ ized for their physiological roles in cancer and other diseases [41, 42]. Comparisons between siRNA and miRNA have been listed in the Table 4.


**Table 4.** Comparative characteristics of siRNA and miRNA

#### **5.3. Tiny noncoding RNAs (tncRNAs)**

while modifying the 3′ end of the sense strand did not affect the RNAi silencing [39]. These findings support the model that each strand of siRNA has different functions in the RNAi process, and the 3′ hydroxylated end of the antisense strand may prime the amplification. Ambros et al. discovered endogenous siRNA in more than 500 genes in wild-type *C. elegans* [39]. This suggests that siRNA may be a globally conserved and common molecule among

miRNAs are 19–25-nt small RNA species produced by Dicer-mediated cleavage of endogenous ~70-nt noncoding stem-loop precursors. The miRNAs, while allowing mismatches, can either repress the target mRNA translation (mostly in mammals) or facilitate mRNA destruction (mostly in plants) [40]. miRNAs *lin*-4 and *let-*7 were the first ones to be identified in *C. elegans* [40]. So far, about 2000 different miRNAs have been identified in plants, animals, and lower species. While some miRNAs are evolutionarily conserved, others are specific for some developmental stages or are species-specific. Different terminologies are referred to in literature. According to one terminology, the miRNAs with well-characterized functions (e.g., *lin-4 and let-7*) are referred to as small temporal RNAs (stRNAs), while other similar small RNAs of unknown functions are called miRNAs [40]. Multiple miRNAs have been character‐ ized for their physiological roles in cancer and other diseases [41, 42]. Comparisons between

1. The siRNAs require processing from long dsRNAs. 1. The miRNAs require processing from stem-loop precursors

that are ~70 nt long.

nucleotides.

proliferation and death.

3. The siRNAs mediate target mRNA cleavage by RISC. 3. The miRNAs can either block target mRNA translation by

1. The miRNAs are single-stranded structures.

2. The miRNAs can function even with a few mismatched

binding to it or mediate target mRNA cleavage by RISC.

4. The miRNAs are constitutively expressed cellular RNA moieties with potential roles in development, and cell

species.

28 RNA Interference

**Resemblences**

**Disparities**

bind and cleave.

**5.2. Micro RNAs (miRNAs)**

siRNA and miRNA have been listed in the Table 4.

**siRNA miRNA**

**siRNA miRNA**

1. The siRNAs are double-stranded structures with 2-nt 3′ overhangs that are formed during cleavage by Dicer.

2. The siRNA require high homology with the mRNA to

4. The siRNAs are usually triggered by transgene incorporation, viral infection, or active transposons.

**Table 4.** Comparative characteristics of siRNA and miRNA

2. An RNase III enzyme Dicer is required for processing. 2. Dicer is required.

3. The siRNAs are usually ~22 nt long. 3. The miRNAs are also ~22 nt long.

Ambros and his colleagues discovered the first tncRNAs in *C. elegans*. They identified and characterized 33 new tncRNAs in *C. elegans* by performing cDNA sequencing and comparative genetics [40]. The tncRNAs are very similar to miRNAs with regard to their size, singlestranded structure, and lack of a precise complementarity to a given mRNA. However, they are distinct with regard to their lack of processing from a "miRNA-like hairpin precursor", and phylogenetic nonconservation. Similarly to miRNA, tncRNAs are transcribed from noncoding sequences. However, their developmental role is not fully understood. According to Ambros and his colleagues, it is plausible that some of the miRNAs might be processed from noncoding mRNAs in the course of RNAi [40].

#### **6. Evolutionary relevance of RNAi in the immunological responses**

RNAi may provide a systemic way to immunize an organism against the invasive nucleic acids from viruses and transposons via inducing the RNAi responses. Virus-induced gene silencing (VIGS) in plants is accomplished by RNAi. Multiple genetic links between RNAi and virulence are known. Many plant viruses code for viral suppressors of gene silencing (VSGS). VSGS acts as a virulence determinant, and hence, is required for developing antivirulence response in the host. In response to the virulence, the host can also modify its PTGS/RNAi mechanisms to prevent future infections. RNAi can even target DNA virus amplification in plants [43]. VIGS mechanisms exist not only in plants and nematodes but also in other species; for example, flock house virus (FHV), a virus that infects *Drosophila*, also codes for a potential silencing suppressor (b2) [24]. Nonetheless, the precise function of RNAi in mammalian antiviral defense is not clear.

RNAi also plays a crucial role in the development process of multicellular organisms. When mutated, *CARPEL FACTORY*, a Dicer homologue in *Arabidopsis*, can cause developmentally defective leaves and induce overproliferation of floral meristems. Inactivation of Dicer by mutations causes developmental problems and sterility in *C. elegans*. Mutations in AGO protein influence normal development in *Drosophila* as well. Hence, components of RNAi pathway play a significant role in normal development, but such components and the affiliated gene products play crucial roles in related but distinct gene regulation pathways [23].

A potential role of RNAi and human disease pathogenesis has been proposed due to associa‐ tion of RNA binding proteins with RISC complex, such as Vasa intronic gene (VIG) and the fragile X mental retardation protein (FMRP) *Drosophila* homologue [36].

#### **7. RNAi as a functional genomics tool and its applications for therapy**

RNAi technology is applicable for gene silencing in many species. RNAi has been used extensively in *C. elegans* for functional genomics. High-throughput investigation of most of the ~19,000 genes has been accomplished. Ahringer and his colleagues produced an RNAi library, representing ~86% of the genes of *C. elegans*[15]. This strategy has been successfully attempted in multiple other model organisms, including human [44].

RNAi has also been utilized successfully in mammalian cells [44]. Various methods have been employed for siRNA knockdown of specific genes in mammalian cells. DNA-vector-mediated RNAi silences genes transiently in mammalian cells, while other expression systems are used for stable silencing. The promoters of RNA polymerase (pol) II and III (U6 and H1, alone or together) have been used for stable silencing. Furthermore, tRNA promoter-based systems have been used for this purpose. However, pol III-based short hairpin RNA (shRNA) expres‐ sion systems (e.g., H1 RNA pol-based pSuper vector) are suitable choices. Retroviral-vectorbased delivery of siRNAs has also been utilized for more efficient silencing. Two classes of retrovirus vectors have been employed: (1) HIV-1-derived lentivirus vectors and (2) Oncore‐ trovirus-based vectors, such as Moloney murine leukemia virus (MoMuLV) and Murine stem cell virus (MSCV). Transgenic mice have been established with germline transmission of a shRNA expression cassette for silencing of genes not targeted by homologous recombinationbased approaches [45]. Desirable applications of this technique include inducible and cell typespecific expression patterns.

The use of RNAi is not limited to the determination of mammalian gene function, and also could be used for treating viral infections and cancer [46, 47]. Viral and human genes that are needed for viral replication can be attacked to generate viral-resistant host cells or to treat viral infections [47]. Oncogenes, which accelerate cancer growth, can be targeted by RNAi [48, 49]. Targeting of molecules important for neovascularization could prevent tumor growth [50]. This book presented several chapters with detailed discussions of therapeutic aspects of the RNAi in immune, blood, cancer, and brain diseases. We refer readers to those chapters (by Hu et al.; Gu; and Cho and Kim) rather to continue repeated information here.

#### **8. Conclusions**

Fast progress in RNAi technology has shown promise for use in reverse genetics and therapy. However, mechanistic complexities of this technology still need to be determined. RNAi has now been established as a revolutionary tool for functional genomics in organisms. Multiple studies have defined the role of RNAi in mammalian and plant defense systems. A plethora of studies have utilized RNAi technology to modulate gene expression. RNAi-based full genomic screens have allowed identification of specific genes, controlling a given trait with high accuracy. Further studies will continue to unravel the unlimited potential of RNAi to serve humankind.

#### **Author details**

~19,000 genes has been accomplished. Ahringer and his colleagues produced an RNAi library, representing ~86% of the genes of *C. elegans*[15]. This strategy has been successfully attempted

RNAi has also been utilized successfully in mammalian cells [44]. Various methods have been employed for siRNA knockdown of specific genes in mammalian cells. DNA-vector-mediated RNAi silences genes transiently in mammalian cells, while other expression systems are used for stable silencing. The promoters of RNA polymerase (pol) II and III (U6 and H1, alone or together) have been used for stable silencing. Furthermore, tRNA promoter-based systems have been used for this purpose. However, pol III-based short hairpin RNA (shRNA) expres‐ sion systems (e.g., H1 RNA pol-based pSuper vector) are suitable choices. Retroviral-vectorbased delivery of siRNAs has also been utilized for more efficient silencing. Two classes of retrovirus vectors have been employed: (1) HIV-1-derived lentivirus vectors and (2) Oncore‐ trovirus-based vectors, such as Moloney murine leukemia virus (MoMuLV) and Murine stem cell virus (MSCV). Transgenic mice have been established with germline transmission of a shRNA expression cassette for silencing of genes not targeted by homologous recombinationbased approaches [45]. Desirable applications of this technique include inducible and cell type-

The use of RNAi is not limited to the determination of mammalian gene function, and also could be used for treating viral infections and cancer [46, 47]. Viral and human genes that are needed for viral replication can be attacked to generate viral-resistant host cells or to treat viral infections [47]. Oncogenes, which accelerate cancer growth, can be targeted by RNAi [48, 49]. Targeting of molecules important for neovascularization could prevent tumor growth [50]. This book presented several chapters with detailed discussions of therapeutic aspects of the RNAi in immune, blood, cancer, and brain diseases. We refer readers to those chapters (by Hu

Fast progress in RNAi technology has shown promise for use in reverse genetics and therapy. However, mechanistic complexities of this technology still need to be determined. RNAi has now been established as a revolutionary tool for functional genomics in organisms. Multiple studies have defined the role of RNAi in mammalian and plant defense systems. A plethora of studies have utilized RNAi technology to modulate gene expression. RNAi-based full genomic screens have allowed identification of specific genes, controlling a given trait with high accuracy. Further studies will continue to unravel the unlimited potential of RNAi to

et al.; Gu; and Cho and Kim) rather to continue repeated information here.

in multiple other model organisms, including human [44].

specific expression patterns.

30 RNA Interference

**8. Conclusions**

serve humankind.

Devi Singh1\*, Sarika Chaudhary2 , Rajendra Kumar3 , Preeti Sirohi1 , Kamiya Mehla4 , Anil Sirohi1 , Shashi Kumar5 , Pooran Chand1 and Pankaj Kumar Singh4

\*Address all correspondence to: devisingh11@gmail.com

1 Molecular Biology laboratory, Department of Genetics and Plant Breeding, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, India

2 Institute of Genomics and Integrative Biology, New Delhi, India

3 Department of Agri-Biotechnology, SardarVallabhbhai Patel University of Agricultureand Technology, Meerut, India

4 Eppley Institute for Research in Cancer and Allied Diseases, UNMC, Omaha, Nebraska, USA

5 International Centre for Genetic Engineering and Biotechnology, New Delhi, India

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## **The Role of Immune Modulatory MicroRNAs in Tumors**

Barbara Seliger, Anne Meinhardt and Doerte Falke

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61805

#### **Abstract**

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Tumors could evade the control of CD8+ T and/or NK cell-mediated surveillance by dis‐ tinct immune escape strategies. These include the aberrant expression of HLA class I anti‐ gens, coinhibitory or costimulatory molecules, and components of the interferon (IFN) signal transduction pathway. In addition, alterations of the tumor microenvironment could interfere with a proper antitumoral immune response by downregulating or inhib‐ iting the frequency and/or activity of immune effector cells and professional antigen pre‐ senting cells. Based on the identification as major mediators of the posttranscriptional silencing of gene expression, microRNAs (miRNAs) have been suggested to play a key role in many biological processes known to be involved in neoplastic transformation. In‐ deed, miRNA expression is frequently deregulated in many cancer types and could have tumor-suppressive as well as oncogenic potential. This review focused on the characteri‐ zation of miRNAs, which are involved in the control of the immune surveillance or im‐ mune escape of tumors and their use as potential diagnostic and prognostic biomarkers as well as therapeutic targets. Moreover, miRNAs can have dual activities by affecting the neoplastic and immunogenic phenotype of tumors.

**Keywords:** APM, IFN, immune escape, microRNA, tumor microenvironment

#### **1. Introduction**

Tumors have developed different strategies to evade immune recognition by cytotoxic T lymphocytes (CTLs) as well as natural killer (NK) cells. This is caused by alterations of the tumor itself, changes of the tumor microenvironment (TME), reduced frequency, and impaired function of diverse immune subpopulations. The processes leading to immune evasion of tumors are diverse and could be associated with structural alterations and/or deregulation of genes/proteins from tumor cells, but also from different immune cells important for recogni‐ tion and killing of tumor cells or in the induction of immune suppression. The identification of microRNAs (miRNAs), involved in the RNA interference (RNAi)-based control of these

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

immune modulatory molecules, clarified the complexity of the mechanisms conditioning tumor immune escape. This review is focused on the identification and characterization of immune modulatory miRNAs (im-miRNAs) in tumors, thereby altering the antitumoral immune response by miRNAi-mediated RNAi.

#### **2. The MHC class I antigen processing and presentation machinery (APM)**

The major histocompatibility complex class I (MHC) molecules present an array of peptide epitopes for surveillance by CD8+ T cells. These peptides are classically derived from proteins synthesized in the cytosol. Upon proteasomal degradation of ubiquitinated proteins, the yielded peptides are then transported into the endoplasmic reticulum (ER) via the heterodi‐ meric peptide transporter associated with antigen processing (TAP). The peptide transport into the ER is ATP dependent and sequence specific. The TAP heterodimer associates in ER with a number of other proteins to form the peptide loading complex (PLC). These include the chaperone tapasin, which recruits MHC class I heavy chain (HC)/β2-microglobulin (β2-m) dimers and calreticulin. The peptides are either trimmed by ER-resident aminopeptidases or directly loaded onto MHC class I molecules [1]. Upon peptide loading, the PLC dissociates from the trimer consisting of the MHC class I HC, β2-m, and peptide is then transported via the trans Golgi to the cell surface and exposed to CD8+ CTLs [1].

#### **3. Immune stimulatory and immune inhibitory molecules and immune response**

An effective T-cell response requires two signals. The first is mediated by the interaction with MHC class I antigens on the antigen-presenting cells (APC), and the second is mediated by the interaction of B7 family members on APC with CD28 or CTLA4 on T cells. The prototypes of B7 family members are B7-1 (CD80) and B7-2 (CD86). During the last years, the B7 family was growing consisting of B7-H1 (PDL-1), B7-H2, B7-H3, B7-H4, and B7-H6 molecules [2, 3]. While B7-H1 and B7-H4 represent coinhibitory molecules, B7-H2 was identified as costimu‐ latory molecule, which is mainly expressed on B cells, monocytes and dendritic cells (DC): B7- H2 binds to the receptor ICOS, which results in activation of T cells through phosphatidylinositol-3-kinase-dependent signal transduction pathways and in the induction of Th2 cell-mediated immune response, proliferation, and cytokine production [4]. The role of B7-H3 is currently controversially discussed and depends on the cell types analyzed, demon‐ strating either costimulatory or coinhibitory activity. Regarding B7-H4, its expression is primarily restricted to activated T cells, B cells, monocytes, and DCs [5, 6]. B7-H4 is not detected in the majority of normal tissues and cells but is overexpressed in a variety of tumor tissues. B7-H6 has been identified as ligand for the NK cell receptor NKp30 and is detectable on surface or in the cytosol of tumor cells and as soluble factor in the peritoneal fluid [7], while it is not expressed on healthy cells. The interaction of B7-H6 with NKp30 is involved in NK cell responses [8]. It is noteworthy that many other coinhibitory molecules have also been identi‐ fied and their role on immune responses is currently under investigation [2, 3].

#### **4. Features of the interferon-γ-mediated signal transduction**

immune modulatory molecules, clarified the complexity of the mechanisms conditioning tumor immune escape. This review is focused on the identification and characterization of immune modulatory miRNAs (im-miRNAs) in tumors, thereby altering the antitumoral

**2. The MHC class I antigen processing and presentation machinery (APM)**

The major histocompatibility complex class I (MHC) molecules present an array of peptide epitopes for surveillance by CD8+ T cells. These peptides are classically derived from proteins synthesized in the cytosol. Upon proteasomal degradation of ubiquitinated proteins, the yielded peptides are then transported into the endoplasmic reticulum (ER) via the heterodi‐ meric peptide transporter associated with antigen processing (TAP). The peptide transport into the ER is ATP dependent and sequence specific. The TAP heterodimer associates in ER with a number of other proteins to form the peptide loading complex (PLC). These include the chaperone tapasin, which recruits MHC class I heavy chain (HC)/β2-microglobulin (β2-m) dimers and calreticulin. The peptides are either trimmed by ER-resident aminopeptidases or directly loaded onto MHC class I molecules [1]. Upon peptide loading, the PLC dissociates from the trimer consisting of the MHC class I HC, β2-m, and peptide is then transported via

**3. Immune stimulatory and immune inhibitory molecules and immune**

An effective T-cell response requires two signals. The first is mediated by the interaction with MHC class I antigens on the antigen-presenting cells (APC), and the second is mediated by the interaction of B7 family members on APC with CD28 or CTLA4 on T cells. The prototypes of B7 family members are B7-1 (CD80) and B7-2 (CD86). During the last years, the B7 family was growing consisting of B7-H1 (PDL-1), B7-H2, B7-H3, B7-H4, and B7-H6 molecules [2, 3]. While B7-H1 and B7-H4 represent coinhibitory molecules, B7-H2 was identified as costimu‐ latory molecule, which is mainly expressed on B cells, monocytes and dendritic cells (DC): B7- H2 binds to the receptor ICOS, which results in activation of T cells through phosphatidylinositol-3-kinase-dependent signal transduction pathways and in the induction of Th2 cell-mediated immune response, proliferation, and cytokine production [4]. The role of B7-H3 is currently controversially discussed and depends on the cell types analyzed, demon‐ strating either costimulatory or coinhibitory activity. Regarding B7-H4, its expression is primarily restricted to activated T cells, B cells, monocytes, and DCs [5, 6]. B7-H4 is not detected in the majority of normal tissues and cells but is overexpressed in a variety of tumor tissues. B7-H6 has been identified as ligand for the NK cell receptor NKp30 and is detectable on surface or in the cytosol of tumor cells and as soluble factor in the peritoneal fluid [7], while it is not expressed on healthy cells. The interaction of B7-H6 with NKp30 is involved in NK cell

CTLs [1].

immune response by miRNAi-mediated RNAi.

the trans Golgi to the cell surface and exposed to CD8+

**response**

38 RNA Interference

Interferons (IFN) are a group of pleiotropic cytokines that play a key role in the intercellular communication during innate and adaptive immune responses, in particular in the host defense against viral and bacterial infections and neoplastic transformation [9]. The IFN family could be classified into type I and type II IFNs, which differ in their activity regarding immune modulation [10].

IFN-γ belongs to the type II IFN and is a central regulator of immune responses by controlling and modulating the expression of targets essential for cell-cell communication and cellular interactions. It is secreted by activated T cells, NK cells, and macrophages and induced by DC and monocytes stimulated with bacterial cell wall components [11]. IFN-γ exerts its activity by binding to its heterodimeric receptor consisting of IFN-γ-R1 and IFN-γ-R2 subunits [12, 13]. This results in the dimerization of the receptor subunits followed by activation (trans‐ phosphorylation) of the receptor-associated tyrosine kinases JAK1 and JAK2 belonging to the Janus kinase (JAK) family and phosphorylation and dimerization of the JAK-associated STAT1 transcription factor. The activated STAT1 is translocated into the nucleus and recruited to the IFN-γ-activated sequence (GAS) element of the promoters of the STAT1 target genes leading to their transcriptional activation.

IFN-γ-regulated genes can be classified into primary and secondary responsive genes. Primary responsive genes are induced early due to the binding of STAT dimers to the GAS element in the promoter region of target genes, like IRF1, CXCL9, and CXCL10 [14]. IRF1 binds to IFNstimulated response elements (ISRE) and modulates gene induction of the secondary respon‐ sive genes. IFN-γ induced the transcription of MHC class I and class II antigens and of many APM components and at high concentrations could lead to a caspase-dependent apoptosis. In addition, IFN-γ is involved in amplifying toll-like receptor (TLR) signaling by increasing or inhibiting the transcription of TLRs, chemokines, and cytokines [15, 16]. Furthermore, IFN-γ promotes the induction of SOCs proteins (suppressor of cytokine signaling), which inhibit IFNγ signaling by a negative feedback loop, resulting in the inactivation of JAK1 and JAK2 [17]. Moreover, IFN-γ signaling is controlled by inhibiting JAK1, JAK2, and IFN-γ-R1 via dephos‐ phorylation mediated by SH2-domain-containing protein tyrosine phosphatase 2 [18], by proteasomal degradation of JAK1 and JAK2 [18] and by inhibition of STAT1, which is mediated by the protein inhibitor of activated STAT1 [19].

#### **5. Distinct levels of tumor immune escape**

Tumors have developed different strategies to escape immune surveillance, which could occur at the level of immune cells, tumor micromilieu, and the tumor itself (Figure 1). The frequency, activity, and function of CD8+ and CD4+ T lymphocytes, DC, NK cells, and B cells are often downregulated in peripheral blood of tumor patients, while the number of immune-suppres‐ sive myeloid-derived suppressor cells (MDSC), NKT cells, and regulatory T cells (Treg) is upregulated [20-23].

**Figure 1.** Tumor immune escape mechanisms containing (A) loss of tumor antigen expression; (B) variations in tumor antigen processing; (C) defects in the peptide transporter TAP, chaperone tapasin, and protease ERAP1; (D) defects in expression of MHC class I heavy chain and β2-microglobulin; (E) release of anti-inflammatory cytokines; (F) downre‐ gulation of IFN-γ-R1, JAK1, JAK2, STAT1, and STAT2; (G) altered phosphorylation states of STAT1; (H) impaired up‐ regulation of IFN-γ regulated genes (MHC class I, APM); (I) upregulated expression of HLA-G; (J) altered methylation pattern of IRF; (K) overexpression of SOCS1; and (L) protection from T-cell-mediated apoptosis.

The tumor microenvironment (TME) consists of various cellular and soluble factors and is of clinical relevance since its composition significantly correlates with the tumor patients' outcome. These include different cellular components, such as fibroblasts, blood vessels, immune cells, stroma cells, extracellular matrix, and soluble factors such as immune-suppres‐ sive cytokines, like interleukin (IL)-10, transforming growth factor (TGF)-β, metabolites, arginase and prostaglandin, hypoxia, and pH, which negatively interfere with the antitumoral immune responses.

In tumors, an aggressive and deregulated growth of neoplastic transformed cells, which overexpress proangiogenic factors, such as the vascular endothelial growth factor (VEGF), leads to the development of organized blood vessels. These blood vessels are fundamentally different from the normal vasculature. Tumor-associated fibroblasts (TAF) represent the major constituents of the tumor stroma and produce growth factors, including the VEGF and inhibitory cytokines that activate extracellular matrix thereby contributing to the tumor growth.

Furthermore, cancer is often driven by inflammation mediated by monocytes and tumorassociated macrophages (TAM), which belong to the innate immune cells. Macrophages could be classified in type 1 and 2 macrophages. While M1 macrophages express a series of proin‐ flammatory cytokines, chemokines and effector molecules, the M2 macrophages express a wide array of anti-inflammatory molecules, including IL-10, IL-35, TGF-β, and adenosine. TAMs are mainly of the M2 phenotype and secrete different cytokines, chemokines and proteases, which promote tumor angiogenesis, growth, metastasis as well as immune sup‐ pression.

In addition to TAM and TAF, MDSC represent a heterogeneous population derived from myeloid progenitors [20]. They can promote tumor growth by enhancing angiogenesis or suppression of innate and adaptive immune responses. Regarding the innate immune cells, MDSCs suppress NK cell cytotoxicity, promote M2 macrophage differentiation, and modulate the priming activity of mature DC [24]. Moreover, MDSCs suppress T-cell responses by induction of apoptosis, secretion of immune modulatory factors, modulation of amino acid metabolism, restriction of T-cell homing, and induction of Treg [25-28]. Tregs suppress the activity of immune cells and maintain immune tolerance to self-antigens. They express CD4, CD25 and FoxP3 [22]. The elevated numbers of Tregs in cancer is due to their efficient migration into the tumor sites [29], local expansion in the tumor environment[29], and *de novo* generation within the tumor [30].

#### **5.1. Alterations of the tumors**

activity, and function of CD8+

upregulated [20-23].

40 RNA Interference

immune responses.

and CD4+

downregulated in peripheral blood of tumor patients, while the number of immune-suppres‐ sive myeloid-derived suppressor cells (MDSC), NKT cells, and regulatory T cells (Treg) is

**Figure 1.** Tumor immune escape mechanisms containing (A) loss of tumor antigen expression; (B) variations in tumor antigen processing; (C) defects in the peptide transporter TAP, chaperone tapasin, and protease ERAP1; (D) defects in expression of MHC class I heavy chain and β2-microglobulin; (E) release of anti-inflammatory cytokines; (F) downre‐ gulation of IFN-γ-R1, JAK1, JAK2, STAT1, and STAT2; (G) altered phosphorylation states of STAT1; (H) impaired up‐ regulation of IFN-γ regulated genes (MHC class I, APM); (I) upregulated expression of HLA-G; (J) altered methylation

The tumor microenvironment (TME) consists of various cellular and soluble factors and is of clinical relevance since its composition significantly correlates with the tumor patients' outcome. These include different cellular components, such as fibroblasts, blood vessels, immune cells, stroma cells, extracellular matrix, and soluble factors such as immune-suppres‐ sive cytokines, like interleukin (IL)-10, transforming growth factor (TGF)-β, metabolites, arginase and prostaglandin, hypoxia, and pH, which negatively interfere with the antitumoral

pattern of IRF; (K) overexpression of SOCS1; and (L) protection from T-cell-mediated apoptosis.

T lymphocytes, DC, NK cells, and B cells are often

Immune escape mechanisms include loss or downregulation of HLA class I antigens and/or components of the antigen processing machinery (APM), upregulation of nonclassical HLA-G and HLA-E antigens, and coinhibitory molecules, including PDL-1, as well as alterations of signaling transduction cascades, including in particular the IFN signaling pathway [31]. The frequency of these different mechanisms highly varied between tumor (sub)types and is often correlated with a worse prognosis and reduced survival of tumor patients.

