**4. Physiological functions of RNA modifications**

Although we do not have full knowledge on the effects of RNA modification on physiological function, there is increasing evidence that they play critical roles in the regulation of gene expression, cellular functions, and development. Disruptions of RNA modification mechanisms have also been associated with disease. We present here a few examples, which demonstrate the importance of RNA modification on physiological function.

As stated earlier, m<sup>6</sup> A modifications are commonly found throughout eukaryotes, as demonstrated by multiple m<sup>6</sup> A‐seq studies. Human m<sup>6</sup> A‐seq analyses revealed 12,769 putative m6 A sites within 6990 and 250 protein‐coding and non‐coding transcripts, respectively [26], whereas, in mice, 4513 m<sup>6</sup> A peaks were identified in 3376 and 66 protein‐coding and non‐coding transcripts, respectively [26]. The m<sup>6</sup> A consensus motif, RRACU, was identified with a median distance from m<sup>6</sup> A peaks of 24 nucleotides [26]. Interestingly, the majority of m<sup>6</sup> A sites were conserved between both mouse and human transcriptomes and enriched further within long internal exons and around stop codons, suggesting strong evolutionary selection [26, 36]. m<sup>6</sup> A‐LAIC‐seq showed that methylated transcripts utilized proximal alternative polyadenylation (APA) sites, which resulted in shorter 3′ untranslated regions, whereas non‐methylated transcripts tended to use distal APA sites [37]. This observation correlated with the finding that m<sup>6</sup> A‐modified transcripts had both significantly shorter RNA half lives and slightly lower translational efficiencies than unmarked transcripts [44].

In vitro and in vivo genetic depletion of the m<sup>6</sup> A writer, *Mettl3*, in both mouse and human, led to the absence of m<sup>6</sup> A modification within *Nanog* mRNA which encodes a pluripotency factor. The absence of m<sup>6</sup> A marks extended Nanog expression throughout differentiation and inhibited embryonic stem cell exit from self‐renewal towards lineage differentiation [44]. m6 A‐seq in mouse naïve embryonic stem cells (ESCs), 11‐day‐old embryoid bodies (EBs), and mouse embryonic fibroblasts (MEFs) revealed m<sup>6</sup> A marks in naïve pluripotency‐promoting genes reduced mRNA stability of key pluripotency‐promoting transcripts and facilitated differentiation [45]. These findings suggest that m<sup>6</sup> A modification provides the flexibility of the stem cell transcriptome required to differentiate into different lineages [44]. NANOG is also important in both the maintenance and specification of cancer stem cells which can metastasize and form primary tumors. The exposure of breast cancer cells to hypoxia induced the expression of the eraser ALKBH5 which resulted in m<sup>6</sup> A demethylation in the 3ʹ UTR of *NANOG* mRNA and the increased half life of *NANOG* mRNA, thereby promoting the breast cancer stem cell (BCSC) phenotype [46]. The m<sup>6</sup> A reader YTHDF2 protects the 5′ UTR of stress‐induced transcripts from demethylation. Cap‐independent translation initiation was enhanced by 5′ UTR methylation [47]. m<sup>6</sup> A modification is critical for the regulation of HIV‐1 replication and HIV‐1ʹs effect on the host immune system [48]. HIV‐1 viral infection induced m6 A modification in both host and viral mRNAs. HIV‐1 coding, non‐coding, and splicing regulatory regions contained a total of 14 m<sup>6</sup> A methylation peaks. In addition, methylation of two highly conserved m<sup>6</sup> A target sites in the HIV‐1 rev response element (RRE) stem loop II region enriched the binding of the HIV‐1 rev protein to the RRE in vivo and enhanced nuclear export of HIV‐1 RNA [48]. The long non‐coding RNA X‐inactive specific transcript (XIST) regulates transcriptional silencing of genes on the X chromosome. XIST is heavily modified with at least 78 m<sup>6</sup> A sites. Knockdown of METTL3 leads to decreased XIST m<sup>6</sup> A marks and impairs XIST‐mediated gene silencing [49].

cyanoethylated with acrylonitrile to form *N*<sup>1</sup>

284 Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

**4. Physiological functions of RNA modifications**

onstrate the importance of RNA modification on physiological function.

A‐seq studies. Human m<sup>6</sup>

group of ce1

with N<sup>3</sup>

As stated earlier, m<sup>6</sup>

majority of m<sup>6</sup>

transcripts [44].

m6

onstrated by multiple m<sup>6</sup>

[26], whereas, in mice, 4513 m<sup>6</sup>

evolutionary selection [26, 36]. m<sup>6</sup>

tified with a median distance from m<sup>6</sup>

non‐coding transcripts, respectively [26]. The m<sup>6</sup>

observation correlated with the finding that m<sup>6</sup>

Watson‐Crick base pairing of I with C is inhibited by the newly formed *N*<sup>1</sup>

‐cyanoethylinosine (ce1

I. Thus, cyanoethylation of I blocks cDNA synthesis by preventing extension

of the cDNA that bears a cytosine (C) corresponding to the editing site during reverse transcription. However, I will be replaced by guanosine (G) [42] (**Figure 2B**). To detect RNA pseudouridylation, several groups developed Pseudo‐seq (Ѱ‐seq). RNA is treated

‐[N‐cyclohexyl‐Nʹ‐β‐(4‐methylmorpholinium) ethylcarbodiimide‐Ѱ (N<sup>3</sup>

which binds covalently to U, G, and Ѱ residues and then exposed to alkaline pH to reduce stable U‐CMC and G‐CMC adducts. Reverse transcription will pause at the remaining intact Ѱ‐CMC sites, allowing for the mapping of Ѱ‐modifications [16, 17] (**Figure 2C**). Comparison of mapping reads from CMC‐treated samples versus non‐treated controls, Ѱ will be detected as the sites with an increased proportion of reads supporting reverse transcription termination. NAD captureSeq (**Figure 2D**) requires the chemo‐enzymatic modification of NAD which is capping the 5ʹ end of RNA. The first step, the transglycosylation of NAD, is catalyzed by ADP‐ribosyl cyclase (ADPRC) from *Aplysia californica* in the presence of an alkynyl alcohol. In the second step, the