#### *5.1.1. MHC class I abnormalities*

The classical MHC class I pathway and the APM components are involved in eradication of developing tumors [32]. Since CD8+ CTLs recognize and eliminate cells presenting tumor antigens via HLA class I molecules, loss of HLA class I expression results in evasion of CTLmediated cell death [33]. Abnormalities of HLA class I antigens are often due to downregula‐ tion of various components of the MHC class I APM, in particular of TAP, tapasin, β2-m, and MHC class I HC. Structural alterations of these components are rare, while MHC class I defects are mainly due to deregulation of the different components, which could be controlled at the transcriptional, epigenetic (methylation, acetylation), posttranscriptional (e.g., microRNAs, protein degradation), or posttranslational (phosphorylation) level.

HLA-G has been demonstrated as a nonclassical HLA class I antigen, which is in general only expressed on immune privileged organs, but also on many tumors of distinct origin [34]. The overexpression of HLA-G or secretion of soluble HLA-G are directly associated with tumor progression and reduced patients' survival. It suppresses antitumoral immune responses by binding to receptors of various immune populations, thereby inhibiting the sensitivity to CTLand NK cell-mediated lysis in particular [34]. In contrast, tumor cells with deficient expression of classical HLA class I molecules are eradicated by NK cells.

#### *5.1.2. Check points as important regulators of immune response*

During carcinogenesis, members of the B7-family play a key regulatory role of both stimulato‐ ry and inhibitory T-cell responses, which depends on the available B7 ligand and receptor on the respective target and immune cells [35, 36]. Interestingly, B7-H1 and B7-H4 were often overexpressed on tumors leading to impaired immune recognition. By interaction with these coinhibitory molecules, the intensity of the T-cell responses is reduced by raising the thresh‐ old of activation, halting proliferation, enhancing apoptosis, and inhibiting the differentia‐ tion of effector cells [37].

#### *5.1.3. Role of IFN-γ in cancer immunogenicity*

Abnormalities of MHC class I expression on tumor cells due to the downregulation or loss of APM component expression are common mechanisms, by which tumor cells can escape from anti-tumor-specific immunity [38, 39]. In addition, tumor cells are often not susceptible to treatment with IFN-γ, which could be due to structural alterations or deregulation of constit‐ uents of the IFN signal pathway. Several studies confirmed that defects in the IFN-γ receptor signaling cascade could be occur at multiple steps of this pathway, including lack of the expression of the IFN-γ-R1, abnormal forms of JAK2, lack of expression of JAK1 [40], altered phosphorylation, repressed STAT1 expression, and overexpression of SOCS1. The latter results in an increased negative feedback regulation of the IFN-γ signal cascade. The defects in the IFN-γ receptor signaling cascade caused impaired expression of IFN-γ regulated genes.

Previous studies demonstrated that IFN-γ responsive genes are frequently downregulated in tumor cells due to impaired IRF1 expression as well as defective transcriptional and posttran‐ scriptional regulation of components involved in the IFN-γ signal transduction pathway. The loss of the IFN-γ-mediated upregulation of TAP in a renal cell carcinoma is associated with the lack of IRF1 and STAT1 binding activities as well as JAK1, JAK2, and STAT1 phosphory‐ lation [41]. This impaired IFN-γ-mediated phosphorylation could not be restored by JAK1 and/ or JAK2 gene transfer. Furthermore, an impaired STAT1-phosphorylation associated with the loss of IFN-γ-mediated MHC class I upregulation was also reported in melanoma and colorectal carcinoma cells [42].

IFN-γ treatment is able to restore the expression of many genes belonging to the MHC class I APM [43, 44]. As a consequence, anti-tumor-specific immune responses can be induced, suggesting that IFN-γ acts as key regulator of immunogenicity [45]. Its antitumoral activity includes also the induction of apoptosis and inhibition of cell proliferation by STAT1 activa‐ tion, which induces expression of cell cycle inhibitor, CDKN1A [46]. In addition, the IFN-γmediated upregulation of MHC class I antigens could be due to DNA demethylation of MHC class I APM genes, suggesting that IFN-γ acts as an epigenetic modifier of APM components [47]. Therefore, IFN-γ is a major player in the regulatory network combating tumor cell proliferation and tumor survival.

#### **6. Features of miRNAs**

are mainly due to deregulation of the different components, which could be controlled at the transcriptional, epigenetic (methylation, acetylation), posttranscriptional (e.g., microRNAs,

HLA-G has been demonstrated as a nonclassical HLA class I antigen, which is in general only expressed on immune privileged organs, but also on many tumors of distinct origin [34]. The overexpression of HLA-G or secretion of soluble HLA-G are directly associated with tumor progression and reduced patients' survival. It suppresses antitumoral immune responses by binding to receptors of various immune populations, thereby inhibiting the sensitivity to CTLand NK cell-mediated lysis in particular [34]. In contrast, tumor cells with deficient expression

During carcinogenesis, members of the B7-family play a key regulatory role of both stimulato‐ ry and inhibitory T-cell responses, which depends on the available B7 ligand and receptor on the respective target and immune cells [35, 36]. Interestingly, B7-H1 and B7-H4 were often overexpressed on tumors leading to impaired immune recognition. By interaction with these coinhibitory molecules, the intensity of the T-cell responses is reduced by raising the thresh‐ old of activation, halting proliferation, enhancing apoptosis, and inhibiting the differentia‐

Abnormalities of MHC class I expression on tumor cells due to the downregulation or loss of APM component expression are common mechanisms, by which tumor cells can escape from anti-tumor-specific immunity [38, 39]. In addition, tumor cells are often not susceptible to treatment with IFN-γ, which could be due to structural alterations or deregulation of constit‐ uents of the IFN signal pathway. Several studies confirmed that defects in the IFN-γ receptor signaling cascade could be occur at multiple steps of this pathway, including lack of the expression of the IFN-γ-R1, abnormal forms of JAK2, lack of expression of JAK1 [40], altered phosphorylation, repressed STAT1 expression, and overexpression of SOCS1. The latter results in an increased negative feedback regulation of the IFN-γ signal cascade. The defects in the IFN-γ receptor signaling cascade caused impaired expression of IFN-γ regulated genes.

Previous studies demonstrated that IFN-γ responsive genes are frequently downregulated in tumor cells due to impaired IRF1 expression as well as defective transcriptional and posttran‐ scriptional regulation of components involved in the IFN-γ signal transduction pathway. The loss of the IFN-γ-mediated upregulation of TAP in a renal cell carcinoma is associated with the lack of IRF1 and STAT1 binding activities as well as JAK1, JAK2, and STAT1 phosphory‐ lation [41]. This impaired IFN-γ-mediated phosphorylation could not be restored by JAK1 and/ or JAK2 gene transfer. Furthermore, an impaired STAT1-phosphorylation associated with the loss of IFN-γ-mediated MHC class I upregulation was also reported in melanoma and

protein degradation), or posttranslational (phosphorylation) level.

of classical HLA class I molecules are eradicated by NK cells.

*5.1.2. Check points as important regulators of immune response*

tion of effector cells [37].

42 RNA Interference

colorectal carcinoma cells [42].

*5.1.3. Role of IFN-γ in cancer immunogenicity*

miRNAs are small noncoding ~22 nucleotide long regulatory RNAs encoded in the human genome, which control the posttranscriptional gene expression by binding to the 3′ untranslated region (UTR) of mRNA of target genes, thereby affecting their stability and/or their translation [48]. An individual miRNA could target numerous cellular mRNAs, while single miRNA can be regulated by several proteins [49, 50]. miRNAs have emerged as key players in the posttranscriptional control of gene expression and based on their predic‐ tion appear to be directly involved in the expression of at least 50% of all protein-coding genes in mammals [51].

A strong relationship between miRNAs and human cancer has been developed during the past years. High throughput analysis allows the comparison of miRNA expression pattern in normal and tumor tissues demonstrating global changes within the miRNA expression in different malignancies. Interestingly, the miRNA genes were frequently located at fragile sites and cancer-associated chromosomal regions. The deregulation of the biogenesis and expres‐ sion of miRNAs is involved in the initiation as well as progression of tumors, metastasis formation, and therapy resistance [52]. Furthermore, miRNAs can participate in reprogram‐ ming components of the tissue tumor microenvironment (TME) in order to promote tumor‐ genicity [53]. In the following sections, miRNAs are described as powerful RNAi inducing regulators of immune modulatory genes involved in escape from immune surveillance. Moreover, this review highlights some miRNAs and their roles in immune escape and discusses these miRNAs as putative targets for (immune) therapy (Figure 2).

#### **6.1. Antigen processing and presentation machinery and miRNAs**

Recent studies showed identified miRNAs able to affect the expression of APM components. Microarray analysis of miRNA-9 overexpressing nasopharyngeal carcinoma cells demonstrat‐ ed that miRNA-9 controls the expression of components of the classical MHC class I pathway. miRNA-9 targets many IFN-induced genes and MHC class I APM molecules, such as the proteasome subunits PSMB8 and PSMB10, TAP1, β2-m, HLA-B, HLA-C, and the nonclassical HLA-F and HLA-H antigens [54]. However, the binding of miRNA-9 to the 3′-UTR of these molecules has not yet been shown. miRNA-9 is involved in the cellular differentiation [55] and

**Figure 2.** Scale of miRNAs targeting immune pathways in cancer exhibit an imbalance of tumor-suppressive miRNAs, which occur more frequent than oncogenic miRNAs as per knowledge from today. Oncogenic miRNA are highly ex‐ pressed in cancer, while tumor-suppressive miRNAs possess a reduced expression.

aberrantly expressed in many cancer types breast cancer[56], colon cancer [57], nasopharyng‐ eal carcinoma [58], and melanoma [59], suggesting that the decreased miRNA-9 expression is associated with tumor suppressor activity. In contrast, miRNA-9 expression is increased in brain cancer [60] and in Hodgkin's lymphoma [61], implying an oncomir potential. Further‐ more, miRNA-9 has been shown to regulate the proliferation [56, 60-62] epithelial-mesenchy‐ mal transition (EMT), invasion and metastasis [62-64], apoptosis [56], tumor angiogenesis [63-64], and evasion of immune surveillance in many cancer types [54]. Although the function of miRNA-9 in the classical MHC class I pathway has still to be characterized in extent, miRNA-9-mediated regulation of APM deficiencies might be at least partially responsible for the T cell-mediated immune escape.

Besides miRNA-9, the ER stress-induced miRNA-346 modulates the expression of APM components and IFN-induced genes as shown by miRNA arrays. Functional studies revealed that TAP1 is a direct target of miRNA-346 using overexpression and RNAi knockdown experiments with miRNA mimic and miRNA inhibitors. The ER stress-mediated MHC class I-associated antigen presentation decrease might be explained by increased miRNA-346 expression [65], although the function of miRNA-346 in cancer has not yet been fully analyzed.

The inflammation and overexpression of miRNA-451 are associated with the carcinogenesis of lung cancer. A decrease in the proliferation, invasion, and metastatic potential of lung cancer cells was detected after the overexpression of miRNA-451. In addition, the proteasome subunit PSMB8 has been identified as a direct target of miRNA-451 using both bioinformatics and dual luciferase reporter assays. This was confirmed by miRNA-451-overexpressing lung cancer cells, demonstrating a reduced PSMB8 protein expression. These data suggest that miRNA-451 inhibits the development and metastasis of lung cancer [66].

There are variations that exist in the 3′-UTR of HLA-C, which modulate the miRNAbinding capacity and consequently the HLA-C surface expression. miR-148a has been shown to bind to the HLA-C 3′-UTR. Next to cancer, the miRNA-148a expression is associated with the control of HIV [67-70]. Furthermore, miRNA-181a is upregulated in Hepatitis B virus infected cells and has a binding site in the 3′-UTR of the HLA-A gene, which might be a target of miRNA-181a [71]. Moreover, viral DNA or RNA can encode miRNAs, e.g., miRNA-US4-1 from the human cytomegalovirus, which targets the amino‐ peptidase ERAP1, thereby blocking CTL response [72].

#### **6.2. Control of HLA-G and MHC-related proteins by miRNAs**

Recently, a number of HLA-G-specific miRNAs have been identified, which belong to the miRNA-148 family consisting of three members, miRNA-148a, miRNA-148b, and miRNA-152. These miRNAs have been shown to act as tumor suppressors in many tumors, including prostate, ovarian, endometrial, and colorectal cancer [69, 73, 74]. In addition, other miRNAs such as miRNA-133, miRNA-548, and miRNA-628 have been identified to inhibit HLA-G expression. The HLA-C-regulating miRNAs are involved in inducing T and/or NK cell responses and have a tumor-suppressive capacity. Moreover, some of the HLA-G-regulated miRNAs are inversely expressed when compared to HLA-G in tumor lesions and are associ‐ ated with disease progression [74].

The cytotoxicity of NK cells is determined by activating and inactivating signals. The ligands of the activating NK cell receptor NKG2D are the major histocompatibility complex class Irelated molecules (MIC) A and B and the human cytomegalovirus UL16-binding proteins (ULBP) [73]. The expression of MICA and MICB is controlled by several oncogenic miRNAs, like miRNA-10b, miRNA-17-5p, miRNA-20a, miRNA-25, miRNA-93, and miRNA-106b, which increase the proliferative, invasive, and angiogenic potential of tumors [73, 75-83] and affect the NK cell cytotoxicity. The tumor-suppressive miRNA-302c, miRNA-376a, and miRNA-433-3p showed a reduced expression in cancer and target the 3′-UTR of MIC [73, 84-87]. Furthermore, the expression of ULBP is regulated by many tumor-suppressive miRNAs, e.g., miRNA-34a/c, miRNA-140-5p, miRNA-302c, miRNA-409-3p, and miRNA-433 p, and by the oncogenic miRNA-650 [73, 83, 85, 87].

#### **6.3. Control of B7 family members by miRNA**

aberrantly expressed in many cancer types breast cancer[56], colon cancer [57], nasopharyng‐ eal carcinoma [58], and melanoma [59], suggesting that the decreased miRNA-9 expression is associated with tumor suppressor activity. In contrast, miRNA-9 expression is increased in brain cancer [60] and in Hodgkin's lymphoma [61], implying an oncomir potential. Further‐ more, miRNA-9 has been shown to regulate the proliferation [56, 60-62] epithelial-mesenchy‐ mal transition (EMT), invasion and metastasis [62-64], apoptosis [56], tumor angiogenesis [63-64], and evasion of immune surveillance in many cancer types [54]. Although the function of miRNA-9 in the classical MHC class I pathway has still to be characterized in extent, miRNA-9-mediated regulation of APM deficiencies might be at least partially responsible for

**Figure 2.** Scale of miRNAs targeting immune pathways in cancer exhibit an imbalance of tumor-suppressive miRNAs, which occur more frequent than oncogenic miRNAs as per knowledge from today. Oncogenic miRNA are highly ex‐

pressed in cancer, while tumor-suppressive miRNAs possess a reduced expression.

Besides miRNA-9, the ER stress-induced miRNA-346 modulates the expression of APM components and IFN-induced genes as shown by miRNA arrays. Functional studies revealed that TAP1 is a direct target of miRNA-346 using overexpression and RNAi knockdown experiments with miRNA mimic and miRNA inhibitors. The ER stress-mediated MHC class I-associated antigen presentation decrease might be explained by increased miRNA-346 expression [65], although the function of miRNA-346 in cancer has not yet been fully analyzed.

The inflammation and overexpression of miRNA-451 are associated with the carcinogenesis of lung cancer. A decrease in the proliferation, invasion, and metastatic potential of lung cancer cells was detected after the overexpression of miRNA-451. In addition, the proteasome subunit

the T cell-mediated immune escape.

44 RNA Interference

The expression of B7 family members is subject to the regulatory control of miRNAs: B7-H1 could act as a costimulatory molecule, which is expressed on B cells, T cells, macrophages, and DCs [88], and acts as a ligand for PDL-1. miRNA-513 targets B7-H1 and inhibits its expression by translational repression [89]. In this context, Tamura and coworkers identified an associa‐ tion between low expression of B7-H2 and the escape from immune surveillance indicating that B7-H2 has a potential role in tumorigenesis. B7-H2 [90] is a direct target of miRNA-24, which inhibits B7-H3 expression and therefore is involved in cancer immune evasion. B7-H3 is an immune regulatory molecule, which is often overexpressed in different cancers and associated with metastasis and poor prognosis [91, 92]. Its expression could be posttranscrip‐ tionally regulated by miRNA-29c. The miRNA-29c-mediated downregulation of B7-H3 expression was found in breast cancer and acts therefore as tumor-suppressive miRNA [93]. Furthermore, another B7-H3-regulating miRNA, miRNA-187, has been identified in clear renal cell carcinoma, and its expression is downregulated in this disease [94]. The coinhibitor B7-H4 functions as a negative mediator of immune responses. So far, no information exists about the role of miRNAs in the regulation of B7-H4 expression. In addition, miRNAs binding to the 3′- UTR of B7-H6 have not yet been identified.

#### **6.4. Control of the IFN-γ pathway by miRNAs**

The regulation of IFN-γ signaling includes negative as well as positive regulators, such as kinases and phosphatases as well as transcription factors. A main regulatory role of IFN-γ signaling is attributed to miRNAs, affecting genes involved in proliferation, differentiation, signal transduction, immune response, and carcinogenesis [50, 95].

IFN-γ can modulate the expression levels of miRNAs and to regulate miRNAs at the level of miRNA biogenesis [96], whereas miRNAs can inhibit IFN expression directly or indirectly. In addition, studies have confirmed that miRNAs are able to target components of the IFN-γ signaling pathway and components of the JAK/STAT-pathway can regulate miRNAs simul‐ taneously. The latter has been described by controlling miRNA expression via transcription factors, such as c-myc, the hypoxia-induced factor (HIF), and STATs [97]. The contribution and regulatory role of miRNAs in IFN-γ signaling is still under investigation and an emerging research area. Here, to highlight the regulatory function of miRNAs in the IFN-γ signaling pathway, the functional role of miRNA-155 has been described in more detail.

miRNA-155 proceeding from the non-protein-coding transcript of the *BIC* gene RNA is required for the normal function of B, T, and DC [98. 99], and its expression is increased during B cell, T cell, macrophage, and DC activation [100]. miRNA-155 has been shown to regulate IFN-γ production in NK cells, while its disruption or knockdown suppressed IFN-γ induction of NK cells [101]. Additional studies reported that miRNA-155 also downregulates IFN-γ-R expression [102]. Furthermore, STAT1 upregulates miRNA-155, which in turn downregulates SOCS1, a negative inhibitor of JAK1 [103]. These findings illustrate that a single miRNA can regulate several target mRNAs of the IFN cascade and miRNAs can be regulated by a number of targets.

miRNA regulating components of IFN-γ signaling pathway mainly act as tumor-suppressive miRNAs. An antiproliferative effect of miRNA-375, which affects JAK2 protein expression, has been recently described [104, 105]. Furthermore, miRNA-135a expression was downregu‐ lated in gastric cancer cell lines, while its overexpression results in inhibition of gastric cancer cell proliferation by targeting JAK2 [106]. Thus, miRNA-135a may function as tumor suppres‐ sor by regulating JAK2 expression in gastric cancer cells [107]. Several studies confirmed other miRNAs targeting JAK2, including miRNA-216a, which is known to inhibit cell growth and promote apoptosis of pancreatic cancer cells by regulating JAK2/STAT3 signaling pathway [108, 109], as well as miRNA-101, which promotes apoptosis of breast cancer cells by targeting JAK2 [110]. Similar results were found for STAT1 and miRNA-145 [111]. miRNA-145 is reported to be downregulated in several cancers [112, 113] and has STAT1 as direct target [111]. Moreover, STAT1 is able to upregulate miRNA-29 family members in melanoma cells, which inhibit melanoma cell proliferation by downregulating CDK6 [114].

Further studies confirmed that miRNA-223 and miRNA-150 are equally involved in IFN-γ signaling, but their role in cancer cells is still controversially discussed. Both miRNA-150 and miRNA-223 could exert oncogenic or tumor-suppressive activity. In hepatocellular carcinoma, acute myeloid leukemia (AML) [115] and gastric mucosa-associated lymphoid tissue lympho‐ ma miRNA-223 expression is repressed [116], while an upregulation of miRNA-223 has been recently described in T-cell acute lymphocytic leukemia (T-ALL) [117]. In this context, Moles and coworkers [118] demonstrated that both miRNA-223 and miRNA-150 target STAT1 3′- UTR and reduce STAT1 expression, which in turn results in reduced expression of IFN-γregulated genes. The expression of miRNA-150 is upregulated in CD19+ B cells from chronic lymphocytic leukemia [119, 120], while in chronic myeloid leukemia [121, 122], ALL [123] and mantel cell carcinoma miRNA-150 is downregulated. Moreover, miRNA-150 is upregulated in adult T-cell leukemia/lymphoma cells. This discrepant expression pattern of miRNA-223 and miRNA-150 suggests that both miRNAs could act as oncogenic as well as tumor suppres‐ sor miRNAs, which are dependent on the cellular context.

#### **6.5. Role of miRNAs in immune cell function**

that B7-H2 has a potential role in tumorigenesis. B7-H2 [90] is a direct target of miRNA-24, which inhibits B7-H3 expression and therefore is involved in cancer immune evasion. B7-H3 is an immune regulatory molecule, which is often overexpressed in different cancers and associated with metastasis and poor prognosis [91, 92]. Its expression could be posttranscrip‐ tionally regulated by miRNA-29c. The miRNA-29c-mediated downregulation of B7-H3 expression was found in breast cancer and acts therefore as tumor-suppressive miRNA [93]. Furthermore, another B7-H3-regulating miRNA, miRNA-187, has been identified in clear renal cell carcinoma, and its expression is downregulated in this disease [94]. The coinhibitor B7-H4 functions as a negative mediator of immune responses. So far, no information exists about the role of miRNAs in the regulation of B7-H4 expression. In addition, miRNAs binding to the 3′-

The regulation of IFN-γ signaling includes negative as well as positive regulators, such as kinases and phosphatases as well as transcription factors. A main regulatory role of IFN-γ signaling is attributed to miRNAs, affecting genes involved in proliferation, differentiation,

IFN-γ can modulate the expression levels of miRNAs and to regulate miRNAs at the level of miRNA biogenesis [96], whereas miRNAs can inhibit IFN expression directly or indirectly. In addition, studies have confirmed that miRNAs are able to target components of the IFN-γ signaling pathway and components of the JAK/STAT-pathway can regulate miRNAs simul‐ taneously. The latter has been described by controlling miRNA expression via transcription factors, such as c-myc, the hypoxia-induced factor (HIF), and STATs [97]. The contribution and regulatory role of miRNAs in IFN-γ signaling is still under investigation and an emerging research area. Here, to highlight the regulatory function of miRNAs in the IFN-γ signaling

miRNA-155 proceeding from the non-protein-coding transcript of the *BIC* gene RNA is required for the normal function of B, T, and DC [98. 99], and its expression is increased during B cell, T cell, macrophage, and DC activation [100]. miRNA-155 has been shown to regulate IFN-γ production in NK cells, while its disruption or knockdown suppressed IFN-γ induction of NK cells [101]. Additional studies reported that miRNA-155 also downregulates IFN-γ-R expression [102]. Furthermore, STAT1 upregulates miRNA-155, which in turn downregulates SOCS1, a negative inhibitor of JAK1 [103]. These findings illustrate that a single miRNA can regulate several target mRNAs of the IFN cascade and miRNAs can be regulated by a number

miRNA regulating components of IFN-γ signaling pathway mainly act as tumor-suppressive miRNAs. An antiproliferative effect of miRNA-375, which affects JAK2 protein expression, has been recently described [104, 105]. Furthermore, miRNA-135a expression was downregu‐ lated in gastric cancer cell lines, while its overexpression results in inhibition of gastric cancer cell proliferation by targeting JAK2 [106]. Thus, miRNA-135a may function as tumor suppres‐ sor by regulating JAK2 expression in gastric cancer cells [107]. Several studies confirmed other miRNAs targeting JAK2, including miRNA-216a, which is known to inhibit cell growth and

UTR of B7-H6 have not yet been identified.

of targets.

46 RNA Interference

**6.4. Control of the IFN-γ pathway by miRNAs**

signal transduction, immune response, and carcinogenesis [50, 95].

pathway, the functional role of miRNA-155 has been described in more detail.

Cancer cells upregulate and downregulate different miRNAs in immune cells to limit the antitumor response. It is well known that tumor cells reprogram the myeloid compartment to evade the immune system and promote tumorigenesis. This might be partially mediated by alterations in the miRNA expression pattern. The miRNA-155 modulates the immune response mediated by T cells, NK cells, B cells, and antigen presenting cells, such as macrophages and DC [124]. Furthermore, miRNA-155 expression has been found to be downregulated in TAMs [125], but also in hepatocellular carcinoma. The restoration of miRNA-155 in macrophages leads to enhanced T-cell function by targeting the suppressor of cytokine signaling. Other miRNAs, like miRNA-142-3p, miRNA-125b, and miRNA-19a-3p, are often downregulated in TAMs, thereby limiting the tumor infiltration of macrophages and reducing the therapeutic effect of adoptive transfer. The restoration of miRNA-125b in macrophages enhances antitu‐ mor response by targeting the IFN-regulatory factor 4, which promotes the M2 macrophage phenotype [126].

Recently, miRNAs have been identified to play a role in MDSC that regulate immune sup‐ pression within the tumor microenvironment. miRNA array analysis identified a number of deregulated miRNAs, e.g., miRNA-494, which suppresses the antitumor CD8+ T-cell responses due to response to TGF-β. miRNA-494 targets PTEN in MDSC, which is responsible for the enhanced immune suppression of CD8+ T cells [127]. Furthermore, a number of other miRNAs are downregulated in MDSC [128], which promote the differentiation of myeloid cells and regulate immune-suppressive signaling pathways.

In addition, miRNAs have been demonstrated in tumor-infiltrating lymphocytes. The sup‐ pression of T-cell activity is due to different mechanisms, including the dysregulation of miRNA expression. In CD4+ T cells from tumor bearing mice and tumor patients, the expres‐ sion of miRNA-17-92 family members was reduced, while T cells derived from miRNA-17-9 transgenic mice demonstrated a superior type 1 phenotype [129]. Furthermore, the expression of miRNA-155 was shown to promote antitumor responses. miRNA-155 in combination with miRNA-146a could upregulate the IFN-γ production of T cells. Furthermore, cancer cells could regulate miRNAs in T cells in order to modulate antitumor T-cell responses. In order to escape immune surveillance, cancer cells alter the expression of transcription factors, surface recep‐ tors, soluble chemokines/cytokines, and miRNAs to support the immune system. The down‐ regulation of miRNA-124 increases Treg infiltration and reduces cytokines production through an altered expression of STAT3, which represents a target of miRNA-124. In contrast, tumorsecreted miRNA-214 induces Treg. Regarding NK cells, the TGF-β-inducible miRNA-183 affects NK cell activity [130]. Thus, the regulation of miRNAs within the cancer cell alters the TME through manipulation.

#### **7. Conclusion and future perspectives**

Taken together, during the past years, the posttranscriptional control of gene expression by miRNAshasgainedrelevance askeyregulatorinawidevarietyofphysiological andpathophy‐ siological processes due to the role of miRNA-mediated RNAi not only in differentiation, proliferation, apoptosis, immune responses but also in viral and bacterial infections as well as neoplastic transformation (Table 1).Aderegulated expression of miRNAs has been often found in tumors of distinct origin, which have been classified into oncogenic or tumor-suppressive miRNAs known to play an essential role in cancer initiation and progression. Therefore, these miRNAs could act as potential biomarkers and therapeutic targets in cancer. *In silico* predic‐ tion analysis further proposed that many miRNAs could target different immune modulato‐ ry molecules expressed either on tumor cells or on different immune cell subpopulations.

As summarized, an emerging relevance of miRNAs in mounting the tumor immune escape by altering the communication between cancer cells, immune cells, and other components of the TME has been demonstrated. This leads to another level of complexity due to the involve‐ ment of miRNAs in the interaction between cancer cells and immune cells. These miRNAs might not only provide new insights into tumor growth and progression as well as antitumoral immune responses but also represent promising therapeutic targets for (immune) therapy. To date, many cancer-deregulated miRNAs have been identified in particular in cancer cells and also in components of the TMA. However, their role in modulating the antitumor immune responses has not yet been characterized in detail. Although the majority of the miRNA alterations detected are dedicated to cancer cells, there is already evidence that miRNAs of infiltrating immune cells also particularly influence tumorgenicity. The identification of further im-miRNAs as well as their functional characterization might lead to a plethora of novel candidate biomarkers for monitoring of immune responses, which might be also potentially used for targeted RNAi therapy.


In addition, miRNAs have been demonstrated in tumor-infiltrating lymphocytes. The sup‐ pression of T-cell activity is due to different mechanisms, including the dysregulation of miRNA expression. In CD4+ T cells from tumor bearing mice and tumor patients, the expres‐ sion of miRNA-17-92 family members was reduced, while T cells derived from miRNA-17-9 transgenic mice demonstrated a superior type 1 phenotype [129]. Furthermore, the expression of miRNA-155 was shown to promote antitumor responses. miRNA-155 in combination with miRNA-146a could upregulate the IFN-γ production of T cells. Furthermore, cancer cells could regulate miRNAs in T cells in order to modulate antitumor T-cell responses. In order to escape immune surveillance, cancer cells alter the expression of transcription factors, surface recep‐ tors, soluble chemokines/cytokines, and miRNAs to support the immune system. The down‐ regulation of miRNA-124 increases Treg infiltration and reduces cytokines production through an altered expression of STAT3, which represents a target of miRNA-124. In contrast, tumorsecreted miRNA-214 induces Treg. Regarding NK cells, the TGF-β-inducible miRNA-183 affects NK cell activity [130]. Thus, the regulation of miRNAs within the cancer cell alters the

Taken together, during the past years, the posttranscriptional control of gene expression by miRNAshasgainedrelevance askeyregulatorinawidevarietyofphysiological andpathophy‐ siological processes due to the role of miRNA-mediated RNAi not only in differentiation, proliferation, apoptosis, immune responses but also in viral and bacterial infections as well as neoplastic transformation (Table 1).Aderegulated expression of miRNAs has been often found in tumors of distinct origin, which have been classified into oncogenic or tumor-suppressive miRNAs known to play an essential role in cancer initiation and progression. Therefore, these miRNAs could act as potential biomarkers and therapeutic targets in cancer. *In silico* predic‐ tion analysis further proposed that many miRNAs could target different immune modulato‐ ry molecules expressed either on tumor cells or on different immune cell subpopulations.

As summarized, an emerging relevance of miRNAs in mounting the tumor immune escape by altering the communication between cancer cells, immune cells, and other components of the TME has been demonstrated. This leads to another level of complexity due to the involve‐ ment of miRNAs in the interaction between cancer cells and immune cells. These miRNAs might not only provide new insights into tumor growth and progression as well as antitumoral immune responses but also represent promising therapeutic targets for (immune) therapy. To date, many cancer-deregulated miRNAs have been identified in particular in cancer cells and also in components of the TMA. However, their role in modulating the antitumor immune responses has not yet been characterized in detail. Although the majority of the miRNA alterations detected are dedicated to cancer cells, there is already evidence that miRNAs of infiltrating immune cells also particularly influence tumorgenicity. The identification of further im-miRNAs as well as their functional characterization might lead to a plethora of novel candidate biomarkers for monitoring of immune responses, which might be also

TME through manipulation.

48 RNA Interference

**7. Conclusion and future perspectives**

potentially used for targeted RNAi therapy.

**Table 1.** Identified miRNAs involved in the tumor immune escape and their tumor–associated function. Controversially discussed miRNAs are found as tumor suppressors in some cancer types, while exhibiting oncogenic properties in other cancer types. n.d., no data.

#### **8. Abbreviations**

APC, antigen presenting cell; APM, antigen processing machinery; β2-m, β2-microglobulin; CDKN, cyclin-dependent kinase inhibitor; CTL, cytotoxic T lymphocyte; CXCL, chemokine (CXC motif) ligand; DC, dendritic cell; GAS, IFN-γ-activated sequence; HC, heavy chain; HLA, human leukocyte antigen; IFN, interferon; IL, interleukin; IRF, interferon regulatory factor; ISRE, IFN-stimulated response element; im-miRNA, immune modulatory miRNA; JAK, janus kinase; LMP, low molecular mass polypeptide; MAPK, MAP kinase; MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; MIC, major histocompatibility complex class I-related molecule; miRNA, microRNA; NK, natural killer cell; PD, programmed death; PDL, PD ligand; PLC, peptide loading complex; STAT, signal transducer and activator of transcription; SOCS, suppressor of cytokine signaling; TAF, tumor-associated fibroblast; TAM, tumor-associated macrophage; TAP, transporter associated with antigen processing; TGF, transforming growth factor; TLR, toll-like receptor; TME, tumor microenvironment; Treg, regulatory T cell; ULBP, human cytomegalovirus UL16-binding protein; UTR, untrans‐ lated region; and VEGF, vascular endothelial growth factor.

#### **Acknowledgements**

We would like to thank Sylvi Magdeburg for excellent secretarial help. This work was supported by a grant from the DFG SE585/22-1, GRK1591, GIF (I 1187-69), and Cancer Research Aid (110703).

#### **Author details**

Barbara Seliger\* , Anne Meinhardt and Doerte Falke

\*Address all correspondence to: barbara.seliger@uk-halle.de

Institute of Medical Immunology, Martin-Luther University Halle-Wittenberg, Germany

#### **References**


[3] Maj T, Wei S, Welling T, Zou W. T cells and costimulation in cancer. Cancer J. 2013;19(6):473. DOI: 10.1097/PPO.0000000000000002.

**8. Abbreviations**

50 RNA Interference

**Acknowledgements**

Aid (110703).

**Author details**

Barbara Seliger\*

**References**

APC, antigen presenting cell; APM, antigen processing machinery; β2-m, β2-microglobulin; CDKN, cyclin-dependent kinase inhibitor; CTL, cytotoxic T lymphocyte; CXCL, chemokine (CXC motif) ligand; DC, dendritic cell; GAS, IFN-γ-activated sequence; HC, heavy chain; HLA, human leukocyte antigen; IFN, interferon; IL, interleukin; IRF, interferon regulatory factor; ISRE, IFN-stimulated response element; im-miRNA, immune modulatory miRNA; JAK, janus kinase; LMP, low molecular mass polypeptide; MAPK, MAP kinase; MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; MIC, major histocompatibility complex class I-related molecule; miRNA, microRNA; NK, natural killer cell; PD, programmed death; PDL, PD ligand; PLC, peptide loading complex; STAT, signal transducer and activator of transcription; SOCS, suppressor of cytokine signaling; TAF, tumor-associated fibroblast; TAM, tumor-associated macrophage; TAP, transporter associated with antigen processing; TGF, transforming growth factor; TLR, toll-like receptor; TME, tumor microenvironment; Treg, regulatory T cell; ULBP, human cytomegalovirus UL16-binding protein; UTR, untrans‐

We would like to thank Sylvi Magdeburg for excellent secretarial help. This work was supported by a grant from the DFG SE585/22-1, GRK1591, GIF (I 1187-69), and Cancer Research

Institute of Medical Immunology, Martin-Luther University Halle-Wittenberg, Germany

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## **Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation**

Utpal Bhadra, Debabani Roy Chowdhury, Tanmoy Mondal and Manika Pal Bhadra

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61975

#### **Abstract**

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5500. DOI: 10.4049/jimmunol.1103505.

DOI: 10.1186/1479-5876-8-17.

62 RNA Interference

10.1073/pnas.1319269111.

General complexities in versatile animals are not always proportional to their genome size. A notable example is that the salamander genome size is 15-fold larger than that of human, which mostly contains unfolded "junk DNA." A vast portion of this non-proteincoding unfolded DNA undergoes transcriptional regulation and produces a large num‐ ber of long noncoding RNAs (lncRNAs). LncRNAs play key roles in gene expression and therapies of different human diseases. Recently, novel lncRNAs and their function on the silencing or activation of a particular gene(s) are regularly being discovered. Another im‐ portant component of gene regulation is high packing of chromatin, which is composed of mainly repetitive sequences with negligible coding potential. In particular, an epige‐ netic marker determines the state of the gene associated with it, whether the gene will be expressed or silenced. Here, we elaborately discuss the biogenesis pathway of lncRNAs as well as their mechanism of action and role in gene silencing and regulation, including RNA interference. Moreover, several lncRNAs are the common precursors of small regu‐ latory RNAs. It is thus becoming increasingly clear that lncRNAs can function via numer‐ ous paradigms as key regulatory molecules in different organisms.

**Keywords:** Transcriptional silencing, long noncoding RNA, cancer, neurological disor‐ der, *Drosophila*

#### **1. Introduction**

Since the earliest days of molecular biology, RNA-mediated gene regulation was known to the researchers, and it was first suggested that noncoding RNA (ncRNA) might have a role in gene regulation by interacting with promoters [1, 2]. After more than four decades of research, the discovery of RNA interference (RNAi) has revolutionized our perception of the mechanism of

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

gene regulation, organization of chromosomes, and epigenetic regulations. Important clues to ncRNA regulatory mechanisms came from homology-dependent gene silencing in plants, which can be initiated by transgenes and recombinant viruses [3]. Studies on the nematode *Caenorhabditis elegans*, the fruit fly *Drosophila melanogaster* [4], fungi mainly yeast, mammalian cells, and plants revealed transcriptional silencing mechanisms involving RNAi, chromatin, and its various modifications [3]. RNAi operates mainly posttranscriptionally; however, its components are associated with transcriptional gene silencing and heterochromatin forma‐ tion, too [5].

Recent findings have made it clear that transcriptional gene silencing (TGS), posttranscrip‐ tional gene silencing (PTGS), and chromatin modifications are utilized by eukaryotic cells to bring about endogenous gene regulation, chromosome organization, and nuclear clustering. The RNA interference mechanisms mainly target the transposable elements, which are abundant and perhaps a defining component of heterochromatin. The role of ncRNA in dosage compensation, inactivation of X chromosome, genomic imprinting, polycomb silencing, and blocking of interactions between enhancers and promoters by chromatin insulators is well proven. Although the studies strongly point towards the involvement of RNAi, its role has not been demonstrated directly [6].

The non-protein-coding transcripts longer than 200 nucleotides are known as long noncoding RNAs to differentiate superficially this class of ncRNAs from microRNAs, short interfering RNAs, piwi-interacting RNAs, small nucleolar RNAs, etc [7]. LncRNAs have emerged as important regulators of cell physiology and pathology. Different studies have come up with an increasing number of lncRNAs showing tissue-specific expression; however, the exact mechanism of action of only a few lncRNAs has been elucidated *in vivo* [8–14]. The biological functions and mechanisms of action of the majority of lncRNAs still remain unknown. LncRNAs can interact with a wide range of molecules and can form RNA-RNA, RNA-DNA, or RNA-protein complexes through specific RNA functional domains [15], resulting in extensive functional diversities. Recent research focuses on lncRNAs and divulges the association of lncRNAs with epigenetic machinery to control chromatin structure, nuclear clustering, and gene expression. The studies reveal that lncRNAs may act together with many histone- and DNA-modifying enzymes to modify the histones or DNA. In addition, a recent discovery of a cardioprotective lncRNA showed a targeting mechanism through ATPdependent chromatin remodeling factors [16], indicating an extensive role of lncRNAs in chromatin structure and regulation. The mechanisms of how lncRNAs control chromatin by covalent modifications are extensively reviewed in the literature [17–20].

The study of lncRNAs has taken the center stage for the researchers working with epigenetic regulations, and there is a report of a new lncRNA regulating a disease, or transcriptome studies come up with a new class of noncoding RNA, or we are introduced to hitherto unknown mechanisms by which an lncRNA regulates a particular gene almost on a weekly basis. These are all possible due to the introduction of many advanced, high-throughput genomic technologies such as microarrays and next-generation sequencing (NGS). There are a huge number of reported lncRNAs that are not derived from protein-coding genes, and in spite of this vast number of reports on lncRNA, we have just started getting a clear picture about how lncRNAs function, how many different types of lncRNAs exist, and how many of the reported lncRNAs are biologically important.

#### **2. The C-value enigma and junk DNA**

gene regulation, organization of chromosomes, and epigenetic regulations. Important clues to ncRNA regulatory mechanisms came from homology-dependent gene silencing in plants, which can be initiated by transgenes and recombinant viruses [3]. Studies on the nematode *Caenorhabditis elegans*, the fruit fly *Drosophila melanogaster* [4], fungi mainly yeast, mammalian cells, and plants revealed transcriptional silencing mechanisms involving RNAi, chromatin, and its various modifications [3]. RNAi operates mainly posttranscriptionally; however, its components are associated with transcriptional gene silencing and heterochromatin forma‐

Recent findings have made it clear that transcriptional gene silencing (TGS), posttranscrip‐ tional gene silencing (PTGS), and chromatin modifications are utilized by eukaryotic cells to bring about endogenous gene regulation, chromosome organization, and nuclear clustering. The RNA interference mechanisms mainly target the transposable elements, which are abundant and perhaps a defining component of heterochromatin. The role of ncRNA in dosage compensation, inactivation of X chromosome, genomic imprinting, polycomb silencing, and blocking of interactions between enhancers and promoters by chromatin insulators is well proven. Although the studies strongly point towards the involvement of RNAi, its role has not

The non-protein-coding transcripts longer than 200 nucleotides are known as long noncoding RNAs to differentiate superficially this class of ncRNAs from microRNAs, short interfering RNAs, piwi-interacting RNAs, small nucleolar RNAs, etc [7]. LncRNAs have emerged as important regulators of cell physiology and pathology. Different studies have come up with an increasing number of lncRNAs showing tissue-specific expression; however, the exact mechanism of action of only a few lncRNAs has been elucidated *in vivo* [8–14]. The biological functions and mechanisms of action of the majority of lncRNAs still remain unknown. LncRNAs can interact with a wide range of molecules and can form RNA-RNA, RNA-DNA, or RNA-protein complexes through specific RNA functional domains [15], resulting in extensive functional diversities. Recent research focuses on lncRNAs and divulges the association of lncRNAs with epigenetic machinery to control chromatin structure, nuclear clustering, and gene expression. The studies reveal that lncRNAs may act together with many histone- and DNA-modifying enzymes to modify the histones or DNA. In addition, a recent discovery of a cardioprotective lncRNA showed a targeting mechanism through ATPdependent chromatin remodeling factors [16], indicating an extensive role of lncRNAs in chromatin structure and regulation. The mechanisms of how lncRNAs control chromatin by

The study of lncRNAs has taken the center stage for the researchers working with epigenetic regulations, and there is a report of a new lncRNA regulating a disease, or transcriptome studies come up with a new class of noncoding RNA, or we are introduced to hitherto unknown mechanisms by which an lncRNA regulates a particular gene almost on a weekly basis. These are all possible due to the introduction of many advanced, high-throughput genomic technologies such as microarrays and next-generation sequencing (NGS). There are a huge number of reported lncRNAs that are not derived from protein-coding genes, and in spite of this vast number of reports on lncRNA, we have just started getting a clear picture

covalent modifications are extensively reviewed in the literature [17–20].

tion, too [5].

64 RNA Interference

been demonstrated directly [6].

It has long been known that developmental complexity or size of an animal does not correspond with C-value or the amount of DNA in the haploid genome [21–23]. The lower animal in the evolution ladder, salamander, has a genome size 15 times larger than that of humans [21], and this discrepancy is known as the "C-value paradox" [23]. Since the introns were discovered, we started to presume that the C-value paradox was now solved [24]. We are almost sure that humans have about 25,000–35,000 protein-coding genes unlike the overestimates of 50,000–100,000 from the initial days of the Human Genome Project [25]. The remaining huge amount of noncoding DNA was termed as "junk DNA" [24, 26] due to the presence of transposons, pseudogenes, and simple repeats, which occupies about 50– 70% of the human genome [27]. C-value enigma poses a discrepancy in genome size and number of protein-coding genes. Phylogenetically close genera may vary in C-value by around four- to five fold [28].

In spite of their "junk" status, scientists were always curious to study them and even realized that "being junk doesn't mean it is entirely useless" [26]. It was hypothesized that the junk DNA might be useful in chromosomal pairing, genome integrity, gene regulation, mRNA processing, and serving as a reservoir for evolutionary innovation. We are now pleasantly surprised at their foresight. In the 1970s, it was already thought that noncoding RNA products, such as rRNAs, tRNAs do not make up the whole transcribed genome.

The scale of "pervasive transcription," however, was not fully appreciated until the late 1990s and early 2000s. After the arrival of whole-genome technologies, from microarray hybridiza‐ tion and deep sequencing analysis techniques, it was recently shown that 70–90% of our genome is transcribed at some point during embryogenesis [29]. Some recently identified transcripts may be present at as low as 0.0006 copies per cell [30]. Another concern is that tiling microarrays can come up with false positives, low dynamic range, resolution, and low concordance between studies [31]. The existence of noncoding transcription in intergenic regions is evident from correlations with chromatin signatures, such as DNase1 hypersensi‐ tivity, and histone modifications such as H3K9ac, H3K4me3, and H3K36me3 [31]. Although these studies report novel and conserved lncRNAs, that is not enough to explain the function of 70–90% of the genome and biological functionality of the ncRNAs. In 1969, Britten and Davidson presented a model for regulation of gene expression in eukaryotic cells where ncRNAs have important roles as regulatory intermediaries to convey signals from sensory to receptor elements [1]. Some of the first examples of gene-specific regulatory roles of lncRNAs were revealed with the discovery of lncRNAs involved in epigenetic regulation, such as H19 [32] and X-inactive specific transcript (Xist) [33, 34].

#### **3. Stand-alone lncRNAs**

These lncRNAs are located as separate units and do not overlap protein-coding genes. Some of these are known as lincRNAs for large intergenic (or intervening) noncoding RNAs (lincRNAs) [35]. Many of the lincRNAs were identified through chromatin signatures for actively transcribed genes (H3K4me3 at the promoter and H3K36me3 along the transcribed length). Many of the characterized lncRNAs are transcribed by RNA Pol II, polyadenylated, and spliced and have an average length of 1 kb.

#### **4. Natural antisense transcripts**

In this study, transcription occurs in the antisense strand of annotated transcription units; about 70% of sense transcripts have reported antisense counterparts [36]. The overlap between these sense/antisense pairs can be a complete sequence, but natural antisense transcripts are mostly found to be enriched around the 5′ promoter or 3′ terminator ends of the sense transcript. The most extensively studied example of sense/antisense pairing is Xist/Tsix (lncRNA antisense to Xist), with two RNAs that control X chromosome inactivation [37]. In addition, many imprinted regions contain coding/noncoding sense/antisense pairs, such as Kcnq1 (potassium channel, voltage-gated KQT-like subfamily Q, member 1)/Kcnq1ot1 (Kcnq1 overlapping transcript 1) [38] and Igf2r (insulin-like growth factor 2 receptor)/Air (antisense Igf2r RNA) [39]. These pairs are generally less spliced or polyadenylated when compared to mRNAs or stand-alone lncRNAs.

#### **5. Long intronic ncRNAs**

Introns have long been known to contain small ncRNAs such as small nucleolar RNAs (snoRNAs) and microRNAs (miRNAs). However, by large-scale transcriptomic or computa‐ tional analyses, many long transcripts have been reported to be encoded within the introns of known genes [40]. Although they have differential expression patterns and respond to the environmental stimuli differently, only a few have been extensively studied to date. One such example is cold-assisted intronic noncoding RNA (COLDAIR) that has been implicated in plant vernalization, which is located in the first intron of the flowering repressor locus FLC [41].

#### **6. Identification of long noncoding RNAs**

LncRNAs are identified by transcripts that map to genomic regions outside the boundaries of protein-coding genes. It is difficult to ascertain the function of a transcript that overlaps a protein-coding gene using targeted knockout or knockdown approaches. Thus, most experi‐ mental investigations of lncRNAs have been focused on those that are located in intron sequences. It is also very difficult to ascertain whether an lncRNA locus is entirely intergenic because lncRNA transcripts are often incomplete and they can originate from a protein-coding gene's promoter or enhancer on either strand [42]. Tiling microarray technique is often useful to detect intergenic transcripts [43]. However, controversial results were found here and these experiments can be ruled out [31]. Early lncRNA collections relied primarily on sequenced cDNA and EST clones [44]. More recently, RNA-Seq has come up with a number of lncRNAs derived from whole transcriptome sequencing. RNA-Seq generates millions of 35–100 nt sequences read in parallel, and it has been confirmed that a large chunk of intergenic sequences are transcribed into lncRNAs [45]. The high-throughput and impartial nature of this technique is being utilized for the detailed assessment of the contribution of lncRNAs to a variety of tissue and/or species under different conditions.

To accurately distinguish noncoding from coding transcripts, sophisticated approaches have beendeveloped.Forexample,theCodingPotentialCalculator[46]takesintoaccountsixfeatures of a transcript, including the proportion of the transcript enclosed by the candidate peptideencoding region, and the sequence similarity to known proteins. An evolutionary approach, followed in phyloCSF, predicts ncRNAs when their sequence differences among species do not show preference as to whether they disrupt or not putatively encode peptides [47].

Experimentally determined transcripts always are relied on more than predicted ones. The availability of large proteomic databases can be utilized to investigate whether a specific RNA molecule is translated into a protein. *In vitro* translation assays have been used, too, but they do not necessarily reflect *in vivo* biology. A true lncRNA should not bind with translation machinery, and this approach is also adopted in the identification of candidate lncRNA. However, a study has reported that 50% of a set of putative lncRNAs are ribosome associated [48], leaving in doubt whether this test is accurate in separating coding from noncoding transcripts. To assign an lncRNA, an experimental determination of the function of a transcript will be necessary. Nevertheless, some transcripts possess both RNA- and coding-sequencedependent functions [49] and demarcating them will be difficult. A computational or experi‐ mental method has not yet been developed that discriminates accurately between coding and noncoding transcripts. For the time being, we can rely on *in silico* screens for the protein-coding potential of putative lncRNAs but be aware that these will contain false-positive predictions, too, especially for genes that encode short polypeptides.

Although many genomes contain a substantial number of lncRNA loci, we still do not know the proportion and number of these that are biologically functional. Because the functional mechanisms of most noncoding transcripts or transcript regions are unknown, it is difficult to design point mutation or deletion experiments and their results are difficult to interpret. Even RNAi techniques are not being helpful to assign the functionality of the ncRNAs.

#### **7. Mechanisms of action**

**3. Stand-alone lncRNAs**

66 RNA Interference

and spliced and have an average length of 1 kb.

**4. Natural antisense transcripts**

mRNAs or stand-alone lncRNAs.

**5. Long intronic ncRNAs**

**6. Identification of long noncoding RNAs**

These lncRNAs are located as separate units and do not overlap protein-coding genes. Some of these are known as lincRNAs for large intergenic (or intervening) noncoding RNAs (lincRNAs) [35]. Many of the lincRNAs were identified through chromatin signatures for actively transcribed genes (H3K4me3 at the promoter and H3K36me3 along the transcribed length). Many of the characterized lncRNAs are transcribed by RNA Pol II, polyadenylated,

In this study, transcription occurs in the antisense strand of annotated transcription units; about 70% of sense transcripts have reported antisense counterparts [36]. The overlap between these sense/antisense pairs can be a complete sequence, but natural antisense transcripts are mostly found to be enriched around the 5′ promoter or 3′ terminator ends of the sense transcript. The most extensively studied example of sense/antisense pairing is Xist/Tsix (lncRNA antisense to Xist), with two RNAs that control X chromosome inactivation [37]. In addition, many imprinted regions contain coding/noncoding sense/antisense pairs, such as Kcnq1 (potassium channel, voltage-gated KQT-like subfamily Q, member 1)/Kcnq1ot1 (Kcnq1 overlapping transcript 1) [38] and Igf2r (insulin-like growth factor 2 receptor)/Air (antisense Igf2r RNA) [39]. These pairs are generally less spliced or polyadenylated when compared to

Introns have long been known to contain small ncRNAs such as small nucleolar RNAs (snoRNAs) and microRNAs (miRNAs). However, by large-scale transcriptomic or computa‐ tional analyses, many long transcripts have been reported to be encoded within the introns of known genes [40]. Although they have differential expression patterns and respond to the environmental stimuli differently, only a few have been extensively studied to date. One such example is cold-assisted intronic noncoding RNA (COLDAIR) that has been implicated in plant vernalization, which is located in the first intron of the flowering repressor locus FLC [41].

LncRNAs are identified by transcripts that map to genomic regions outside the boundaries of protein-coding genes. It is difficult to ascertain the function of a transcript that overlaps a protein-coding gene using targeted knockout or knockdown approaches. Thus, most experi‐ mental investigations of lncRNAs have been focused on those that are located in intron

We do not know yet the mechanistic detail of the enormous number of reported lncRNAs. However, a few that have been thoroughly studied provide clues regarding how lncRNAs might carry out gene regulation (Figure 1). In addition, many lncRNAs blur the line of different categories and employ several different mechanisms. The discovery of new lncRNAs and more thorough characterization of those already known will reveal additional modes of action.

It has been found that a major role of lncRNA is to recruit regulatory proteins for the regulation of chromatin states [50]. This kind of lncRNAs may act in *cis*, on adjacent or nearby genes, or they might act in *trans*, regulating genes located in distant domains or chromosomes. Polycomb repressive complex 2 (PRC2) interacts with a large number of lncRNAs [51–54]. The *Drosophi‐ la* polycomb proteins, first discovered as homeotic gene, express during development [55, 56]. These include enhance of zeste homolog 2 (Ezh2, catalytic subunit in PRC2), which is a key H3K27 methyltransferase, and the Pc/Chromobox (Cbx) family proteins in PRC1, chromodo‐ main-containing proteins that can bind trimethylated H3K27 [55, 56]. Observed interactions of polycomb proteins with lncRNAs suggest that polycomb recruitment is RNA directed in mammals. HOX transcript antisense RNA (HOTAIR) in the homeobox (HOX) C cluster is reported to repress transcription of HOXD in *trans* through interaction with PRC2 [57]. Xist RNA-containing repeat A (RepA) has been found to recruit PRC2 [58]. RepA targets PRC2 to the Xist promoter resulting in Xist up-regulation. The interesting fact is that RepA/Xist interaction with PRC2 may be blocked by the antisense Tsix transcript, also interacting with PRC2 and competitively inhibiting the painting of Xist on inactive X chromosome [58].

Other epigenetic complexes interact with lncRNAs as well, such as the H3K9 methyltransferase G9a interacting with the imprinted lncRNA Air [59]. Kcnq1ot1 has been hypothesized to recruit both PRC2 and G9a to the promoter of Kcnq1 [60] acting as a scaffold. On the other hand, antisense ncRNA in the INK4 locus (ANRIL), associated with p15/INK4 (inhibitors of CDK4 family) B-p16/INK4A-p14/ARF tumor suppressor gene cluster, interacts with both the PRC1 component Cbx7 and the PRC2 component Suz1 [61, 62]. HOTAIR also interacts with the lysine-specific demethylase 1 (LSD1)/corepressor protein of LSD1 (CoREST)/repressor for element 1-silencing transcription factor (REST) complex in addition to PRC2 to prevent gene activation [63].

LncRNAs can also act by recruiting factors involved in gene activation. Such factors from the HOXA (homeotic gene A cluster), two lncRNAs, Mistral (Mira), and HOXA transcript at the distal tip (HOTTIP) have been involved in recruiting the mixed lineage leukemia (MLL) complex in *cis* regulation [64, 65].

An H3K4 trimethylase, myeloid/lymphoid or mixed-lineage leukemia (MLL), is a member of the Trithorax group of developmentally important gene-activating proteins in flies [66]. Using 3C or chromosome conformation capture technique, it was found that multiple loci, which are 40 kb apart in the HOXA cluster, are in close physical proximity, enabling MLL to regulate their expression. Other than histone modifications, lncRNAs also impact epigenetic regulation by modulating DNA methylation at CpG dinucleotides, which has an important role in the stable repression of genes [67]. During embryogenesis, methylation markers are first to be found on previously unmethylated DNA by the DNA (cytosine-5-)-methyltransferase 3α (Dnmt3a) and 3β (Dnmt3b) and later maintained through DNA replication by Dnmt1. Tsix might be converted to Xist by utilizing Dnmt3a activity to methylate and finally silence the Xist promoter [68, 69]. In the same way, Kcnq1ot1 may recruit Dnmt1 [70].

Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation http://dx.doi.org/10.5772/61975 69

**Figure 1.** Mechanisms of lncRNA function (modified from Kung et al. [129]).

might carry out gene regulation (Figure 1). In addition, many lncRNAs blur the line of different categories and employ several different mechanisms. The discovery of new lncRNAs and more thorough characterization of those already known will reveal additional modes of action. It has been found that a major role of lncRNA is to recruit regulatory proteins for the regulation of chromatin states [50]. This kind of lncRNAs may act in *cis*, on adjacent or nearby genes, or they might act in *trans*, regulating genes located in distant domains or chromosomes. Polycomb repressive complex 2 (PRC2) interacts with a large number of lncRNAs [51–54]. The *Drosophi‐ la* polycomb proteins, first discovered as homeotic gene, express during development [55, 56]. These include enhance of zeste homolog 2 (Ezh2, catalytic subunit in PRC2), which is a key H3K27 methyltransferase, and the Pc/Chromobox (Cbx) family proteins in PRC1, chromodo‐ main-containing proteins that can bind trimethylated H3K27 [55, 56]. Observed interactions of polycomb proteins with lncRNAs suggest that polycomb recruitment is RNA directed in mammals. HOX transcript antisense RNA (HOTAIR) in the homeobox (HOX) C cluster is reported to repress transcription of HOXD in *trans* through interaction with PRC2 [57]. Xist RNA-containing repeat A (RepA) has been found to recruit PRC2 [58]. RepA targets PRC2 to the Xist promoter resulting in Xist up-regulation. The interesting fact is that RepA/Xist interaction with PRC2 may be blocked by the antisense Tsix transcript, also interacting with PRC2 and competitively inhibiting the painting of Xist on inactive X chromosome [58].

Other epigenetic complexes interact with lncRNAs as well, such as the H3K9 methyltransferase G9a interacting with the imprinted lncRNA Air [59]. Kcnq1ot1 has been hypothesized to recruit both PRC2 and G9a to the promoter of Kcnq1 [60] acting as a scaffold. On the other hand, antisense ncRNA in the INK4 locus (ANRIL), associated with p15/INK4 (inhibitors of CDK4 family) B-p16/INK4A-p14/ARF tumor suppressor gene cluster, interacts with both the PRC1 component Cbx7 and the PRC2 component Suz1 [61, 62]. HOTAIR also interacts with the lysine-specific demethylase 1 (LSD1)/corepressor protein of LSD1 (CoREST)/repressor for element 1-silencing transcription factor (REST) complex in addition to PRC2 to prevent gene

LncRNAs can also act by recruiting factors involved in gene activation. Such factors from the HOXA (homeotic gene A cluster), two lncRNAs, Mistral (Mira), and HOXA transcript at the distal tip (HOTTIP) have been involved in recruiting the mixed lineage leukemia (MLL)

An H3K4 trimethylase, myeloid/lymphoid or mixed-lineage leukemia (MLL), is a member of the Trithorax group of developmentally important gene-activating proteins in flies [66]. Using 3C or chromosome conformation capture technique, it was found that multiple loci, which are 40 kb apart in the HOXA cluster, are in close physical proximity, enabling MLL to regulate their expression. Other than histone modifications, lncRNAs also impact epigenetic regulation by modulating DNA methylation at CpG dinucleotides, which has an important role in the stable repression of genes [67]. During embryogenesis, methylation markers are first to be found on previously unmethylated DNA by the DNA (cytosine-5-)-methyltransferase 3α (Dnmt3a) and 3β (Dnmt3b) and later maintained through DNA replication by Dnmt1. Tsix might be converted to Xist by utilizing Dnmt3a activity to methylate and finally silence the

Xist promoter [68, 69]. In the same way, Kcnq1ot1 may recruit Dnmt1 [70].

activation [63].

68 RNA Interference

complex in *cis* regulation [64, 65].

LncRNA-directed methylation has also been implicated in the regulation of rDNA. Ribosomal DNA exists in the genome as tandem repeat units [71]. Each unit encodes a polycistronic transcript consisting various rRNAs, and each unit is separated by intergenic spacers (IGSs) transcribed by RNA Pol I [72]. Recently, it was reported that IGS transcripts undergo proc‐ essing into 150- to 300-nt fragments called promoter (p)RNAs, which act as scaffolds to recruit poly (ADP ribose)-polymerase-1 (PARP1) [73], the ATP-dependent nucleolar chromatin remodeling complex (NoRC) [74], and Dnmt3b [75]. A conserved hairpin structure is formed by pRNA that binds both PARP1 and the TIP5 subunit of NoRC, leading to TIP5 conformation change resulting in the recruitment of NoRC to the nucleolus, where rDNA is located [74, 76]. The interesting fact is that the recruitment of Dnmt3b by pRNA is dependent on DNA:RNA triplexing, possibly via Hoogsteen base pairing, between the 5′ end of pRNA and the rDNA promoter [75]. The DNA:RNA triplex formation might be a general mechanism by which lncRNAs recruit *trans* factors to specific DNA loci. LncRNAs are intrinsically bound to chromatin during transcription and transcribed from a single locus in the genome, so they have a direct allele- and locus-specific control in *cis* unlike transcription factors. The length of lncRNAs is also suitable to reach out and capture epigenetic marks. This *cis*-acting mechanism resembles transcriptional gene silencing seen in the yeast *Schizosaccharomyces pombe* in assembling centromeric heterochromatin [77, 78].

The nucleus is always in the dynamic state and is the center for most of the essential functions of an organism [79]. Recent studies indicate that lncRNAs are the key regulators of nuclear compartments. The structure and function of several nuclear bodies seem to be controlled by RNA. One example is nuclear-enriched abundant transcript 1 (NEAT1) that maintains the stability of paraspeckles, which participate in the nuclear retention of mRNAs after adenosineto-inosine hyperediting [80, 81]. NEAT1 interacts with paraspeckle proteins, such as p54/ NONO and PSP [80–82] and recruits these proteins to form paraspeckles. This is an active process where continuous transcription of NEAT1 is required [84]. The related molecules, NEAT2 or metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), are involved in the localization of serine/arginine (SR) splicing factors to nuclear speckles where they can be stored and later modified by phosphorylation [85]. MALAT1 directs these splicing factors to sites of transcription, ultimately controlling the alternative splicing of certain mRNA precursors [86]. MALAT1 interacts with the PRC1 subunit Cbx4/Pc2 and participates in the transportation of genes between nuclear compartments for silencing and activation. Extracel‐ lular growth signals help unmethylated Cbx4 to bind MALAT1 and localize its target genes, along with Lysine (K)-specific demethylase 1A (LSD1) to interchromatin granules that usually cluster around nuclear speckles. However, Cbx4 gets methylated in the absence of extracellular signal and instead binds another lncRNA TUG1, then binds with Ezh2, and translocates to silencing compartments called polycomb bodies [87]. Although these recent observations have started to open up an avenue to understand lncRNA and their mechanisms of action, we are still way behind. The function of an overwhelming number of lncRNAs that are being discovered almost daily is unknown until now.

#### **8. Epigenetic regulation**

The two most abundant modes of action of lncRNAs are the modulation of chromatin by recruiting histone proteins and transcription factors within specific chromatin-modifying complexes. A very good example of recruitment of specific histones is X chromosome inacti‐ vation (XCI), which is caused by "Xist" as described in the earlier section [58]. A similar event is genomic imprinting, where genes are expressed from the allele of only one parent. One of the first and best studied lncRNAs is H19, which is mutually imprinted with insulin-like growth factor 2 (Igf2). This lncRNA is highly expressed, but its deletion has no phenotypic outcome, and it is anticipated to function as a microRNA precursor [88]. Other lncRNAs (e.g., Air, Kcnq1ot1, and HOTAIR) show modulatory activities both in *cis* or in *trans* and regulating gene expression through partnering with chromatin-modifying complexes [70, 89]. Specifical‐ ly, HOTAIR is a *trans*-acting lncRNA that serves as a scaffold for two histone modification complexes: it binds both to PRC2 and to LSD1 [63]. In the *Arabidopsis* plant, it was found that different environmental conditions are able to induce the transcription of related NATs (i.e., COOLAIR) that eventually silence a flower repressor locus, flowering locus c (FLC) [90]. Recently, it was discovered that lncRNA, namely COLDAIR, bearing minor differences from COOLAIR (transcribed in the sense direction relative to FLC mRNA transcription), interacts on its own with PRC2 and targets it to FLC [41]. Other *trans*-acting lncRNAs have different functions, some of which remain incompletely defined. There are several poorly defined *trans*-acting lncRNAs, such as the p21-associated ncRNA DNA damage activated (PANDA), which is induced upon DNA damage in a p53-dependent manner and it controls the expression of proapoptotic genes [91].

#### **9. Transcriptional regulation**

resembles transcriptional gene silencing seen in the yeast *Schizosaccharomyces pombe* in

The nucleus is always in the dynamic state and is the center for most of the essential functions of an organism [79]. Recent studies indicate that lncRNAs are the key regulators of nuclear compartments. The structure and function of several nuclear bodies seem to be controlled by RNA. One example is nuclear-enriched abundant transcript 1 (NEAT1) that maintains the stability of paraspeckles, which participate in the nuclear retention of mRNAs after adenosineto-inosine hyperediting [80, 81]. NEAT1 interacts with paraspeckle proteins, such as p54/ NONO and PSP [80–82] and recruits these proteins to form paraspeckles. This is an active process where continuous transcription of NEAT1 is required [84]. The related molecules, NEAT2 or metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), are involved in the localization of serine/arginine (SR) splicing factors to nuclear speckles where they can be stored and later modified by phosphorylation [85]. MALAT1 directs these splicing factors to sites of transcription, ultimately controlling the alternative splicing of certain mRNA precursors [86]. MALAT1 interacts with the PRC1 subunit Cbx4/Pc2 and participates in the transportation of genes between nuclear compartments for silencing and activation. Extracel‐ lular growth signals help unmethylated Cbx4 to bind MALAT1 and localize its target genes, along with Lysine (K)-specific demethylase 1A (LSD1) to interchromatin granules that usually cluster around nuclear speckles. However, Cbx4 gets methylated in the absence of extracellular signal and instead binds another lncRNA TUG1, then binds with Ezh2, and translocates to silencing compartments called polycomb bodies [87]. Although these recent observations have started to open up an avenue to understand lncRNA and their mechanisms of action, we are still way behind. The function of an overwhelming number of lncRNAs that are being

The two most abundant modes of action of lncRNAs are the modulation of chromatin by recruiting histone proteins and transcription factors within specific chromatin-modifying complexes. A very good example of recruitment of specific histones is X chromosome inacti‐ vation (XCI), which is caused by "Xist" as described in the earlier section [58]. A similar event is genomic imprinting, where genes are expressed from the allele of only one parent. One of the first and best studied lncRNAs is H19, which is mutually imprinted with insulin-like growth factor 2 (Igf2). This lncRNA is highly expressed, but its deletion has no phenotypic outcome, and it is anticipated to function as a microRNA precursor [88]. Other lncRNAs (e.g., Air, Kcnq1ot1, and HOTAIR) show modulatory activities both in *cis* or in *trans* and regulating gene expression through partnering with chromatin-modifying complexes [70, 89]. Specifical‐ ly, HOTAIR is a *trans*-acting lncRNA that serves as a scaffold for two histone modification complexes: it binds both to PRC2 and to LSD1 [63]. In the *Arabidopsis* plant, it was found that different environmental conditions are able to induce the transcription of related NATs (i.e., COOLAIR) that eventually silence a flower repressor locus, flowering locus c (FLC) [90]. Recently, it was discovered that lncRNA, namely COLDAIR, bearing minor differences from

assembling centromeric heterochromatin [77, 78].

70 RNA Interference

discovered almost daily is unknown until now.

**8. Epigenetic regulation**

The discovery and characterization of promoter-associated RNAs opened up a new under‐ standing on how genes are regulated during transcription. These RNAs are localized within the promoter and consist of various sizes of RNA molecules [92]. The long ones are found at a single-gene level and are associated with the modification of DNA methylation and deme‐ thylation patterns [93] as mentioned earlier. Interestingly, long (antisense) pRNAs generally form double-stranded molecules that are processed into endo-siRNAs, and since they have sequence complementarity with the promoter, they induce transcriptional gene silencing [20, 94–96] or activation [97–99].

LncRNAs sometimes affect transcription by acting as coregulators or by regulating the association and activity of coregulators. One example is embryonic ventral forebrain-2 (Evf-2) that functions as a coactivator for the homeobox transcription factor distal-less homeobox 2 (Dlx2) [100].

#### **10. Posttranscriptional regulation**

lncRNAs not only have a role in transcription but also they function in splicing, mRNA stability, and translation. Antisense lncRNA sometimes bind to the sense RNA, conceal the splice sites, and thereby modify the balance between splice variants. Antisense transcript RevErbAα modifies the splicing of thyroid hormone receptor alpha genes (TRα) TRα1 and TRα2 mRNAs [101].

The terminal differentiation-induced ncRNA (TINCR) associates with Staufen 1 but not with the complex between TINCR-NA, which is a differentiation factor [102].

LncRNAs have also been implicated in translational regulation. An example is the antisense for PU1 mRNA. Its translation is inhibited by an antisense polyadenylated lncRNA with a halflife longer than the original transcript [103]. Another example is the lncRNA Uchl1, which is controlled by mammalian target of rapamycin (mTOR) pathway, shuttles from the nucleus to the cytoplasm, and controls the translation of the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) mRNA by promoting its association with polysomes [104].

**Figure 2.** Posttranscriptional gene silencing by lncRNA and miRNA (adapted from Gomes et al. [130]).

#### **11. Role of lncRNAs in cancer and other human diseases**

The genome-wide association studies identify several cancer risk loci outside of proteincoding regions. Of 301 single-nucleotide polymorphisms currently linked to cancer, only 12 (3.3%) modify the amino acid sequence of the protein, and most of the loci are located in the introns (40%) or intergenic regions (44%) [105]. These facts and the observations that miRNA and lncRNAs are involved in differentiation and development point towards the fact that alterations in their expression profiles could be correlated with cancer develop‐ ment. Reports suggested that lncRNAs have tissue-specific expression and is found to be deregulated in distinct types of cancers. For example, overexpression of miR-155 was reported in hematopoietic, breast, lung, and colon cancers [106], whereas miR-21 is overexpressed in glioblastoma [107]. In addition, lymphoproliferative disorders were found in transgenic mice overexpressing miR-17-92 [108]. Incidences of lung, colon, and gastric cancers were found to be correlated with the overexpression of miR-17-92 cluster [109]. LncRNAs have been associated with cancer development likewise. The lncRNA MALAT1 is up-regulated in several cancer types, resulting in an increase in cell proliferation and migration in lung and colorectal cancer cells [105]. The role of MALAT1 in controlling alternative splicing of pre-mRNAs [86] can be deduced from this. A more recent study indicates that MALAT1 may also participate in the regulation of gene expression by a mechanism other than alternative splicing in lung metastasis [110].

Other studies have shown that miRNA and lncRNAs both can function as tumor suppressor genes or oncogenes. The tumor suppressor gene p53 regulates the three gene members of the Long Noncoding RNAs are Frontier Breakthrough of RNA World and RNAi-based Gene Regulation http://dx.doi.org/10.5772/61975 73

**Figure 3.** Relationship among various noncoding RNAs and different disorders caused by them (adapted from Gomes et al. [130].

**11. Role of lncRNAs in cancer and other human diseases**

72 RNA Interference

**Figure 2.** Posttranscriptional gene silencing by lncRNA and miRNA (adapted from Gomes et al. [130]).

mechanism other than alternative splicing in lung metastasis [110].

The genome-wide association studies identify several cancer risk loci outside of proteincoding regions. Of 301 single-nucleotide polymorphisms currently linked to cancer, only 12 (3.3%) modify the amino acid sequence of the protein, and most of the loci are located in the introns (40%) or intergenic regions (44%) [105]. These facts and the observations that miRNA and lncRNAs are involved in differentiation and development point towards the fact that alterations in their expression profiles could be correlated with cancer develop‐ ment. Reports suggested that lncRNAs have tissue-specific expression and is found to be deregulated in distinct types of cancers. For example, overexpression of miR-155 was reported in hematopoietic, breast, lung, and colon cancers [106], whereas miR-21 is overexpressed in glioblastoma [107]. In addition, lymphoproliferative disorders were found in transgenic mice overexpressing miR-17-92 [108]. Incidences of lung, colon, and gastric cancers were found to be correlated with the overexpression of miR-17-92 cluster [109]. LncRNAs have been associated with cancer development likewise. The lncRNA MALAT1 is up-regulated in several cancer types, resulting in an increase in cell proliferation and migration in lung and colorectal cancer cells [105]. The role of MALAT1 in controlling alternative splicing of pre-mRNAs [86] can be deduced from this. A more recent study indicates that MALAT1 may also participate in the regulation of gene expression by a

Other studies have shown that miRNA and lncRNAs both can function as tumor suppressor genes or oncogenes. The tumor suppressor gene p53 regulates the three gene members of the miR-34 family. Curiously, the microRNA-34 (miR-34) activation resembles p53 activity, such as the induction of cell cycle arrest and promotion of apoptosis, and p53-mediated apoptosis becomes defective in the absence of miR-34 [111].

LncRNAs that recruit epigenetic modifiers to specific loci such as ANRIL, XIST, HOTAIR, and KCNQ1OT1 are found to have altered expression in a variety of cancers [112]. Another lncRNA called TERRA binds telomerase, inhibiting its activity *in vitro* [113], and is observed to be downregulated in many cancer cells, linking it with the longevity of cancer cells.

Chromatin remodeling by lncRNA is linked to other diseases such as facioscapulohumeral muscular dystrophy (FSHD) [97], lethal lung developmental disorder [114], and the HELLP syndrome, a pregnancy-associated disease [114] in addition to cancer. The HELLP stands for H = hemolysis (breakdown of red blood cells), EL = elevated liver enzymes (liver function), and LP = low platelet counts (platelets help the blood clot). These examples directly link lncRNA and miRNAs in cancer biology and other human diseases and indicate the involve‐ ment of a complex interplay among their biogenesis pathways, their regulatory mechanisms, and their targets.

#### **12. Dosage compensation and X inactivation**

X chromosome inactivation (XCI) occurs in females during embryogenesis, where either the maternal or paternal X chromosome is randomly silenced. The molecular mechanisms of XCI are not yet fully understood. However, it is known that a 500-kb stretch of DNA at Xq13 known as the X-inactivation centre (XIC) is the site for initiation of X inactivation. There are several lncRNAs, including X-inactive specific transcript (Xist), its antisense transcript Tsix, Xinactivation intergenic transcription elements (Xite), Jpx transcript, and Xist activator (Jpx), and others play pivotal roles in XCI [115]. Xist was one of the first to be identified and best studied lncRNAs. It is a ~17-kb transcript (~19 kb in humans) expressed from the future inactive X chromosome (Xi) [116]. Tsix is a ~40-kb antisense transcript to Xist. It negatively regulates Xist. Recent studies indicate that Xite is a transcriptional enhancer of Tsix [115], and likewise, Jpx RNA appears to help in Xist expression [117].

When two homologous X chromosomes are brought at close proximity, Tsix and Xite initiate the inactivation process by counting, and this is associated with the presence of RNA poly‐ merase II (RNAPII) [118, 119]. The chromatin insulator CTCF, which binds to Tsix and Xite genomic loci [120], play an important role. The transcription factor OCT4 is then hypothesized to bind with Tsix promoters of one of the X chromosomes, which then converts to active X chromosome (Xa) due to increased transcription of Tsix [120]. Thereafter, Dnmt3a is recruited to the Xa and establishes stable silencing of Xist on the Xa [115, 118].

#### **13. LncRNA in genomic imprinting**

In mammals, genomic imprinting is an epigenetic marker in a way that their expression occurs specifically in parental origin manner. This occurs during early gametogenesis in nearly 1% of protein-coding genes. To date, we have identified around 150 imprinted genes in mice. Imprinted genes are often located in clusters of size from a few kilobases to 2 to 3 Mb. LncRNAs are present in all the identified and elucidated imprinted clusters as their partners. The expression of lncRNAs is reciprocally linked with corresponding proteincoding genes [121–123].

Genomic imprinting mainly happens by chromatin insulators [124–126] and lncRNAs [38, 127]. LncRNAs repress flanking gene promoters in *cis* action (Kcnq1ot1 and Airn lncRNAs [115]). However, several reports indicate that lncRNAs function as a major force in the regulation of parent-of-origin-specific expression. Today, we know that the human genome contains more than 58,648 lncRNA expressed genes compared to only 21,313 protein-coding genes [128]. The majority of the lncRNAs act by interacting with chromatin-modifying complexes such as PRC2, G9a, hnRNPK, and SWI/SNF, recruiting them sequentially to silence genes in *cis* or *trans* action [57, 60].

#### **14. Perspectives**

**12. Dosage compensation and X inactivation**

74 RNA Interference

Jpx RNA appears to help in Xist expression [117].

**13. LncRNA in genomic imprinting**

coding genes [121–123].

genes in *cis* or *trans* action [57, 60].

to the Xa and establishes stable silencing of Xist on the Xa [115, 118].

X chromosome inactivation (XCI) occurs in females during embryogenesis, where either the maternal or paternal X chromosome is randomly silenced. The molecular mechanisms of XCI are not yet fully understood. However, it is known that a 500-kb stretch of DNA at Xq13 known as the X-inactivation centre (XIC) is the site for initiation of X inactivation. There are several lncRNAs, including X-inactive specific transcript (Xist), its antisense transcript Tsix, Xinactivation intergenic transcription elements (Xite), Jpx transcript, and Xist activator (Jpx), and others play pivotal roles in XCI [115]. Xist was one of the first to be identified and best studied lncRNAs. It is a ~17-kb transcript (~19 kb in humans) expressed from the future inactive X chromosome (Xi) [116]. Tsix is a ~40-kb antisense transcript to Xist. It negatively regulates Xist. Recent studies indicate that Xite is a transcriptional enhancer of Tsix [115], and likewise,

When two homologous X chromosomes are brought at close proximity, Tsix and Xite initiate the inactivation process by counting, and this is associated with the presence of RNA poly‐ merase II (RNAPII) [118, 119]. The chromatin insulator CTCF, which binds to Tsix and Xite genomic loci [120], play an important role. The transcription factor OCT4 is then hypothesized to bind with Tsix promoters of one of the X chromosomes, which then converts to active X chromosome (Xa) due to increased transcription of Tsix [120]. Thereafter, Dnmt3a is recruited

In mammals, genomic imprinting is an epigenetic marker in a way that their expression occurs specifically in parental origin manner. This occurs during early gametogenesis in nearly 1% of protein-coding genes. To date, we have identified around 150 imprinted genes in mice. Imprinted genes are often located in clusters of size from a few kilobases to 2 to 3 Mb. LncRNAs are present in all the identified and elucidated imprinted clusters as their partners. The expression of lncRNAs is reciprocally linked with corresponding protein-

Genomic imprinting mainly happens by chromatin insulators [124–126] and lncRNAs [38, 127]. LncRNAs repress flanking gene promoters in *cis* action (Kcnq1ot1 and Airn lncRNAs [115]). However, several reports indicate that lncRNAs function as a major force in the regulation of parent-of-origin-specific expression. Today, we know that the human genome contains more than 58,648 lncRNA expressed genes compared to only 21,313 protein-coding genes [128]. The majority of the lncRNAs act by interacting with chromatin-modifying complexes such as PRC2, G9a, hnRNPK, and SWI/SNF, recruiting them sequentially to silence

LncRNA has diversified tentacles for functions. Those include an alteration of transcriptional profiles, controlling of protein expression, complex structural or organizational roles, RNA processing or RNA editing and role of being the precursor of small RNAs. Because a very small fraction of lncRNA have been molecularly characterized to date, many more yet to be discov‐ ered that fit into this diversified functional paradigms. Future work will definitely ask many more questions about the interplay of lncRNA transcripts and whether it is sufficient to have fundamental sequence of events or not. Many lncRNAs play intermediate roles in *cis*regulation that gets represented in ectopic expression in *trans* regulation.

Most recent challenges are to identify how the molecular function of each type of lncRNA results in different diseases of the organism. LncRNA appears to expose numerous develop‐ mental events such as the generation of photoreceptor cells in retina development, control of cell surveillance, cell cycle progression of mammary gland development, and finally genera‐ tion of knockout animal development. Many lncRNAs are not eliminated as transcriptional noise in the genome but are useful for normal developmental processes.

LncRNA has a tremendous impact on disease development due to its flawless miscegenation. In tumor formation, the expression of lncRNAs is very important. They function like specific markers of tumor formation. However, the exact mechanism by which tumor initiation, formation, and progression would occur is not fully understood. It is true that the interplay and significant role of lncRNA in different disease research is really an unexplored area, which is eventually determining the new therapeutic targets. Recently, it was found that lncRNA may form β-amyloid plaques in Alzheimer's disease. This possibility suggested that noncoding transcript might serve as an attractive drug target for Alzheimer's disease.

Most conventionally, genetic information may run through protein-coding sequences, but it is now found that transcription is pervasive through the nucleic acid content of eukaryotic genome, which generated a numerous number of lncRNA, which are possibly the key regulators of protein-coding sequences. We anticipate that many more surprises are yet to be explored in the coming decades. Therefore, future research might provide more pleasant but unexpected surprises in the lncRNA function.

#### **15. Conclusion**

The above description exhibits a brief survey of the current status of knowledge regarding the identification, localization, functions, and mechanisms of actions of lncRNAs related to different human diseases. A fraction of genomic nucleic acid is transcribed to protein, but an overwhelming majority of the genome sectors of the organisms contain lncRNA with unknown functional efficacy. Some are nuclear or cytoplasmic and are highly overexpressed, and others are rarely detected. Truly, it is impossible to discern the important criteria such as stability, conservation, and time of expression related to human diseases. LncRNA in the Xic is only found in placental mammals and is not conserved in other mammals. However, this limited conservation might not be essential in other higher animals. The true test for real function lies in the mechanism, genetic pathway, and tissue-specific activity for each lncRNA. The genome of an organism is not always streamlined by the natural selection. Thus, here, we really tried to avoid the speculative statements about localization, function, and dissecting mechanism regarding long noncoding RNA. Truly, we have just begun to scratch the skin of LncRNA in the human body. The lncRNA world is so galactically vast that we have an enormous task to completely learn about it. We feel that additional discoveries of lncRNA may provide a real exciting phase in the study of RNA world.

#### **Acknowledgements**

We thank the lab members and Sohini Bose for the extensive discussion in the subjects and help in writing. This work is sponsored by the CSIR Net work grant (BSC-0121) of UB and DBT grant to M.P.B. (GAP 0362). DRC work supported by DST (GAP0432) grant.

#### **Author details**

Utpal Bhadra1\*, Debabani Roy Chowdhury1 , Tanmoy Mondal2 and Manika Pal Bhadra2

\*Address all correspondence to: utpal@ccmb.res.in

1 Functional Genomics and Gene Silencing Group, Centre for Cellular and Molecular Biology, Hyderabad, India

2 Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad, India

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found in placental mammals and is not conserved in other mammals. However, this limited conservation might not be essential in other higher animals. The true test for real function lies in the mechanism, genetic pathway, and tissue-specific activity for each lncRNA. The genome of an organism is not always streamlined by the natural selection. Thus, here, we really tried to avoid the speculative statements about localization, function, and dissecting mechanism regarding long noncoding RNA. Truly, we have just begun to scratch the skin of LncRNA in the human body. The lncRNA world is so galactically vast that we have an enormous task to completely learn about it. We feel that additional discoveries of lncRNA may provide a real

We thank the lab members and Sohini Bose for the extensive discussion in the subjects and help in writing. This work is sponsored by the CSIR Net work grant (BSC-0121) of UB and DBT

, Tanmoy Mondal2

and Manika Pal Bhadra2

grant to M.P.B. (GAP 0362). DRC work supported by DST (GAP0432) grant.

1 Functional Genomics and Gene Silencing Group, Centre for Cellular and Molecular

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2 Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad, India

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Utpal Bhadra1\*, Debabani Roy Chowdhury1

\*Address all correspondence to: utpal@ccmb.res.in

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86 RNA Interference

S0960-9822(00)00442-5

S0960-9822(00)00597-2

DOI: 10.1038/ng.3192

## **Noncanonical Synthetic RNAi Inducers**

O.V. Gvozdeva and E.L. Chernolovskaya

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61685

#### **Abstract**

This review focuses on current strategies of development of noncanonical synthetic RNA interference (RNAi) inducers with structural modifications for promoting better gene si‐ lencing with low risk of side effects. A particular focus is on longer RNA duplexes 25–30 nucleotides (nt) in length that mimic Dicer substrates to improve interaction of RNAi in‐ ducers with RNAi machinery. Various design strategies of efficient Dicer substrate smallinterfering RNA (siRNA) are described. It was found that the length, chemical modifications, and overhang structure influence the gene silencing activity and RNA-in‐ duced silencing complex (RISC) assembly. Special attention is paid to the long doublestranded RNA duplexes that induce effective gene silencing in Dicer-dependent or Dicerindependent mode. Some structural variants of shorter siRNAs, including hairpin and dumbbell siRNAs and fork-siRNA (fsiRNA) with several nucleotide substitutions at the 3′ end of the sense strand, are also analyzed. These structural modifications provide effi‐ ciently increased gene silencing of targets with unfavorable duplex thermodynamic asymmetry. Recent data remove the length and structure limits for the design of RNAi effectors, and add another example in the list of novel RNAi-inducing molecules differ‐ ing from the classical siRNA, which is discussed in this chapter.

**Keywords:** RNAi, siRNA, fsiRNA, dsiRNA, tsiRNA, structural modifications, mechanism of action

#### **1. Introduction**

RNA interference is a conserved mechanism of a sequence-specific posttranscriptional gene silencing triggered by double-stranded RNAs homologous to the silenced gene [1, 2]. Long double-stranded RNA(dsRNAs) are cleaved in the cell by RNase III class endonuclease Dicer into short fragments 21–22 nucleotides (nt) in length with 2–3-nt 3′ overhangs at both ends [3, 4]. These fragments (small-interfering RNAs, siRNAs) enter RNA-induced silencing complex (RISC) and associate with core proteins belonging to Argonaute (AGO) family [5]. AGO

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

unwinds the duplex and cuts one of the strands in the middle, and then this strand (designated as "passenger") dissociates from the complex and is degraded by cellular ribonucleases. The other strand (designated as "guide") remains in activated RISC and recognizes the cellular mRNA complementary to the guide strand. The configuration of the complex determines which strand remains in the complex and which strand leaves and degrades. Active RISC complex containing the guide strand binds to the complementary mRNA and induces its cleavage. When the cleaved mRNA is released, RISC is recycled for a new round of cleavage [4, 6]. The details of the RNAi mechanism are well reviewed in literature [7–9].

Synthetic small-interfering RNAs have become an advanced and powerful tool for specific gene silencing and could be considered as promising class of therapeutics for the treatment of diseases associated with overexpression of specific genes [10–12]. However, therapeutic applications of canonical non-modified siRNAs are limited by their sensitivity to ribonucleas‐ es, possibility of unfavorable guide strand selection, and activation of innate immune system by siRNA containing immunostimulatory motives in the sequence, which can lead to poor gene silencing efficiency [13, 14]. Different structural variations of the RNAi inducers together with chemical modification were developed to overcome these problems.

This review focuses on current strategies of development of siRNA structural modifications for promoting better gene silencing with low risk of side effects, with particular focus on longer siRNA duplexes 25–30 nt in length that mimic Dicer substrates (Dicer substrate siRNA (dsiRNA)) [15–20]. Special attention has been paid to the long double-stranded RNA duplexes, which induced effective gene silencing and did not require Dicer-mediated processing of the substrate into smaller units: trimer RNA (tsiRNA) with 63 nt in length and tripartite-interfering RNA (tiRNA) with 38 nt in length [21, 22]. Applications of some structure variations of shorter siRNAs and the potential of different synthetic RNAi inducers in different applications have also been reviewed and summarized.

#### **2. Dicer substrate interfering RNAs**

Long dsRNAs homologous to the targeted mRNA were successfully used for silencing of gene expression in nonmammalian species [1, 4]. Early attempts to use long dsRNAs in mammalian cells triggering of RNAi failed due to activation of innate immune system by dsRNA [15]. Although activation of innate immunity may be beneficial for the therapy in some cases, uncontrolled induction of the interferon response results in global changes in gene expression profile and, in some cases, in cells death [23–25]. It was found that chemically synthesized 21 mer RNA duplexes with 2-nt 3′ overhangs at both ends, which directly mimic the products produced by Dicer, efficiently suppressed gene expression in mammalian cells [4, 26]. These duplexes, referred to as canonical siRNAs, are widely used in biomedical research [11]. Later, it was found that RNA duplexes, smaller than 30 nt in length but longer than siRNAs, were significantly more efficient than canonical siRNAs and did not induce interferon response in a variety of cell lines [15]. It was established experimentally that 27-mer duplexes possess maximal silencing activity, longer duplexes demonstrated reduced silencing activity, and 40– 45-mer duplexes were inactive. At the same time, 27-mer duplexes, named as Dicer substrate siRNA (dsiRNA), were efficiently cleaved by Dicer producing a variety of 21-nt-long distinct products. High potency of 27-mer duplexes initially was explained by the formation of siRNA pool containing functional siRNAs with extremely high silencing activity. Some of 27-mer duplexes were significantly more potent at nanomolar or picomolar concentrations than the specific 21-mer siRNA selected according to the current computational algorithms [15]. However, further experiments demonstrated that none of the synthetic 21-nt siRNAs, included in the corresponding set to all possible products of Dicer processing of 27-mer duplexes, demonstrated the same level of silencing activity as 27-mers at low concentrations [15].

unwinds the duplex and cuts one of the strands in the middle, and then this strand (designated as "passenger") dissociates from the complex and is degraded by cellular ribonucleases. The other strand (designated as "guide") remains in activated RISC and recognizes the cellular mRNA complementary to the guide strand. The configuration of the complex determines which strand remains in the complex and which strand leaves and degrades. Active RISC complex containing the guide strand binds to the complementary mRNA and induces its cleavage. When the cleaved mRNA is released, RISC is recycled for a new round of cleavage

Synthetic small-interfering RNAs have become an advanced and powerful tool for specific gene silencing and could be considered as promising class of therapeutics for the treatment of diseases associated with overexpression of specific genes [10–12]. However, therapeutic applications of canonical non-modified siRNAs are limited by their sensitivity to ribonucleas‐ es, possibility of unfavorable guide strand selection, and activation of innate immune system by siRNA containing immunostimulatory motives in the sequence, which can lead to poor gene silencing efficiency [13, 14]. Different structural variations of the RNAi inducers together

This review focuses on current strategies of development of siRNA structural modifications for promoting better gene silencing with low risk of side effects, with particular focus on longer siRNA duplexes 25–30 nt in length that mimic Dicer substrates (Dicer substrate siRNA (dsiRNA)) [15–20]. Special attention has been paid to the long double-stranded RNA duplexes, which induced effective gene silencing and did not require Dicer-mediated processing of the substrate into smaller units: trimer RNA (tsiRNA) with 63 nt in length and tripartite-interfering RNA (tiRNA) with 38 nt in length [21, 22]. Applications of some structure variations of shorter siRNAs and the potential of different synthetic RNAi inducers in different applications have

Long dsRNAs homologous to the targeted mRNA were successfully used for silencing of gene expression in nonmammalian species [1, 4]. Early attempts to use long dsRNAs in mammalian cells triggering of RNAi failed due to activation of innate immune system by dsRNA [15]. Although activation of innate immunity may be beneficial for the therapy in some cases, uncontrolled induction of the interferon response results in global changes in gene expression profile and, in some cases, in cells death [23–25]. It was found that chemically synthesized 21 mer RNA duplexes with 2-nt 3′ overhangs at both ends, which directly mimic the products produced by Dicer, efficiently suppressed gene expression in mammalian cells [4, 26]. These duplexes, referred to as canonical siRNAs, are widely used in biomedical research [11]. Later, it was found that RNA duplexes, smaller than 30 nt in length but longer than siRNAs, were significantly more efficient than canonical siRNAs and did not induce interferon response in a variety of cell lines [15]. It was established experimentally that 27-mer duplexes possess maximal silencing activity, longer duplexes demonstrated reduced silencing activity, and 40–

[4, 6]. The details of the RNAi mechanism are well reviewed in literature [7–9].

with chemical modification were developed to overcome these problems.

also been reviewed and summarized.

90 RNA Interference

**2. Dicer substrate interfering RNAs**

Based on the earlier observations that Dicer participates both in the cleavage of dsRNAs and in the incorporation of the products of cleavage into RISC complex in *Drosophila melanogaster*, it has been suggested that Dicer could participate in direct loading of siRNA into RISC and in RISC assembly [16, 27]. It has been experimentally proved that dsiR‐ NAs form the RISC loading complex (RLC) in vitro more efficiently than the canonical 21 mer siRNA duplexes [18]. Because Dicer does not form complexes with 21-base pair (bp) duplexes, it was assumed that Dicer facilitates RLC formation after dsRNA cleavage without dissociation from the cleavage product. These findings become a basis for the develop‐ ment of a new class of RNAi inducers [16, 17, 28].

The silencing activity of dsiRNA depends on its structure. At the first step of recognition, PAZ (Piwi Argonaut and Zwille) domain of Dicer predominantly "anchors" two ribonucleo‐ tides on 3′ overhangs because those blunt 27-mer duplexes are not good substrates for Dicer. PAZ domain plays a vital role in the orientation of bound RNA in the active site of the enzyme and determines the cleavage position on RNA for AGO protein. Unlike 21 mer siRNA, where two-base 3′-deoxynucleotide overhangs are often used regardless of their complementarity to the target mRNA sequence (mostly dTdT), the overhang sequen‐ ces are important for the properties of dsiRNA. Incorporation of deoxynucleotides at the 3′ ends of dsiRNA strands has an adverse effect on dsiRNA processing [19]. The se‐ quence of 3′ terminal overhangs could control dicing polarity and strand selection into RISC. Thus, Dicer preferentially binds with purine/purine (GG, AA) nucleotides [19]. Protruding nucleotides added to the 3′ terminal of the antisense strand facilitate its preferential loading into RISC [19]. Hence, asymmetric duplexes with one 2-nt 3′ over‐ hang and DNA residues on the blunt end of the duplex provide a single favorable PAZ binding site and reduce heterogeneity of cleavage products (Figure 1) [16–18, 29, 30].

The stability of dsiRNA in physiological fluids is extremely an important factor for its applications in vivo [31]. Although dsRNAs are more stable in comparison with singlestranded RNAs and 21-bp siRNA, they still rapidly degrade in the serum [32]. It was found that bonds with 3′ pyrimidine nucleotides are cleaved faster than bonds with 3′ purines. Kubo and his colleagues demonstrated that degradation rate of dsiRNAs correlated with the amount of pyrimidines at the 3′ end [31]. At the same time, degradation rate of dsiRNAs also correlates with the presence of AU-rich domains that might be related to low thermal stability, easy dissociation, and faster cleavage by both endo- and exonucleases. Chemical modifications can improve nuclease stability and reduce off-target effects [33–36]. Fluorescein modification of 3′

**Figure 1.** The scheme of dsiRNA design (according to [17]). N – ribonucleic acids, d - deoxyribonucleic acids, short arrows – the site of Dicer cleavage.

end of 27-mer RNA duplexes significantly reduces RNAi activity because 3′ ends are important for interaction with Dicer and should be available for the proper recognition [15]. On the other hand, dsiRNAs with chemical modifications of the 5′ end possess high nuclease stability and RNAi activity in the cell-cultured medium. Thus, 5′-end amino-modified dsiRNA demon‐ strated improved RNAi activity and stability in the cell-cultured medium [31].

Incorporation of 2′-O-methyl modifications is an efficient and inexpensive method to improve nuclease resistance of synthetic RNA duplexes [33, 37–40]. However, dsiRNA duplexes with modifications of all or the majority of nucleotides in both sense and antisense strands are practically inactive, because extensive modification blocks the cleavage of duplex by Dicer [17]. Limiting the modifications to incorporation of only 9–11 modified bases into the antisense strand and avoiding modifications in the site of Dicer cleavage prevents this undesired effect [17]. Mass spectrometry analysis of in vitro dicing reactions showed that modified duplexes produce a mixture of 21- and 22-nt species, whereas unmodified duplexes are processed only into 21-mer species. If modifications were spaced further away from the dicing site, a prefer‐ ential accumulation of 21-mer species was observed [17]. However, it was noted that in some cases, usually, a good modification pattern may decrease the silencing activity of dsiRNA. This phenomenon has been related to the newly unidentified sites in the sequence context of some chemically modified dsiRNAs, contributing to impairment of dsiRNAs silencing effect [17]. These observations could explain differences in the efficiency of various dsiRNAs and made possible the creation of the modification patterns compatible with dicing.

Accumulation of experimental data revealed that 27–30-bp dsRNAs, including dsiRNA and, in some cases, even 21-bp siRNA, could stimulate innate immune system and induce interferon response in certain cell types [23]. Toll-like receptors (TLRs) 7 and 8 appear to be the main molecules responsible for the immune recognition of siRNA and dsiRNA, whereas toll-like receptor 3 recognizes longer than 30-bp dsRNA [41, 42]. Activation of innate immune system through TLRs results in the production of interferon α, tumor necrosis factors α, and inter‐ leukins IL6 and IL12 [42]. Immunostimulatory properties of siRNA are sequence dependent; TLR7 and TLR8 receptors recognize GU-rich sequences of siRNA [43]. Moreover, several immunostimulatory motifs of siRNA enriched in GU nucleotides were identified [44, 45]. It is recommended to avoid these motives in siRNA and dsiRNA design; unfortunately, not all immunostimulatory motifs have been discovered that complicate the design procedure. Earlier, it was demonstrated that chemical modifications involving 2′ position of the ribose ring in siRNA could block the immune response [46]. Incorporation of 2′-O-methyl U and G bases into siRNA significantly reduced immunostimulatory activity of siRNA in vitro and in vivo, containing immune-stimulating motives in the sequence [41]. Moreover, the effective suppression of immunostimulatory activity could be reached by using only a small percentage of modified nucleotides (<10%). Collingwood and his colleagues [17] applied this approach to dsiRNA and demonstrated that limited 2′-O-methyl modifications of uridine and guanosine into antisense strand of dsiRNA efficiently prevent induction of innate immune response in different cell lines.

Another option to reduce nonspecific effects of dsiRNA is to use enzymatically produced pools of Dicer substrate RNA [20]. Dicer from the protozoan parasite *Giardia intestinalis* was used to obtain enzymatically produced dsiRNAs. It cuts long dsRNA into fragments from 25 to 27 nt in length. The sequences-related side effects were decreased in the pool of enzymatically produced dsiRNAs due to the low concentration of individual dsiRNAs with undesirable sequence.

end of 27-mer RNA duplexes significantly reduces RNAi activity because 3′ ends are important for interaction with Dicer and should be available for the proper recognition [15]. On the other hand, dsiRNAs with chemical modifications of the 5′ end possess high nuclease stability and RNAi activity in the cell-cultured medium. Thus, 5′-end amino-modified dsiRNA demon‐

**Figure 1.** The scheme of dsiRNA design (according to [17]). N – ribonucleic acids, d - deoxyribonucleic acids, short

Incorporation of 2′-O-methyl modifications is an efficient and inexpensive method to improve nuclease resistance of synthetic RNA duplexes [33, 37–40]. However, dsiRNA duplexes with modifications of all or the majority of nucleotides in both sense and antisense strands are practically inactive, because extensive modification blocks the cleavage of duplex by Dicer [17]. Limiting the modifications to incorporation of only 9–11 modified bases into the antisense strand and avoiding modifications in the site of Dicer cleavage prevents this undesired effect [17]. Mass spectrometry analysis of in vitro dicing reactions showed that modified duplexes produce a mixture of 21- and 22-nt species, whereas unmodified duplexes are processed only into 21-mer species. If modifications were spaced further away from the dicing site, a prefer‐ ential accumulation of 21-mer species was observed [17]. However, it was noted that in some cases, usually, a good modification pattern may decrease the silencing activity of dsiRNA. This phenomenon has been related to the newly unidentified sites in the sequence context of some chemically modified dsiRNAs, contributing to impairment of dsiRNAs silencing effect [17]. These observations could explain differences in the efficiency of various dsiRNAs and made

Accumulation of experimental data revealed that 27–30-bp dsRNAs, including dsiRNA and, in some cases, even 21-bp siRNA, could stimulate innate immune system and induce interferon response in certain cell types [23]. Toll-like receptors (TLRs) 7 and 8 appear to be the main molecules responsible for the immune recognition of siRNA and dsiRNA, whereas toll-like receptor 3 recognizes longer than 30-bp dsRNA [41, 42]. Activation of innate immune system through TLRs results in the production of interferon α, tumor necrosis factors α, and inter‐

strated improved RNAi activity and stability in the cell-cultured medium [31].

arrows – the site of Dicer cleavage.

92 RNA Interference

possible the creation of the modification patterns compatible with dicing.

In the cases when silencing of more than one gene is required, the transfection of siRNA mixture is used. Co-transfection of different siRNAs may result in different knockdown efficiency of individual targets due to competition between siRNAs for RISC loading depend‐ ing on the thermodynamic asymmetry of the duplexes [30]. Therefore, preliminary testing is required to assess the degree of competition between various siRNAs. Competition between RNAi inducers aimed at different mRNAs could be avoided by using Dicer substrate RNA. Entry of dsiRNAs into RNAi pathway is not limited by RISC loading step, and discrimination of canonical siRNAs based on RISC incorporation is reduced. These beneficial properties of dsiRNAs can provide an effective tool for targeting multiple mRNAs.

Currently, siRNAs have become a powerful tool for effective suppression of expression of target genes in vitro and in vivo applications. Moreover, several compounds are already used in clinical trials. However, the examples of Dicer substrate RNAs usage in vivo are fewer in number. Several studies use dsiRNA to silence therapeutically relevant genes in vivo (Table 1)*.* Frequently, cancer-related genes and genes of viruses [50, 51, 56–59] are chosen as targets for dsiRNAs [47–49]. Several researchers used *TNFα* gene as a target for the treatment of inflammatory and autoimmune diseases [52–54]. Murine models are the most popular animal models among various studies that used dsiRNA in vivo [47–54]; however, there are studies where other animal models, for example, rats, were used [53, 55]. An exciting example of dsiRNA application was described by Doré-Savard and his colleagues, who demonstrated, for the first time, the efficient suppression of target genes in central nervous system (CNS) of rats by dsiRNA [55]. In this study, 27-mer dsiRNAs were used to reduce expression of neurotensin receptor-2 (NTS2) involved in ascending nociception. dsiRNAs were formulated with cationic lipid i-Fect and used in intrathecal spinal cord injection. Extremely low doses of dsiRNA (0.005 mg/kg) efficiently silenced NTS2 mRNA and protein levels for 3–4 days. It is known that administration of high doses of non-modified siRNA increases the risk of activation of innate immune system, especially when siRNA is used together with cationic lipids. Low doses of highly active dsiRNAs could minimize this adverse effect. No apparent toxicity and other offtarget effects were found during the experiment [55]. The dose–response experiments per‐ formed in another study [28] also show that 27-mer Dicer substrate RNA provide improved gene silencing when used at lower concentrations [28]. The silencing activity of canonical 21 mer siRNAs was compared with that of dsiRNA at 1 and 5 nM concentrations. The 27-mer dsiRNA displayed more potent gene silencing at 1 nM concentration, while at 5 nM concen‐ tration, the difference in silencing was less pronounced [28].



lipid i-Fect and used in intrathecal spinal cord injection. Extremely low doses of dsiRNA (0.005 mg/kg) efficiently silenced NTS2 mRNA and protein levels for 3–4 days. It is known that administration of high doses of non-modified siRNA increases the risk of activation of innate immune system, especially when siRNA is used together with cationic lipids. Low doses of highly active dsiRNAs could minimize this adverse effect. No apparent toxicity and other offtarget effects were found during the experiment [55]. The dose–response experiments per‐ formed in another study [28] also show that 27-mer Dicer substrate RNA provide improved gene silencing when used at lower concentrations [28]. The silencing activity of canonical 21 mer siRNAs was compared with that of dsiRNA at 1 and 5 nM concentrations. The 27-mer dsiRNA displayed more potent gene silencing at 1 nM concentration, while at 5 nM concen‐

**tion/dose**

**Biological effect Reference**

[57]

[48]

[57]

[50]

20 nM >80% cell growth inhibition [47]

after second injection

5 nM 99.5% inhibition in luciferase

assay

50 nM >50% reduction of both

mRNA and protein

mRNA and protein

250 nM > 100-fold decrease of viral titer

tumor weight

0.65 nM 50% reduction in plaque assay

10 nM 50% mRNA reduction [53]

Liver cancer 1 nM >90% mRNA level reduction [49]

tration, the difference in silencing was less pronounced [28].

25D/27-mer *cdc20* (mouse) Breast

coding regions of hepatitis C virus: *NS3, NS4B, NS5A, NS5B*

**Structure Target (gene) Disease Concentra-**

cancer

Hepatitis C virus infection

cancer

Respiratory syncytial virus infection

Mice *Hsp27* (mouse) 3 mg/kg >50% reduction of both

Mice *Ctnnb1 (*mouse*)* 5 mg/kg Significant reduction of

cancer

Human metapneum

Mice 2 μg/mouse Tumor growth inhibition

**Experimental system**

94 RNA Interference

MDA-MB-435

Huh7.5 cells 25D/27-mer 5' UTR and

PC-3 cells 25D/27-mer *HSP27* (human) Prostate

*N* gene of respiratory syncytial virus (RSV)

*CTNNB1* (human*)*

human

AY-27 cells 25D/27-mer *Mki-67* (rat) Bladder

25D/27-mer 2'OMe

2'OMe

LLC-MK2 cells 25D/27-mer *N, D,L* genes of

Hela cells 25D/27-mer

cells

HAE cells obtained from bronchi and lungs


**Table 1.** Application of dsiRNA for silencing of disease-related genes (summarized from PubMed). 25/27-mer – dsiRNAs with 25 - base sense strand and 27 – base antisense strand; 25D – 2 bases at the 3'-end are substituted with DNA; 2'OMe – 2' – O methyl modifications as described in [17].

In another study, potent 2′-O-methyl modified dsiRNAs targeted to β-catenin were designed [49]. It is known that β-catenin acts as the transcription factor and its overexpression causes the development of several types of cancer, including liver cancer. At the first step, large-scale screening of 488 dsiRNAs for in vitro mRNA knockdown activity was performed to choose the most efficient dsiRNAs for targeting β-catenin. Then, the absence of immunostimulatory activity attributed to selected dsiRNA was confirmed using the assay based on the ability of an oligonucleotide to induce the production of antibodies to the PEGylated components of the lipid nanoparticles containing oligonucleotides. dsiRNA was administered to mice intrave‐ nously twice a week during 3 weeks after implanting Hep 3B tumor cells. dsiRNAs induced strong β-catenin mRNA knockdown and efficient tumor inhibition. Other examples of dsiRNAs applications as potential therapeutics for inhibition of the disease-related overex‐ pressed genes in vivo and in vitro have been summarized in Table 1.

Beneficial properties of dsiRNAs make these structures popular inhibitors of target genes. At first, dsiRNAs induce more potent silencing of the target genes at lower concentrations than canonical siRNAs. The next advantage of dsiRNAs is longevity of silencing: In some cases, it lasts up to 10 days. Then, the usage of dsiRNAs enables to minimize off-target effects such as toxicity and heterogeneity of processed products. An additional benefit is the high potency of dsiRNAs in silencing of multiple mRNAs where canonical siRNAs due to competition during RISC loading step appear to be less effective. The main disadvantage of dsiRNA is the higher cost of synthesis in comparison with canonical siRNA. However, low dosage of dsiRNA used in experiments eliminates this drawback. On the other hand, dsiRNAs share with siRNAs the same problems in therapeutic applications. The major challenge lies in the delivery of these structures into desired cells, tissues, and organs. To overcome this problem, various ap‐ proaches are developed; however, this question has not been completely answered yet. Nevertheless, dsiRNA as potent inducers of RNAi offers promising strategies for efficient therapy.

#### **3. Interfering RNA with noncanonical duplex structure**

Different variations of siRNA duplex structures were proposed to improve their silencing activity. Here we will consider three types of the most frequently used siRNAs with structural modifications of duplexes: short hairpin RNAs (shRNAs)/microRNA (miRNA) mimics, dumbbell RNAs, and fork-siRNA (Figure 2).

**Experimental system**

96 RNA Interference

therapy.

**Structure Target (gene) Disease Concentra-**

pressed genes in vivo and in vitro have been summarized in Table 1.

**3. Interfering RNA with noncanonical duplex structure**

DNA; 2'OMe – 2' – O methyl modifications as described in [17].

Rat 0.005 mg/kg 86% and 62% mRNA level

**Table 1.** Application of dsiRNA for silencing of disease-related genes (summarized from PubMed). 25/27-mer – dsiRNAs with 25 - base sense strand and 27 – base antisense strand; 25D – 2 bases at the 3'-end are substituted with

In another study, potent 2′-O-methyl modified dsiRNAs targeted to β-catenin were designed [49]. It is known that β-catenin acts as the transcription factor and its overexpression causes the development of several types of cancer, including liver cancer. At the first step, large-scale screening of 488 dsiRNAs for in vitro mRNA knockdown activity was performed to choose the most efficient dsiRNAs for targeting β-catenin. Then, the absence of immunostimulatory activity attributed to selected dsiRNA was confirmed using the assay based on the ability of an oligonucleotide to induce the production of antibodies to the PEGylated components of the lipid nanoparticles containing oligonucleotides. dsiRNA was administered to mice intrave‐ nously twice a week during 3 weeks after implanting Hep 3B tumor cells. dsiRNAs induced strong β-catenin mRNA knockdown and efficient tumor inhibition. Other examples of dsiRNAs applications as potential therapeutics for inhibition of the disease-related overex‐

Beneficial properties of dsiRNAs make these structures popular inhibitors of target genes. At first, dsiRNAs induce more potent silencing of the target genes at lower concentrations than canonical siRNAs. The next advantage of dsiRNAs is longevity of silencing: In some cases, it lasts up to 10 days. Then, the usage of dsiRNAs enables to minimize off-target effects such as toxicity and heterogeneity of processed products. An additional benefit is the high potency of dsiRNAs in silencing of multiple mRNAs where canonical siRNAs due to competition during RISC loading step appear to be less effective. The main disadvantage of dsiRNA is the higher cost of synthesis in comparison with canonical siRNA. However, low dosage of dsiRNA used in experiments eliminates this drawback. On the other hand, dsiRNAs share with siRNAs the same problems in therapeutic applications. The major challenge lies in the delivery of these structures into desired cells, tissues, and organs. To overcome this problem, various ap‐ proaches are developed; however, this question has not been completely answered yet. Nevertheless, dsiRNA as potent inducers of RNAi offers promising strategies for efficient

Different variations of siRNA duplex structures were proposed to improve their silencing activity. Here we will consider three types of the most frequently used siRNAs with structural

**tion/dose**

**Biological effect Reference**

reduction in lumbar dorsal root ganglia and in spinal cord, respectively

**Figure 2.** The different types of interfering RNAs with non-canonical duplex structures.

The identification of a large class of endogenous regulatory RNA molecules – microRNAs (miRNAs) arouse interest in constructing the similar synthetic structures for efficient silencing of target genes. miRNA precursors are generated in the cell as long primary transcripts that are cleaved in nucleus by RNase III class nuclease Drosha [60–62]. Then, they are exported to the cytoplasm and cleaved by Dicer, which is active at processing of complex hairpin structures [63]. It is known that Dicer substrates more effectively enter RISC complex than canonical siRNA and induce more potent RNAi [15–17, 28]. Moreover, shRNAs could interact with particular chaperones that promote recognition of shRNA by Dicer [64]. miRNAs form imperfect complementary complexes containing bulges with 3′ untranslated region of the target mRNA, wherein the position of the loops defines the mechanism of action: target cleavage or the block of translation. In the first case, synthetic miRNA mimics have no advantages over shRNA, and in the second case, they do not act in a catalytic mode. Therefore, synthetic miRNA applications are restricted to exploring the miRNA-regulated pathways involved in the natural processes, or development of replacement therapy for the diseases associated with mutation in specific miRNA. The use of shRNA seems to be more promising.

Although long dsRNA hairpins are prepared synthetically, enzymatically, or endogenously expressed, plasmid or viral vectors could be used in nonmammalian organisms. Long RNA hairpins cannot be applied in mammals for the specific gene silencing because they also induce interferon response in mammals via the same mechanism used by long RNA duplexes [3, 65, 66]. Therefore, length of hairpin RNAs for application in this type of species is limited by 30 bp. shRNAs expressed by different vectors under control of RNA polymerase III and CMV promoters were proved to efficiently trigger RNAi [67, 68].

Applications of viral vector-based expression of shRNAs are limited because of some obstacles such as possibility of insertional mutagenesis, malignant transformation, and host immune response [69]. At the same time, an application of plasmid vectors is safe, but inefficient delivery into cells limits its use only for experimental purposes, where antibiotic resistance genes included in the vector is used for the selection. In contrast to expressed shRNA, synthetic shRNA seems to be more attractive for RNAi-based therapies. It was found that chemically synthesized short hairpin RNAs (shRNAs) with 19–29-base-pair stem, at least 4-nucleotide loop and 2-nucleotide 3′ overhangs are more potent inducers of RNAi than the canonical smallinterfering RNAs targeted to the same sequence in mRNA [64, 70–73]. Two main types of shRNAs with opposite positions of the loops were designed (Figure 3). The right loop structures (R-shRNAs) have sense strand at the 5′ end of the hairpin, whereas the left loop shRNAs (L-shRNAs) have antisense strand at the 5′ end of the hairpin (Figure 3) [71–74]. The majority of studies were carried out using R-hand loop structure.

**Figure 3.** General structures of L-shRNAs and R-shRNA (according to [71]). Red color: sense strand, blue color: anti‐ sense strand.

It was clearly demonstrated that the silencing activity of shRNAs depends on stem length, loop length, sequence, and terminal overhangs [70]. Dicer efficiently cleaves shRNA with certain minimum stem of 19 nt in length forming 22-nt products starting from the free 3′ end of the RNA.

interferon response in mammals via the same mechanism used by long RNA duplexes [3, 65, 66]. Therefore, length of hairpin RNAs for application in this type of species is limited by 30 bp. shRNAs expressed by different vectors under control of RNA polymerase III and CMV

Applications of viral vector-based expression of shRNAs are limited because of some obstacles such as possibility of insertional mutagenesis, malignant transformation, and host immune response [69]. At the same time, an application of plasmid vectors is safe, but inefficient delivery into cells limits its use only for experimental purposes, where antibiotic resistance genes included in the vector is used for the selection. In contrast to expressed shRNA, synthetic shRNA seems to be more attractive for RNAi-based therapies. It was found that chemically synthesized short hairpin RNAs (shRNAs) with 19–29-base-pair stem, at least 4-nucleotide loop and 2-nucleotide 3′ overhangs are more potent inducers of RNAi than the canonical smallinterfering RNAs targeted to the same sequence in mRNA [64, 70–73]. Two main types of shRNAs with opposite positions of the loops were designed (Figure 3). The right loop structures (R-shRNAs) have sense strand at the 5′ end of the hairpin, whereas the left loop shRNAs (L-shRNAs) have antisense strand at the 5′ end of the hairpin (Figure 3) [71–74]. The

**Figure 3.** General structures of L-shRNAs and R-shRNA (according to [71]). Red color: sense strand, blue color: anti‐

sense strand.

promoters were proved to efficiently trigger RNAi [67, 68].

98 RNA Interference

majority of studies were carried out using R-hand loop structure.

Correct 3′ overhangs increase the efficacy and specificity of processing, whereas blunt-end shRNAs produce a set of products [64]. In the case of endogenously expressing regulatory RNAs, 3′ UU overhangs of miRNA precursors generated by Drosha cleavage determine subsequent proper recognition and processing by Dicer [75]. Synthetic shRNA with similar overhangs and mimicking the products of Drosha preprocessing is used. The presence of a 3′- UU overhang improves silencing activity of 19-mer shRNAs as 3′-UU overhangs might provide additional site for PAZ domain of Dicer [70].

The loop length also plays a crucial role in the silencing activity of shRNAs. Thus, it was found that 29-nt stem and 4-nt loop inhibited the target gene expression more efficiently as compared with shRNA with 19-nt stem and loop [76, 70]. In contrast, when 9-nt loop was used for 19-nt stem shRNA, it demonstrated more potent silencing of target genes than longer shRNA with the 4-nt loop. Brummelkamp and his colleagues also demonstrated that shRNAs with 19-nt stem and 9-nt loops possessed the maximum silencing activity as compared with shRNAs with 7-nt loops, while shRNA with 5-nt loops were inactive [68]. All observed differences in the silencing activities of shRNA, divergent in the length of the stem and loop, were more pronounced under low or intermediate concentrations. The silencing activity of different shRNAs, used at high concentrations, did not depend substantially on the loop length [70].

These results may be explained by the fact that the loop length influences the efficiency of processing by Dicer. [64]. Indeed, 4-nt loops of shRNA with 19-nt stem have poorer confor‐ mation flexibility at the junction between the duplex stem and a single strand of the loop. As short loops are set close or at the Dicer cleavage site, the restriction-associated conformational changes made shRNAs stems poor substrates for Dicer. Therefore, shRNAs with short loops and 19-nt stem enter RISC complex in the later stages and remain not processed by Dicer [64]. It was suggested that another single-strand-specific ribonuclease, independent from Dicer, cleaved this type of shRNA. On the other hand, shRNAs with 19-nt stem and longer loops (9– 10 nt) are efficiently processed by Dicer [76].

The potency of 19-nt stem shRNAs, targeted to the same sequence, depends on the position of the loop. Right (R)-shRNAs 19 nt in length are significantly less active than left (L)-shRNAs of the same length [71, 72]. However, the position of the loop (left or right) in longer shRNAs did not affect their activity. It was suggested that low potency of R-shRNA form is related to the fact that 5′ end of the antisense strand must be readily available for the efficient binding of AGO2 in the RISC complex. Otherwise, the 5′ end of sense strand would enter RISC and the target mRNA would not be cleaved. [77]. L-shRNAs with a short loop of 1–2 nt in length could be active. Moreover, L-shRNAs without any loop, where sense strand is directly connected with antisense strand, may be also active. In this case, the sense strand is shorter than antisense strand and the loop is formed by 3′ end of the antisense strand [71].

Moreover, the nature of nucleotides in 3′ overhang does not influence the activity of L-shRNA and 3′ overhangs could be substituted for deoxyribonucleotides [71]. The high efficiency of LshRNAs may be explained by the high energy of binding of antisense strand with AGO2 due to availability of 5′ end of antisense strand in L-shRNA, dominated over the influence of overhangs and loop length. [71].

shRNAs have similar but not identical sequence preferences with siRNAs. Thus, the functional shRNAs have mainly AU nucleotides at position 9 and GC nucleotides at position 11, while these preferences are less significant in functional siRNA. At the same time, the functional shRNAs have the similar thermodynamic asymmetry as functional siRNAs. The computer algorithms for selection of potent shRNAs have been developed [76].

Short hairpin RNAs are a little more resistant to nucleases than siRNAs due to the protection of one end; however, shRNAs still quickly degrade in biological fluids [78]. The elegant method to stabilize non-modified RNA strands was described by Abe et al. [79]. Abe and his colleagues constructed dumbbell-shaped RNA structures and demonstrated their potency as RNAi inducers with stability in the biological fluids [79]. Dumbbell-shaped RNAs were designed by analogy with DNA dumbbells consisted of double-helical stem and closed by two hairpin loops. Dumbbell-shaped RNA structures are used as models for the analysis of local structures in DNA [80]. Local unwinding of duplexes facilitates enzymatic cleavage by nucleases. Two loops at the both ends of dumbbell RNA stabilize the duplex and limit its enzymatic cleavage [79, 81]. Dumbbell structures get processed by Dicer much more slowly in comparison with their linear analogues due to inefficient recognition by Dicer. The rate of processing depends on the stem length, too. For example, dumbbell RNAs with 27-bp-stem length were processed more quickly than the same sequence with 15–19-bp stem length. RNA dumbbells with 23-bp stems and 9-nt loops were found to be the most active. Indeed, 9-nt loops are commonly used in shRNAs as the most effective hairpin loops [67]. The stem length was optimized to keep high potency and reduce interferon response. Silencing activity of these dumbbell RNAs was significantly higher than that induced by linear counterparts and was retained for longer period even at lower concentrations [81]. The introduction of deoxynucleotides into the loop of dumbbell RNAs further significantly increases shRNA stability in biological fluids without loss of silencing activity. Moreover, the loop of dumbbell RNAs can be modified by carriers such as aptamers and peptides [81]. All benefits of dumbbell RNAs make them new potent RNAi inducers. The main disadvantage of these structures is the high cost of their synthesis in comparison with canonical siRNAs. At the same time, the low dosage and prolonged silencing effect can reduce expenses. The detailed scheme of RNA dumbbell synthesis is described by Abe and his colleagues [82].

Another type of RNAi inducer, fork-siRNA, was first introduced by Hohjoh [83]. Fork-siRNAs contain base substitutions in the 3′ end of the sense strand of siRNA, resulting in destabilization of the duplex [83–85]. The effect of fork-siRNAs is explained by the fact that thermodynamic asymmetry of the duplexes determines the orientation of siRNA in RISC. Thermodynamic stability of the terminal regions of the duplex defines which strand is cleaved and dissociated during RISC activation, and another strand remains in the activated RISC and guides target mRNA recognition and cleavage [86]. Antisense strand of siRNA must be included in activated RISC for efficient gene silencing, if activated RISC contains the sense strand no silencing occur.

The selection of active siRNAs may be complicated if a target mRNA is mutated or is a chimerical gene. To address this issue, the favorable asymmetry can be achieved by the introduction of several base substitutions at the 3′ end of the sense strand. Mismatches at the 3′ end of the sense strand, resulting in the formation of unpaired or destabilized regions, increase the silencing activity of siRNA with low or moderate concentrations [83]. The number of mismatches in fork-siRNA also plays a crucial role in its silencing activity [85]. Fork-siRNAs with one to two mismatches at the 3′ end possess silencing activity similar to that of canonical siRNAs, indicating that this number of mismatches is not enough for the efficient silencing. Fork-siRNA with four mismatches is the most potent, whereas fork-siRNA with six mismatch‐ es possesses reduced silencing activity [85].

An optimal number of mismatches depend on the overall thermodynamic stability of the duplex. Computational algorithms for siRNA sequence selection determine the recommended range of *T*m difference between the terminal regions, and four mismatches could work for sequences within the range. On the other hand, mismatches in the 3′ part of the sense strand and long unpaired ends increase the sensitivity of fork-siRNA to nucleases. Consequently, the application of non-modified fork-siRNAs in vivo is limited by the fact that they have reduced stability in biological fluids due to the increased degradation by nucleases [83, 85]. To solve this problem, the algorithm for designing nuclease-resistant fork-siRNAs that contain 2′-Omethyl modifications in nuclease-sensitive sites was developed, which allows obtaining forksiRNAs whose stability is comparable to that of canonical siRNAs [85].

Thereby, fork-siRNAs may improve unfavorable asymmetry of siRNA with low or moderate silencing activity, especially when the selection of functional siRNA is restricted by the sequence content of the corresponding mRNA. It makes sense to use them for silencing of uneasy or precisely located targets.

### **4. Short noncanonical RNA**

Moreover, the nature of nucleotides in 3′ overhang does not influence the activity of L-shRNA and 3′ overhangs could be substituted for deoxyribonucleotides [71]. The high efficiency of LshRNAs may be explained by the high energy of binding of antisense strand with AGO2 due to availability of 5′ end of antisense strand in L-shRNA, dominated over the influence of

shRNAs have similar but not identical sequence preferences with siRNAs. Thus, the functional shRNAs have mainly AU nucleotides at position 9 and GC nucleotides at position 11, while these preferences are less significant in functional siRNA. At the same time, the functional shRNAs have the similar thermodynamic asymmetry as functional siRNAs. The computer

Short hairpin RNAs are a little more resistant to nucleases than siRNAs due to the protection of one end; however, shRNAs still quickly degrade in biological fluids [78]. The elegant method to stabilize non-modified RNA strands was described by Abe et al. [79]. Abe and his colleagues constructed dumbbell-shaped RNA structures and demonstrated their potency as RNAi inducers with stability in the biological fluids [79]. Dumbbell-shaped RNAs were designed by analogy with DNA dumbbells consisted of double-helical stem and closed by two hairpin loops. Dumbbell-shaped RNA structures are used as models for the analysis of local structures in DNA [80]. Local unwinding of duplexes facilitates enzymatic cleavage by nucleases. Two loops at the both ends of dumbbell RNA stabilize the duplex and limit its enzymatic cleavage [79, 81]. Dumbbell structures get processed by Dicer much more slowly in comparison with their linear analogues due to inefficient recognition by Dicer. The rate of processing depends on the stem length, too. For example, dumbbell RNAs with 27-bp-stem length were processed more quickly than the same sequence with 15–19-bp stem length. RNA dumbbells with 23-bp stems and 9-nt loops were found to be the most active. Indeed, 9-nt loops are commonly used in shRNAs as the most effective hairpin loops [67]. The stem length was optimized to keep high potency and reduce interferon response. Silencing activity of these dumbbell RNAs was significantly higher than that induced by linear counterparts and was retained for longer period even at lower concentrations [81]. The introduction of deoxynucleotides into the loop of dumbbell RNAs further significantly increases shRNA stability in biological fluids without loss of silencing activity. Moreover, the loop of dumbbell RNAs can be modified by carriers such as aptamers and peptides [81]. All benefits of dumbbell RNAs make them new potent RNAi inducers. The main disadvantage of these structures is the high cost of their synthesis in comparison with canonical siRNAs. At the same time, the low dosage and prolonged silencing effect can reduce expenses. The detailed scheme of RNA dumbbell synthesis is

Another type of RNAi inducer, fork-siRNA, was first introduced by Hohjoh [83]. Fork-siRNAs contain base substitutions in the 3′ end of the sense strand of siRNA, resulting in destabilization of the duplex [83–85]. The effect of fork-siRNAs is explained by the fact that thermodynamic asymmetry of the duplexes determines the orientation of siRNA in RISC. Thermodynamic stability of the terminal regions of the duplex defines which strand is cleaved and dissociated during RISC activation, and another strand remains in the activated RISC and guides target

algorithms for selection of potent shRNAs have been developed [76].

overhangs and loop length. [71].

100 RNA Interference

described by Abe and his colleagues [82].

siRNA shorter than canonical siRNA could also induce efficient silencing of target genes in mammalian cells acting via RNAi mechanism [87–90]. Short siRNAs have some benefits as inducers of RNAi such as reduction of immune response and decreased cost of the synthesis [76]. Various strategies have been used to design the minimal length for inducing RNA interference. As A-form helix of RNA plays an essential role for inducing RNAi, Chiu and Rana [91] found minimal length of dsRNA A-form helical structure required to enter active RISC complex. They demonstrated that siRNA with 16 bp in length and 2-nt 3′ overhangs repre‐ senting ~ 1.5 helical turns efficiently assembles into catalytically active RISC and was sufficient for silencing of target genes. Indeed, 16-mer siRNAs were more potent in comparison to 19 mer siRNAs, while 15-mer siRNAs silenced gene expression at lower efficacy than 16-mer siRNAs, and 14–13-mer siRNAs were practically inactive [87]. It was demonstrated that the mechanism of target cleavage was different: cleavage sites in 16-mer siRNAs were shifted to 3 nt in comparison with 19-mer siRNAs (Figure 4). The 16-bp siRNAs induced the silencing faster than canonical siRNA due to the higher efficacy of RISC formation [87, 91]. Moreover, asymmetric duplexes with 3′ overhang on the antisense strand only demonstrated reduced off-target silencing in comparison with symmetric duplexes of the same length due to preferential incorporation of the guide strand into the RISC complex [91]. Thus, considering the benefits of 16-mer siRNAs, they possess high potential for using in biomedical studies, but new examples of their use are not available.

**Figure 4.** The mechanism of Dicer cleavage for 19 bp siRNA and short siRNA 16 bp in length. Arrows indicate cleav‐ age sites defined by the 5'-end of guide strand (according to [87]).

Antisense siRNA also have been proposed as RNAi triggers. The sense strand of siRNA duplex is degraded and antisense strand remains in the active RISC to target complemen‐ tary mRNA. Early studies demonstrated that 5′-phosohorylated antisense siRNAs with 22– 40 nt in length were able to induce gene silencing in *Caenorhabditis elegans* [92, 93]. Several studies investigated the possibility of using antisense strand of siRNA 19–29 nt in length for transient knockdown of target genes in mammalian cells [88–90]. Antisense siRNA entered the RISC complex and provided mRNA cleavage but with lower efficiency in comparison with canonical siRNA. Antisense siRNA and canonical siRNA possess similar mRNA target position effects, cleavage fragment production, and tolerance to mutational and chemical modifications. However, antisense siRNA modified at 3′ end by fluorescein group or deoxyribose showed reduced silencing activity compared with canonical siRNA, where silencing activity remained at the same level in spite of 3′ modifications [89]. Moreover, the velocity of mRNA degradation induced by antisense siRNA is higher than that provided by canonical siRNA, but the duration of silencing effect is shorter. Thus, it was assumed that antisense siRNA induces RNAi through similar pathways as doublestranded siRNA but enters the pathway at the intermediate stage [89].

The differences in the silencing activity between antisense and canonical siRNAs may be explained by the low intracellular stability of the single-stranded RNA and by low efficacy of association with RISC [89]. Among the advantages of siRNAs, a lower price of synthesis and no side effects associated with the induction of interferon response should be considered [88]. Partial boranophosphate backbone (BP) modifications were designed to increase the stability and the silencing activity of the antisense siRNA [88]. BP-modified antisense siRNAs possess silencing activity comparable to unmodified double-stranded siRNAs. Partial 2′-O-methyl modification was used for the stabilization of antisense RNA, the activity resulting in singlestranded siRNA was comparable with the activity of double-stranded siRNA when used in high or intermediate concentrations, where in low concentration, canonical siRNAs were more active [85].

Overall, in spite of lower silencing activity compared with canonical siRNA, antisense siRNA may be used in specific situations, for instance, to eliminate off-target silencing of genes in the case when the sense strand has substantial homology to nontarget genes [88].

### **5. Long-interfering RNAs**

3 nt in comparison with 19-mer siRNAs (Figure 4). The 16-bp siRNAs induced the silencing faster than canonical siRNA due to the higher efficacy of RISC formation [87, 91]. Moreover, asymmetric duplexes with 3′ overhang on the antisense strand only demonstrated reduced off-target silencing in comparison with symmetric duplexes of the same length due to preferential incorporation of the guide strand into the RISC complex [91]. Thus, considering the benefits of 16-mer siRNAs, they possess high potential for using in biomedical studies, but

**Figure 4.** The mechanism of Dicer cleavage for 19 bp siRNA and short siRNA 16 bp in length. Arrows indicate cleav‐

Antisense siRNA also have been proposed as RNAi triggers. The sense strand of siRNA duplex is degraded and antisense strand remains in the active RISC to target complemen‐ tary mRNA. Early studies demonstrated that 5′-phosohorylated antisense siRNAs with 22– 40 nt in length were able to induce gene silencing in *Caenorhabditis elegans* [92, 93]. Several studies investigated the possibility of using antisense strand of siRNA 19–29 nt in length for transient knockdown of target genes in mammalian cells [88–90]. Antisense siRNA entered the RISC complex and provided mRNA cleavage but with lower efficiency in comparison with canonical siRNA. Antisense siRNA and canonical siRNA possess similar mRNA target position effects, cleavage fragment production, and tolerance to mutational and chemical modifications. However, antisense siRNA modified at 3′ end by fluorescein group or deoxyribose showed reduced silencing activity compared with canonical siRNA, where silencing activity remained at the same level in spite of 3′ modifications [89]. Moreover, the velocity of mRNA degradation induced by antisense siRNA is higher than that provided by canonical siRNA, but the duration of silencing effect is shorter. Thus, it was assumed that antisense siRNA induces RNAi through similar pathways as double-

The differences in the silencing activity between antisense and canonical siRNAs may be explained by the low intracellular stability of the single-stranded RNA and by low efficacy of association with RISC [89]. Among the advantages of siRNAs, a lower price of synthesis and no side effects associated with the induction of interferon response should be considered [88].

stranded siRNA but enters the pathway at the intermediate stage [89].

new examples of their use are not available.

102 RNA Interference

age sites defined by the 5'-end of guide strand (according to [87]).

Long dsRNAs >30 nt in length efficiently silence the expression of target gene in nonmamma‐ lian cells [1, 4]. The early attempts to use the similar structures for efficient knockdown of target genes in mammalian cells failed due to activation of interferon response [4, 94]. Later, various design strategies have been developed to prevent the induction of interferon response and construct new potent RNAi inducers [21, 95, 96]. Depending on the architecture of duplexes, all long dsRNAs may be divided into linear and branched structures.

Partial 2′-O-methyl modification effectively prevents the activation of interferon response by Dicer-substrate RNAs [17]; therefore, it was proposed to use similar approach for longer linear duplexes [21]. Longer siRNAs containing the sequence of canonical siRNAs repeated two and three times are called dimer (42 nt in length) and trimer (63 nt in length) small-interfering RNA. Selective 2′-O-methyl modifications were introduced into nuclease-sensitive sites of both sense and antisense strands of dimer and trimer siRNAs, the modifications in the sites of potential Dicer cleavage were omitted. Selectively modified dimer and trimer siRNAs, unlike the unmodified ones, did not induce interferon response in cultured cells. The trimers (called tsiRNA) were significantly more active at lower dose-equivalent (per moles of 21 bp) concen‐ trations than their canonical analogues but the silencing effect develops more slowly [21] and acts in a Dicer-independent mode, presumably via direct RISC loading. Although the Dicer cleavage sites were free from modifications, modifications in flanking regions of tsiRNAs could inhibit the Dicer cleavage. The observed mechanism may be associated with a specific pattern of modification, used by the authors, which cannot be excluded such that the change in the pattern will allow the tsiRNA to be processed by Dicer and act through a canonical mechanism.

Targeting single mRNA by RNAi inducer for therapeutic purposes has several limitations: (1) the presence of mutation in the target site reduces the efficiency of silencing, which is especially important for viral genes, and (2) signal pathways involved in cancer cell growth contain duplications of regulatory elements and bypass regulatory pathways [97–99]. Thus, simulta‐ neous inhibition of several genes seems to be an effective strategy. Co-transfection of several siRNAs may be not effective due to competition between siRNAs [30]. Therefore, long linear synthetic siRNAs targeted two or more genes hold great promise in these cases.

Peng and his colleagues designed long linear siRNA at least 30 nt in length (multi-siRNAs) for dual-gene silencing [95]. To avoid undesired interferon response and improve RNAi potency, 2′-O-methyl modifications and gap in either sense or antisense strands were used. 2′-O-methyl modifications were introduced into every second nucleotide of both strands. The gap divided the complementary strand into two equal segments. It was demonstrated that multi-siRNAs with the gap provided more efficient simultaneous silencing of two target genes in comparison with corresponding single-target siRNAs (Figure 5). Interestingly, the simultaneous silencing of two target genes by long siRNA without gap was ineffective. It was supposed that the gap may provide sites for Dicer or facilitate Dicer processing. Because the Dicer substrates have preference in RISC loading, multi-siRNAs could possess more efficient silencing activity than canonical siRNAs [16, 18]. The experiments demonstrated that silencing effects of multi-siRNA was eliminated when AGO2 was downregulated confirming the action through the same RNAi pathway as canonical siRNAs [95]. However, further experiments are required to clarify the exact mechanism of increased activity of these siRNAs.

**Figure 5.** Design of long linear duplexes with gap in either sense or antisense strands. Red color: sense strand, blue color: antisense strand.

Long unmodified siRNAs up to 42 nt in length were used for silencing of gene expression in some specific cell lines without the induction of interferon response [96–101]. For example, direct fusion of two 17- and 19-bp long non-modified siRNAs resulted in efficient silencing of two target genes [96]. Two RNAs were merged "head-to-head" in a way that the 5'-ends of both antisense strands would look outside from the duplex allowing efficient and stereospecific AGO2 binding and efficient silencing of both targets [96]. Heterologous duplexes merged "head-to-tail" of antisense strands demonstrated reduced silencing activity [96]. Similar results were obtained for tandem siRNAs of 40–42 nt in length consisting of 21+21 and 21+23 units [101] as well as 40-nt long duplexes [100]. These results may be explained by the fact that the induction of interferon response depends on the cell type [23]. Indeed, some cell lines may possess reduced immune-sensitivity to the siRNA treatment and the results obtained on the cell cultures cannot be unacceptable for in vivo experiments.

Another class of long siRNAs are various branched structures (Figure 6). Initially, branched oligonucleotides were applied to study mRNA splicing [102–104]. Then diverse branched structures were used as building blocks for self-assembling nanostructures [105–107]. More‐ over, nanostructures of different shapes and sizes have been proposed as an effective delivery system for siRNA, ribozymes, etc. [106, 107]. Recently, it has been demonstrated that branched small RNA structures may also be effective RNAi inducers. Thus, these duplexes can simul‐ taneously inhibit two or more genes and possess improved silencing activity and intracellular delivery properties [96–109]. Different strategies are developed to form branches. Symmetric doubler phosphoramidites are used to construct branches with two or four strands [108]. In another variant, trebler phosphoramidite structure with extended short DNA linker is used as a core for branched small RNA with three arms [110]. Direct annealing was used to design RNAs with three and four arms [111, 109]. However, base pairing close to the junction region may be disturbed and single-stranded nuclease-sensitive region may be formed.

Peng and his colleagues designed long linear siRNA at least 30 nt in length (multi-siRNAs) for dual-gene silencing [95]. To avoid undesired interferon response and improve RNAi potency, 2′-O-methyl modifications and gap in either sense or antisense strands were used. 2′-O-methyl modifications were introduced into every second nucleotide of both strands. The gap divided the complementary strand into two equal segments. It was demonstrated that multi-siRNAs with the gap provided more efficient simultaneous silencing of two target genes in comparison with corresponding single-target siRNAs (Figure 5). Interestingly, the simultaneous silencing of two target genes by long siRNA without gap was ineffective. It was supposed that the gap may provide sites for Dicer or facilitate Dicer processing. Because the Dicer substrates have preference in RISC loading, multi-siRNAs could possess more efficient silencing activity than canonical siRNAs [16, 18]. The experiments demonstrated that silencing effects of multi-siRNA was eliminated when AGO2 was downregulated confirming the action through the same RNAi pathway as canonical siRNAs [95]. However, further experiments are required to clarify

**Figure 5.** Design of long linear duplexes with gap in either sense or antisense strands. Red color: sense strand, blue

Long unmodified siRNAs up to 42 nt in length were used for silencing of gene expression in some specific cell lines without the induction of interferon response [96–101]. For example, direct fusion of two 17- and 19-bp long non-modified siRNAs resulted in efficient silencing of two target genes [96]. Two RNAs were merged "head-to-head" in a way that the 5'-ends of both antisense strands would look outside from the duplex allowing efficient and stereospecific AGO2 binding and efficient silencing of both targets [96]. Heterologous duplexes merged "head-to-tail" of antisense strands demonstrated reduced silencing activity [96]. Similar results were obtained for tandem siRNAs of 40–42 nt in length consisting of 21+21 and 21+23 units [101] as well as 40-nt long duplexes [100]. These results may be explained by the fact that the induction of interferon response depends on the cell type [23]. Indeed, some cell lines may possess reduced immune-sensitivity to the siRNA treatment and the results obtained on the

the exact mechanism of increased activity of these siRNAs.

cell cultures cannot be unacceptable for in vivo experiments.

color: antisense strand.

104 RNA Interference

**Figure 6.** The architecture of various multi-target branched siRNAs. Different colors indicate siRNA units targeted to various genes.

Chang and his colleagues introduced tripartite RNA structure without any linker containing three 19-bp-duplex regions obtained by annealing of three 38-nt single-stranded RNAs [111]. The 5′ end of each antisense strand was directed outside, making seed regions of all antisense strands accessible for AGO2 loading. Single-stranded regions near the strand junction were defended by 2′-O-methyl modifications affecting six nucleotides. It was demonstrated that tripartite small RNA more efficiently silences the expression of three target genes in compar‐ ison with a mixture of corresponding canonical siRNAs due to a more efficient intracellular delivery by Lipofectamine [111]. Specifically, tripartite small RNA was not processed by Dicer possibly due to the influence of 2′-O-methyl modifications introduced into the single-stranded region of tripartite RNA [111]. Similar structures without any modifications, designed by another group of scientists, were efficiently processed by Dicer into 20-nt products [109]. On the other hand, tripartite small RNA without any modifications was unstable in biological fluids and quickly degraded.

Tetramer RNA consisted of four arms 23 bp in length proved to be more stable and also acts in a Dicer-dependent mode. Both trimer and tetramer siRNAs provided prolonged RNAi effect and efficiently inhibited the expression of three or four genes simultaneously. The influence of the structures on the interferon response was not reported [109].

Overall, long linear and branched siRNAs could be efficiently used for simultaneous inhibition of multiple genes. Selective 2′-O-methyl modifications and specific elements of the structure (gaps, nonnucleotide insertions) could reduce undesired interferon response. The application of long RNAi inducers is restricted by the complexity of the design (in the case of branched molecules) and the higher cost of synthesis in comparison with canonical siRNAs; however, recent advances in the synthesis of oligoribonucleotide allows overcoming these problems. Long linear and branched siRNAs could be useful for the development of anticancer and antiviral therapeutics targeting multiple genes.

#### **6. Conclusion**

Small-interfering RNAs provide universal and effective method for the silencing of target genes because almost all genes could be targeted by siRNAs. A large number of diseases, associated with hyperexpression of certain genes or expression of their chimeric or mutated variants, could be treated by inhibition of gene expression; therefore, siRNA has a great potential as a new therapeutic drug. Different design strategies have been used to improve properties of siRNAs and reduce off-target effects. Structural modifications can expand the boundaries of siRNA applications.

At present, synthetic siRNAs structurally mimicking the Dicer substrates (dsiRNAs) are widely used as potent RNAi inducers. The use of dsiRNA may prevent the development of undesired toxicity associated with off-target effects of both the inducer and the transfection reagent or any type of carrier due to the lower effective concentrations and the increase in the longevity of silencing. Therefore, application of dsiRNAs is considered to be extremely promising in anticancer and antiviral therapeutics as well as for the treatment of chronic diseases where multiple administrations are necessary to reach the desired silencing effect. Chemical modification patterns compatible with Dicer processing were designed and suc‐ cessfully applied for prevention of undesired stimulation of immune system and for acquiring nuclease resistance. Single-stranded structured synthetic siRNAs, such as Dicer-processed short hairpin RNA and dumbbell RNA, possess all benefits as Dicer substrates and exhibit additional flexibility in fine-tuning of the stability, kinetics, and silencing duration. Long RNAi inducers, acting in a Dicer-dependent or Dicer-independent mode, effectively silence target genes at low concentrations. Multi-target siRNAs have a great promise in the treatment of complex diseases such as cancer and immune-inflammatory disorders or viral infections [108]. Long linear or branched structures with selective chemical or structural modifications could successfully inhibit the expression of several genes without undesired off-target effects. Currently, however, the complexity and high cost of the synthesis restrict the biomedical application of long small RNAs. Some structural modifications in siRNAs have specific applications. Fork-siRNA are successfully being used for the silencing of genes with restricted selection of sequence content such as chimeric or point-mutated genes.

siRNAs with various structural modifications find a wide application in biomedical research and therapeutics. Some of them have already been used in clinical trials. The great success was achieved in the multi-target therapy that may increase treatment effectiveness. However, the therapeutics applications are limited by the inefficient delivery of these compounds into organs, tissues, and cells. Problem of low bioavailability of siRNA in vivo could be overcome by two ways: the better delivery and the higher activity. Future expansion of the repertoire of RNAi inducers contributes to resolving of both challenges. Although many approaches are developed, more efforts are still needed to improve safety and efficiency of siRNA in vivo.

#### **Acknowledgements**

strands accessible for AGO2 loading. Single-stranded regions near the strand junction were defended by 2′-O-methyl modifications affecting six nucleotides. It was demonstrated that tripartite small RNA more efficiently silences the expression of three target genes in compar‐ ison with a mixture of corresponding canonical siRNAs due to a more efficient intracellular delivery by Lipofectamine [111]. Specifically, tripartite small RNA was not processed by Dicer possibly due to the influence of 2′-O-methyl modifications introduced into the single-stranded region of tripartite RNA [111]. Similar structures without any modifications, designed by another group of scientists, were efficiently processed by Dicer into 20-nt products [109]. On the other hand, tripartite small RNA without any modifications was unstable in biological

Tetramer RNA consisted of four arms 23 bp in length proved to be more stable and also acts in a Dicer-dependent mode. Both trimer and tetramer siRNAs provided prolonged RNAi effect and efficiently inhibited the expression of three or four genes simultaneously. The influence

Overall, long linear and branched siRNAs could be efficiently used for simultaneous inhibition of multiple genes. Selective 2′-O-methyl modifications and specific elements of the structure (gaps, nonnucleotide insertions) could reduce undesired interferon response. The application of long RNAi inducers is restricted by the complexity of the design (in the case of branched molecules) and the higher cost of synthesis in comparison with canonical siRNAs; however, recent advances in the synthesis of oligoribonucleotide allows overcoming these problems. Long linear and branched siRNAs could be useful for the development of anticancer and

Small-interfering RNAs provide universal and effective method for the silencing of target genes because almost all genes could be targeted by siRNAs. A large number of diseases, associated with hyperexpression of certain genes or expression of their chimeric or mutated variants, could be treated by inhibition of gene expression; therefore, siRNA has a great potential as a new therapeutic drug. Different design strategies have been used to improve properties of siRNAs and reduce off-target effects. Structural modifications can expand the

At present, synthetic siRNAs structurally mimicking the Dicer substrates (dsiRNAs) are widely used as potent RNAi inducers. The use of dsiRNA may prevent the development of undesired toxicity associated with off-target effects of both the inducer and the transfection reagent or any type of carrier due to the lower effective concentrations and the increase in the longevity of silencing. Therefore, application of dsiRNAs is considered to be extremely promising in anticancer and antiviral therapeutics as well as for the treatment of chronic diseases where multiple administrations are necessary to reach the desired silencing effect. Chemical modification patterns compatible with Dicer processing were designed and suc‐ cessfully applied for prevention of undesired stimulation of immune system and for acquiring

of the structures on the interferon response was not reported [109].

antiviral therapeutics targeting multiple genes.

boundaries of siRNA applications.

fluids and quickly degraded.

106 RNA Interference

**6. Conclusion**

This work was supported by the Russian Scientific Foundation under the grant #14-14-00697.

#### **Author details**


Laboratory of Nucleic Acids Biochemistry, Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia

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116 RNA Interference


## **Microinjection-Based RNA Interference Method in the Water Flea,** *Daphnia pulex* **and** *Daphnia magna*

Kenji Toyota, Shinichi Miyagawa, Yukiko Ogino and Taisen Iguchi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61485

#### **Abstract**

It is well known that most daphnid species have several attractive life history characteris‐ tics such as cyclical parthenogenesis, environmental sex determination, and predator-in‐ duced defense formation. Recent advances in high-throughput omics technologies make it easy to obtain a huge number of potential candidate factors involved in environmental stimuli-triggered phenotypic alterations. Furthermore, our group has developed a micro‐ injection system to introduce foreign materials such as nucleotides and chemicals into the early-stage (one-cell stage) egg of *Daphnia pulex* and *Daphnia magna*. Consequently, we es‐ tablished a microinjection-based RNAi system that allows arbitrary gene functions to be investigated. However, this microinjection system does not seem to have pervaded in the daphnid research community due to its low throughput and high level of skills required. In this chapter, we review the microinjection method and its RNAi system in water fleas, *D. pulex* and *D. magna*, providing some technical tips and making challenging proposals for the development of novel high-throughput RNAi methods. Finally, we provide an overview of recently developed gene functional analysis methods such as overexpression and genome-editing systems.

**Keywords:** *Daphnia pulex*, *Daphnia magna*, genome editing, microinjection, RNAi-related gene

#### **1. Introduction**

The cladoceran crustacean water fleas are representative zooplankton ubiquitously found in freshwater habitats around the world [1]. Among them, species of the family Daphniidae, particularly genus *Daphnia*, have been well studied. All age classes of daphnids are principle consumers of algae and thus play an important role in the food web in freshwater ecosystems. In addition to this ecological significance, over the last decade, daphnids have drawn consid‐

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

erable attention as a good indicator organism for aquatic toxicology and have thus been used in ecotoxicological studies [2]. Moreover, for over 100 years, they have shown various adaptive phenotypic alterations in response to external environmental stimuli, including environmental sex determination (ESD) [3, 4], cyclical parthenogenesis, in which the mode of reproduction changes between parthenogenesis and sexual reproduction [3, 4], and inducible defense, which is a predator-triggered alteration of body shape [5, 6]. The acquisition of such sophisticated life history strategies has enabled daphnids to prosper around the world. These environmental stimuli-triggered phenomena in daphnid species have attracted many scientists involved in ecological, developmental, and evolutionary biology [4, 7–12]. Although recent progress in sequencing technology facilitates the deciphering of genome and transcriptome information using 'nonmodel organisms', analytical methods for arbitrary gene functions are still insuffi‐ ciently developed. In studies involving daphnids, the whole-genome sequencing project of *D. pulex* has been completed [12]. Furthermore, a microinjection system using early-stage embryos has been established, allowing gene functional analyses, including RNA interference (RNAi), to be possible in daphnids [13, 14]. Even though this microinjection-based experi‐ mental method can be used in two representative daphnid species, *D. pulex* and *D. magna*, some experimental aspects are different between them due to the difference in size of their early-stage embryos.

This chapter introduces *Daphnia* species as attractive models for eco-evo-devo studies and summarizes technical methods and tips for microinjection-based RNAi in *D. pulex* and *D. magna*. Finally, we review recent advances in the application of microinjection methods in daphnids such as genome editing and transgenesis.

#### **1.1. Life cycle**

Daphnids produce offspring either by parthenogenesis or by sexual reproduction in response to external environmental conditions. This mode of reproduction is referred to as cyclical parthenogenesis. They have a short generation period that lasts approximately 1 week under constant laboratory conditions, and their lifetime spans over 2 months or as much as 1 year when reared under colder temperatures [15]. Under optimal growing conditions, daphnids parthenogenetically produce offspring that expand their population consisting almost exclusively of females. This results in the exponential growth of genetically identical clone clusters. Mother daphnids produce several dozen eggs in their own brood chamber as a clutch just a few minutes after molting. Embryonic development occurs in the brood chamber. Subsequently, neonates are released to the outside just before the mother molts. Then indi‐ vidual mothers in the parthenogenetic phase repeat molting, spawning, and the release of neonates throughout their lifetime (Figure 1, parthenogenetic phase).

On the other hand, when environmental conditions deteriorate, for instance, a short daylength, lower temperature, food shortage, overcrowding, and the presence of predators, males are induced by parthenogenesis and are, therefore, genetically identical to their sisters and mother [4, 7–9, 16, 17] (Figure 1, sexual reproductive phase). In other words, an individual parthenogenetic mother has the potential to produce female and male offspring in response to changes in external environmental conditions. After copulation, sexual eggs, referred to as resting eggs that are enclosed in an ephippium (modified carapace that is darkly pigmented by melanin), are produced. Resting eggs can tolerate extreme conditions such as drying, freezing, and digestion by fish and can remain viable for over 100 years [18]. When favorable conditions are restored, female neonates hatch out from resting eggs and reinitiate the parthenogenetic phase, thus building up a new population (Figure 1, sexual reproductive phase).

**Figure 1.** Schematic drawing of cyclical parthenogenesis in daphnids.

erable attention as a good indicator organism for aquatic toxicology and have thus been used in ecotoxicological studies [2]. Moreover, for over 100 years, they have shown various adaptive phenotypic alterations in response to external environmental stimuli, including environmental sex determination (ESD) [3, 4], cyclical parthenogenesis, in which the mode of reproduction changes between parthenogenesis and sexual reproduction [3, 4], and inducible defense, which is a predator-triggered alteration of body shape [5, 6]. The acquisition of such sophisticated life history strategies has enabled daphnids to prosper around the world. These environmental stimuli-triggered phenomena in daphnid species have attracted many scientists involved in ecological, developmental, and evolutionary biology [4, 7–12]. Although recent progress in sequencing technology facilitates the deciphering of genome and transcriptome information using 'nonmodel organisms', analytical methods for arbitrary gene functions are still insuffi‐ ciently developed. In studies involving daphnids, the whole-genome sequencing project of *D. pulex* has been completed [12]. Furthermore, a microinjection system using early-stage embryos has been established, allowing gene functional analyses, including RNA interference (RNAi), to be possible in daphnids [13, 14]. Even though this microinjection-based experi‐ mental method can be used in two representative daphnid species, *D. pulex* and *D. magna*, some experimental aspects are different between them due to the difference in size of their

This chapter introduces *Daphnia* species as attractive models for eco-evo-devo studies and summarizes technical methods and tips for microinjection-based RNAi in *D. pulex* and *D. magna*. Finally, we review recent advances in the application of microinjection methods in

Daphnids produce offspring either by parthenogenesis or by sexual reproduction in response to external environmental conditions. This mode of reproduction is referred to as cyclical parthenogenesis. They have a short generation period that lasts approximately 1 week under constant laboratory conditions, and their lifetime spans over 2 months or as much as 1 year when reared under colder temperatures [15]. Under optimal growing conditions, daphnids parthenogenetically produce offspring that expand their population consisting almost exclusively of females. This results in the exponential growth of genetically identical clone clusters. Mother daphnids produce several dozen eggs in their own brood chamber as a clutch just a few minutes after molting. Embryonic development occurs in the brood chamber. Subsequently, neonates are released to the outside just before the mother molts. Then indi‐ vidual mothers in the parthenogenetic phase repeat molting, spawning, and the release of

On the other hand, when environmental conditions deteriorate, for instance, a short daylength, lower temperature, food shortage, overcrowding, and the presence of predators, males are induced by parthenogenesis and are, therefore, genetically identical to their sisters and mother [4, 7–9, 16, 17] (Figure 1, sexual reproductive phase). In other words, an individual parthenogenetic mother has the potential to produce female and male offspring in response to changes in external environmental conditions. After copulation, sexual eggs, referred to as

early-stage embryos.

120 RNA Interference

**1.1. Life cycle**

daphnids such as genome editing and transgenesis.

neonates throughout their lifetime (Figure 1, parthenogenetic phase).

#### **1.2.** *Daphnia* **as model species for ecological, evolutionary and developmental biology: ecoevo-devo**

Although several details are still controversial, recent molecular phylogenetic analyses of the Arthropoda have revealed that the Crustacea clade is not monophyletic and is divided into at least three clades (Ostracoda, Malacostraca, and Branchiopoda) that include daphnids (Figure 2). Also, the current phylogenetic hypothesis supports the notion that Branchiopoda and Hexapoda form a sister group (Figure 2). This suggests that a growing body of findings involving daphnids has accumulated and can contribute to our understanding of the evolu‐ tionary and developmental aspects of Arthropoda, connecting knowledge between wellstudied Hexapoda and more primitive Arthropoda clades.

**Figure 2.** Phylogenetic tree of the Arthropoda. The branching pattern is based on Oakley *et al.* [19] with some modifica‐ tions [20].

*D. pulex* and *D. magna have* long been used in ecological, evolutionary, developmental, and ecotoxicological studies as representative model daphnid species for the following reasons: *D. pulex* is ubiquitously widespread around the world, including Japan (Figure 3A), and shows striking phenotypic alterations in response to predator-released chemicals, forming 'neckteeth' [5]; *D. magna* has a huge body size among cladoceran species (maximum length of approxi‐ mately 10 mm, Figure 3B); they are easy to maintain and rear under laboratory conditions; they propagate rapidly because of their short generation time and reproductive cycle; embry‐ onic development can be observed *in vitro*; male offspring can be induced by administration of juvenile hormone agonists [21, 22]. In addition, individuals within a single strain are most likely genetically identical due to the diploidy of the parthenogenetic eggs that are maintained by mitosis-like meiosis, which skips a part of the first meiosis [23], allowing the environmental effects on their physiological and developmental processes to be investigated under a constant genetic background. Furthermore, we established a reliable induction system for environ‐ mental sex determination (ESD) studies using the *D. pulex* WTN6 strain, in which the sex of the offspring can be controlled by changing the day-length conditions in which long-day (14 h light:10 h dark) and short-day (10 h light:14 h dark) conditions can induce female and male offspring, respectively [17], and for inducible defense in several *D. pulex* strains where the incidence and number of neckteeth vary in response to different concentrations of *Chaoborus* kairomone [24].

In addition to the aforementioned advantages, useful experimental tools are available, for example, embryonic developmental staging [25, 26], whole-mount *in situ* hybridization and immunostaining using developing embryos [27], immunofluorescence and fluorescence *in situ* hybridization (FISH) [28], an expressed sequence tags (ESTs) database [29], and genetic linkage maps [30–32]. Furthermore, the whole-genome sequencing of *D. pulex* is complete [12, 32], although that of *D. magna* is still ongoing (https://wiki.cgb.indiana.edu/display/magna/ Home). In addition, recent growing omics and bioinformatics technology enables daphnid researchers to investigate cells, tissues, and organisms from a multilevel perspective such as the transcriptome [12, 33–35], proteome [36], or metabolome [37]. Thus, various excellent experimental tools and an increasingly huge accumulation of omics data make *D. pulex* and *D. magna* attractive model organisms for studying the molecular mechanisms underlying phenotypic alterations that depend on external environmental conditions. These reliable induction systems of focal phenotypes are indispensable for analyzing their physiological and developmental mechanisms.

**Figure 2.** Phylogenetic tree of the Arthropoda. The branching pattern is based on Oakley *et al.* [19] with some modifica‐

*D. pulex* and *D. magna have* long been used in ecological, evolutionary, developmental, and ecotoxicological studies as representative model daphnid species for the following reasons: *D. pulex* is ubiquitously widespread around the world, including Japan (Figure 3A), and shows striking phenotypic alterations in response to predator-released chemicals, forming 'neckteeth' [5]; *D. magna* has a huge body size among cladoceran species (maximum length of approxi‐ mately 10 mm, Figure 3B); they are easy to maintain and rear under laboratory conditions; they propagate rapidly because of their short generation time and reproductive cycle; embry‐ onic development can be observed *in vitro*; male offspring can be induced by administration of juvenile hormone agonists [21, 22]. In addition, individuals within a single strain are most likely genetically identical due to the diploidy of the parthenogenetic eggs that are maintained by mitosis-like meiosis, which skips a part of the first meiosis [23], allowing the environmental effects on their physiological and developmental processes to be investigated under a constant genetic background. Furthermore, we established a reliable induction system for environ‐ mental sex determination (ESD) studies using the *D. pulex* WTN6 strain, in which the sex of the offspring can be controlled by changing the day-length conditions in which long-day (14 h light:10 h dark) and short-day (10 h light:14 h dark) conditions can induce female and male offspring, respectively [17], and for inducible defense in several *D. pulex* strains where the incidence and number of neckteeth vary in response to different concentrations of *Chaoborus*

In addition to the aforementioned advantages, useful experimental tools are available, for example, embryonic developmental staging [25, 26], whole-mount *in situ* hybridization and immunostaining using developing embryos [27], immunofluorescence and fluorescence *in situ* hybridization (FISH) [28], an expressed sequence tags (ESTs) database [29], and genetic linkage maps [30–32]. Furthermore, the whole-genome sequencing of *D. pulex* is complete [12, 32], although that of *D. magna* is still ongoing (https://wiki.cgb.indiana.edu/display/magna/ Home). In addition, recent growing omics and bioinformatics technology enables daphnid

tions [20].

122 RNA Interference

kairomone [24].

**Figure 3.** *Daphnia pulex* (A) and *D. magna* (B). Upper and lower parts indicate the adult and embryo just after ovulation, respectively.

#### **2. Microinjection-based RNA interference (RNAi) in daphnid species**

As mentioned above, recent high-throughput sequencing technologies have enabled biologists to use nonmodel organisms to easily access genomic information. However, the development of experimental methods for gene functional analysis still hampers their reverse genetics approach. To date, the RNAi method in *D. pulex* and *D. magna* has been established by a microinjection method using early-stage embryos. Although our previous reports described detailed methodology for microinjection and the tips, tricks, and traps associated with this methodology [13, 14], there are slight differences for each daphnid species. Furthermore, descriptions of genes involved in the RNAi machinery is insufficient. Therefore, we compa‐ ratively summarize the tips to manipulate the microinjection methods in detail prior to focusing on the current status of daphnid RNAi. We then introduce the gene repertoires involved in the RNAi mechanism in *D. pulex* and *D. magna* genomes.

#### **2.1. Microinjection system**

The daphnid microinjection system uses parthenogenetic eggs just after ovulation into the mother's brood chamber. The individual mother begins to lay eggs into the brood chamber a few minutes after releasing neonates and molting. To obtain many healthy eggs, eggs from 2 to 6-week-old daphnids should be collected.

There are two major technical issues in microinjection of daphnids. One is the rapid hardening of the egg membrane just after ovulation [38], which is caused by enzymatic activity of peroxidase. The second problem is a substantial difference between the internal and external osmotic pressures of the egg. The former issue hampers the penetration of the egg membrane by a needle while the latter causes the leakage of egg components after injection. To overcome these problems, Kato et al. [13] and Hiruta et al. [14] established improved protocols for microinjection in *D. magna* and *D. pulex*, respectively. They succeeded in the transient inhibi‐ tion of the egg membrane hardening by ice-cold treatment just after ovulation. They also found the best culture conditions after injection by increasing external osmotic pressures: M4 medium [39] with 80-mM sucrose in *D. magna* [13] and a 2% agar plate covered with 60-mM sucrose dissolved in M4 media in *D. pulex* [14].

In addition, since the fineness of the needle is critical for the success of microinjection, we next describe a detailed preparation method. A glass needle is made from a glass capillary tube (GD-1; Narishige, Tokyo, Japan) by a micropipette puller (P-97/IVF; Sutter Instrument, Novato, CA, USA). The programmed P-97 parameters are as follows: heat: 845; pull: 50; velocity: 120; time: 200; pressure: 500. The value of the 'heat' parameter required for the ramp test is based on the manufacturer's protocol because this value depends on a combination of the filament and the glass capillary. In our case, using a combination of a regular P-97 filament and a GD-1 glass capillary, the 'heat' parameter value ranges between 845 and 870.

Based on the aforementioned information, we describe next the manipulation procedure of microinjection using daphnids eggs.


**5.** Eggs are surgically obtained from the mother daphnid and placed in ice-cold M4-sucrose medium.

**2.1. Microinjection system**

124 RNA Interference

to 6-week-old daphnids should be collected.

dissolved in M4 media in *D. pulex* [14].

microinjection using daphnids eggs.

temperature.

The daphnid microinjection system uses parthenogenetic eggs just after ovulation into the mother's brood chamber. The individual mother begins to lay eggs into the brood chamber a few minutes after releasing neonates and molting. To obtain many healthy eggs, eggs from 2

There are two major technical issues in microinjection of daphnids. One is the rapid hardening of the egg membrane just after ovulation [38], which is caused by enzymatic activity of peroxidase. The second problem is a substantial difference between the internal and external osmotic pressures of the egg. The former issue hampers the penetration of the egg membrane by a needle while the latter causes the leakage of egg components after injection. To overcome these problems, Kato et al. [13] and Hiruta et al. [14] established improved protocols for microinjection in *D. magna* and *D. pulex*, respectively. They succeeded in the transient inhibi‐ tion of the egg membrane hardening by ice-cold treatment just after ovulation. They also found the best culture conditions after injection by increasing external osmotic pressures: M4 medium [39] with 80-mM sucrose in *D. magna* [13] and a 2% agar plate covered with 60-mM sucrose

In addition, since the fineness of the needle is critical for the success of microinjection, we next describe a detailed preparation method. A glass needle is made from a glass capillary tube (GD-1; Narishige, Tokyo, Japan) by a micropipette puller (P-97/IVF; Sutter Instrument, Novato, CA, USA). The programmed P-97 parameters are as follows: heat: 845; pull: 50; velocity: 120; time: 200; pressure: 500. The value of the 'heat' parameter required for the ramp test is based on the manufacturer's protocol because this value depends on a combination of the filament and the glass capillary. In our case, using a combination of a regular P-97 filament and a GD-1

Based on the aforementioned information, we describe next the manipulation procedure of

**1.** The setting of tools for microinjection and surgery are shown in Figure 4A-C. A glass Petri dish is prepared by placing two cover glasses side by side with M4-sucrose at ambient

**2.** The synthesized double-strand RNA (dsRNA) is mixed with an equal amount of 2-mM Chromeo 494 fluorescent dye (Active Motif Chromeon GmbH, Tegernheim, Germany), which is used as a visible marker for injection. When using *D. pulex* eggs, it is possible to confirm whether injection has succeeded by visual observation since the eggs are more

transparent than *D. magna* eggs (Figure 3). A visible marker is thus not essential.

**3.** The dsRNA solution (1.0–1.5 μL) is loaded into the needle. The tip of the needle is then

**4.** Mother daphnids just before molting (brood chamber is empty) are collected and observed until spawning begins. They are transferred to ice-cold M4-sucrose medium just before

glass capillary, the 'heat' parameter value ranges between 845 and 870.

manually cut off using forceps under a stereomicroscope.

spawning is complete (4–5 eggs remain in each ovary).


**Figure 4.** Microinjection of daphnid egg. (A) Experimental equipment. 1: stereomicroscope; 2: micromanipulator (MN-153, Narishige, Tokyo, Japan); 3: electronic microinjector (Femtojet, Eppendorf, Hauppauge, NY, USA). (B) Tools for surgical manipulation. 1: Pasteur pipet (Thomas Scientific, Swedesboro, NJ, USA); 2: glass dish with two cover glasses; 3: plate for blood test; 4: forceps (Dumoxel #5 Biologie). (C) A glass needle is made from a glass capillary tube (GD-1; Narishige, Tokyo, Japan). (D) Overview illustration of microinjection field. (E, F) Magnified view and illustra‐ tion of microinjection using *D. pulex* egg. (G) Schematic illustration of embryo incubation after injection.

#### **2.2. RNAi machinery in daphnid species**

The well-known natural role of RNAi in organisms is the innate immune system against viruses and transposable elements [40]. Using this phenomenon, RNAi induction has been developed as an innovative method for gene functional analysis by exogenous application of dsRNA in *Caenorhabditis elegans* [41]. The dsRNA is first recognized by Dicer protein and cut off into short 21–24 nucleotides referred to as short interfering RNAs (siRNA). These are then invariably incorporated into large Argonaute-containing effector complexes known as RNAinduced silencing complexes (RISCs), after which one-side strand of the dsRNA is cleaved and degraded. Finally, the active Argonaute-containing RISC cleaves the target RNA sequence with the complementary sequence to siRNA [40, 42]. In addition to this core machinery of the RNAi pathway, many eukaryotes have the potential to amplify an amount of siRNA by a hostencoded RNA-dependent RNA polymerase (RdRp). However, RdRp orthologs have not been identified from the Arthropoda genome including *D. pulex* except for the tick genome [42].

In the *D. pulex* and *D. magna* genomes, there are three Dicer and Argonaute paralogs, but the *D. magna* genome contains two copies of Dicer. Previous studies have shown that Dicer paralogs are categorized into two clusters corresponding to the microRNA (miRNA) pathway (Dicer-1) and the siRNA pathway (Dicer-2) [42, 43]. The miRNA is also a short 21–25 RNA, which is generated from a hairpin in pre-mRNA, and plays an important role in translational repression associated with RISC [44]. Phylogenetic analyses found that all Dicer paralogs of *D. pulex* and *D. magna* were classed into the Dicer-1 group [45] (Figure 5A). Moreover, we successfully recruited three Argonaute paralogs from the genome of each daphnid and constructed a phylogenetic tree (Figure 5B). Previous studies revealed that Argonaute family members are key components in different RNA silencing pathways and are categorized into two subfamilies, Argonaute and PIWI (P-element induced wimpy testis). Argonaute subfamily members have been found in widespread taxa, including yeast, plants, and animals and have been identified as Argonaute-1 and Argonaute-2, which are involved in miRNA and siRNA pathways, respectively. In contrast, the PIWI subfamily has been identified only in animals as Argonaute-3, which is involved in PIWI-interacting RNA (piRNA) pathways [42, 46, 47]. Four Argonaute family members were found from *D. pulex* and *D. magna* genome sequences in this study, although only two paralogs have already been previously reported [43]. Phylogenetic analysis revealed that both paralogs fall into the Arogonaute-1 clade of the Argonaute subfamily, whereas each one paralog was categorized into Argonaute-3 and PIWI clades of the PIWI subfamily (Figure 5B). The number of Argonaute family members found in daphnids seems to be as conserved as in insect species [42, 48], although no Argonaute-2 paralogs have yet been identified. Taken together, our results suggest that the genomes of daphnids might lack the Dicer-2 and Argonaute-2 orthologs, which are factors responsible for regulating the siRNA-inducing transcriptional gene-silencing pathway. In other words, our data imply that the RNAi machinery of daphnid species might be distinct from the equivalent well-studied mechanism in insects. To understand the full picture of the RNAi machinery of daphnid species, further studies that examine domain sequence similarity and gene functional analysis will be required.

**2.2. RNAi machinery in daphnid species**

126 RNA Interference

will be required.

The well-known natural role of RNAi in organisms is the innate immune system against viruses and transposable elements [40]. Using this phenomenon, RNAi induction has been developed as an innovative method for gene functional analysis by exogenous application of dsRNA in *Caenorhabditis elegans* [41]. The dsRNA is first recognized by Dicer protein and cut off into short 21–24 nucleotides referred to as short interfering RNAs (siRNA). These are then invariably incorporated into large Argonaute-containing effector complexes known as RNAinduced silencing complexes (RISCs), after which one-side strand of the dsRNA is cleaved and degraded. Finally, the active Argonaute-containing RISC cleaves the target RNA sequence with the complementary sequence to siRNA [40, 42]. In addition to this core machinery of the RNAi pathway, many eukaryotes have the potential to amplify an amount of siRNA by a hostencoded RNA-dependent RNA polymerase (RdRp). However, RdRp orthologs have not been identified from the Arthropoda genome including *D. pulex* except for the tick genome [42].

In the *D. pulex* and *D. magna* genomes, there are three Dicer and Argonaute paralogs, but the *D. magna* genome contains two copies of Dicer. Previous studies have shown that Dicer paralogs are categorized into two clusters corresponding to the microRNA (miRNA) pathway (Dicer-1) and the siRNA pathway (Dicer-2) [42, 43]. The miRNA is also a short 21–25 RNA, which is generated from a hairpin in pre-mRNA, and plays an important role in translational repression associated with RISC [44]. Phylogenetic analyses found that all Dicer paralogs of *D. pulex* and *D. magna* were classed into the Dicer-1 group [45] (Figure 5A). Moreover, we successfully recruited three Argonaute paralogs from the genome of each daphnid and constructed a phylogenetic tree (Figure 5B). Previous studies revealed that Argonaute family members are key components in different RNA silencing pathways and are categorized into two subfamilies, Argonaute and PIWI (P-element induced wimpy testis). Argonaute subfamily members have been found in widespread taxa, including yeast, plants, and animals and have been identified as Argonaute-1 and Argonaute-2, which are involved in miRNA and siRNA pathways, respectively. In contrast, the PIWI subfamily has been identified only in animals as Argonaute-3, which is involved in PIWI-interacting RNA (piRNA) pathways [42, 46, 47]. Four Argonaute family members were found from *D. pulex* and *D. magna* genome sequences in this study, although only two paralogs have already been previously reported [43]. Phylogenetic analysis revealed that both paralogs fall into the Arogonaute-1 clade of the Argonaute subfamily, whereas each one paralog was categorized into Argonaute-3 and PIWI clades of the PIWI subfamily (Figure 5B). The number of Argonaute family members found in daphnids seems to be as conserved as in insect species [42, 48], although no Argonaute-2 paralogs have yet been identified. Taken together, our results suggest that the genomes of daphnids might lack the Dicer-2 and Argonaute-2 orthologs, which are factors responsible for regulating the siRNA-inducing transcriptional gene-silencing pathway. In other words, our data imply that the RNAi machinery of daphnid species might be distinct from the equivalent well-studied mechanism in insects. To understand the full picture of the RNAi machinery of daphnid species, further studies that examine domain sequence similarity and gene functional analysis

**Figure 5.** Phylogenetic trees of Dicer (A) and Argonaute (B). Amino acid sequences were aligned by ClustalW, and the maximum likelihood trees were constructed from these alignments using the JTT model with bootstrap analyses of 1000 replicates along with complete deletion options (818 and 597 amino acid positions were used, respectively) by MEGA6 [49]. Branches with bootstrap support >70% are indicated by numbers at nodes. Both *D. pulex* and *D. magna* are indicated in bold. To obtain the predicted sequences encoding the *D. magna* orthologs of RNAi-related genes, pro‐ tein sequences of *D. pulex* were used in BLAST searches querying *D. magna* Genome BLAST (http://arthro‐ pods.eugenes.org/EvidentialGene/daphnia/daphnia\_magna/BLAST/). The SID protein sequences of *D. pulex* were retrieved from wFleaBase (http://wfleabase.org/). Phylogenetic trees were constructed based on data from McTaggart et al. [43] with some modifications.

#### **2.3. Future challenges for development of high-throughput RNAi method in daphnids**

Despite the availability of a microinjection-based RNAi method in daphnids, as demonstrated by knocking down genes responsible for morphogenesis (*distal-less* and *eyeless*) [13, 14, 50], sexual differentiation (*doublesex1*) [51], endocrine system (*ecdysteroid-phosphate phosphatase*) [52], and neurogenesis (*single-minded homolog*) [53], this method has several experimental limitations. For example, microinjection can only be performed using an early stage (1-cell stage) egg, suggesting that this system cannot be used to perform a functional analysis of genes that act during the adult stage and is unsuitable for large-scale experiments. Moreover, specialized and skillful technical training is necessary to master the microinjection technique in daphnids. To overcome those technical hurdles, we introduce two potential ideas to establish more high-throughput and user-friendly methods for the study of daphnids. One is electroporation, which is a physical transfection method that uses an electrical pulse to increase the permeability of cell membranes, allowing nucleic acids and/or chemicals to be introduced into cells. Recent innovation of the electroporation system has enabled the establishment of rapid functional analysis in various organisms [54–56]. Indeed, our group has successfully developed an *in vivo* electroporation system for the introduction of foreign DNA into neonatal *D. magna* within 6 hours after release from the mother's brood chamber [57]. Therefore, it might be possible to apply this system for RNAi using various stages of daphnids.

The second method is a feeding (oral delivery) RNAi system, which was first developed in *C. elegans*[58]. The feeding RNAi system is the most convenient and inexpensive method for highthroughput screening since bacteria produce the designed dsRNA that are fed to host animals. This system has so far been applied in various insects [59] and decapod crustaceans [60–62]. Unlike the time-consuming and troublesome microinjection method that can only be per‐ formed in the early egg stage in daphnids, the alternative feeding RNAi method may poten‐ tially be applied for manipulating a wide range of genes in many individuals at the same time.

#### **3. Extended microinjection-based applications**

The microinjection system can be widely applied for the development of not only RNAi but also other attractive methods for gene functional analysis. Indeed, several microinjectionbased functional methods have been developed in daphnid species. First, a transient overex‐ pression system for arbitrary genes or reporter constructs was established by microinjection of synthesized mRNA bearing the 5′ cap structure and the 3′ poly(A) tail [51] and a DNA reporter construct [63]. These methods allow for a gain-of-function analysis, although only one example has shown that the phenotype induced by transient overexpression was only observed in the first instar juvenile [51]. However, the aforementioned overexpression and RNAi system in daphndis suffer from several drawbacks such as incomplete gain- or loss-offunction, transient effect, and limited analyzable stages.

To overcome these limitations, a transgenic *D. magna* line was established by using microin‐ jected GFP or DsRED reporter plasmid, although the success rate was quite low (0.67%) [64, 65]. Furthermore, genome editing with engineered endonucleases is rapidly growing as a stable experimental method for generating heritable mutations in not only well-known in model organisms but also in nonmodel organisms. There are three representative methods: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) systems [66]. In order to perform targeted mutagenesis in the *D. pulex* and *D. magna* genomes, TALEN and CRISPR/Cas9 systems have been established by microinjection of these engineered nucleases [50, 65, 67]. Taken together, these genome-editing techniques will enable scientists to accu‐ rately define arbitrary gene functions in daphnid species in future studies.

#### **4. Conclusion**

**2.3. Future challenges for development of high-throughput RNAi method in daphnids**

128 RNA Interference

be possible to apply this system for RNAi using various stages of daphnids.

**3. Extended microinjection-based applications**

function, transient effect, and limited analyzable stages.

The second method is a feeding (oral delivery) RNAi system, which was first developed in *C. elegans*[58]. The feeding RNAi system is the most convenient and inexpensive method for highthroughput screening since bacteria produce the designed dsRNA that are fed to host animals. This system has so far been applied in various insects [59] and decapod crustaceans [60–62]. Unlike the time-consuming and troublesome microinjection method that can only be per‐ formed in the early egg stage in daphnids, the alternative feeding RNAi method may poten‐ tially be applied for manipulating a wide range of genes in many individuals at the same time.

The microinjection system can be widely applied for the development of not only RNAi but also other attractive methods for gene functional analysis. Indeed, several microinjectionbased functional methods have been developed in daphnid species. First, a transient overex‐ pression system for arbitrary genes or reporter constructs was established by microinjection of synthesized mRNA bearing the 5′ cap structure and the 3′ poly(A) tail [51] and a DNA reporter construct [63]. These methods allow for a gain-of-function analysis, although only one example has shown that the phenotype induced by transient overexpression was only observed in the first instar juvenile [51]. However, the aforementioned overexpression and RNAi system in daphndis suffer from several drawbacks such as incomplete gain- or loss-of-

To overcome these limitations, a transgenic *D. magna* line was established by using microin‐ jected GFP or DsRED reporter plasmid, although the success rate was quite low (0.67%) [64,

Despite the availability of a microinjection-based RNAi method in daphnids, as demonstrated by knocking down genes responsible for morphogenesis (*distal-less* and *eyeless*) [13, 14, 50], sexual differentiation (*doublesex1*) [51], endocrine system (*ecdysteroid-phosphate phosphatase*) [52], and neurogenesis (*single-minded homolog*) [53], this method has several experimental limitations. For example, microinjection can only be performed using an early stage (1-cell stage) egg, suggesting that this system cannot be used to perform a functional analysis of genes that act during the adult stage and is unsuitable for large-scale experiments. Moreover, specialized and skillful technical training is necessary to master the microinjection technique in daphnids. To overcome those technical hurdles, we introduce two potential ideas to establish more high-throughput and user-friendly methods for the study of daphnids. One is electroporation, which is a physical transfection method that uses an electrical pulse to increase the permeability of cell membranes, allowing nucleic acids and/or chemicals to be introduced into cells. Recent innovation of the electroporation system has enabled the establishment of rapid functional analysis in various organisms [54–56]. Indeed, our group has successfully developed an *in vivo* electroporation system for the introduction of foreign DNA into neonatal *D. magna* within 6 hours after release from the mother's brood chamber [57]. Therefore, it might

Recent growing innovations in high-throughput omics technologies (e.g., genomics, transcrip‐ tomics, proteomics, and metabolomics) enable us to obtain comprehensive profiles of a huge amount of candidate factors responsible for unique life history features of daphnids [12, 34, 68]. In order to investigate an arbitrary gene function, the establishment of an experimental method for gene functional analysis has been enthusiastically addressed by researchers using nonmodel organisms, even in the postgenomic era. In this chapter, we summarized (1) the experimental procedure with several tips for a microinjection system in *D. pulex* and *D. magna*, (2) information about genes responsible for their RNAi machinery, (3) potential concepts for novel user-friendly high-throughput RNAi systems in daphnids, and (4) other microinjection-based applications in daphnids. Further studies involved in the development of novel experimental methods and investigation of a wide range of gene functions can lead to a better understanding of the overview of the attractive environmental stimuli-dependent phenomenon in daphnids.

#### **Acknowledgements**

We thank members of the Iguchi laboratory for helpful advice and comments. Our work benefits from, and contributes to, the *Daphnia* Genomics Consortium. This work was supported by a JSPS Research Fellowship for Young Scientists to KT (No. 12J05579), a Sasakawa Scientific Research Grant from the Japan Science Society to KT, a Saito Ho-on Kai Scientific Research Grant from the Saito Gratitude Foundation to KT, grants from the Ministry of Education, Culture, Sports, Science, and Technology (TI), the Ministry of the Environment of Japan (TI), a Grant from the National Institute for Basic Biology (TI), and the Research Council of Norway (project 221455), Adverse Outcome Pathways for Endocrine Disruption in *Daphnia magna*, a conceptual approach for mechanistically based risk assessment (TI).

#### **Author details**

Kenji Toyota, Shinichi Miyagawa, Yukiko Ogino and Taisen Iguchi\*

\*Address all correspondence to: taisen@nibb.ac.jp

Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, and National Institutes of Natural Sciences, Higashiyama, Myodaiji, Okazaki, Aichi, Japan

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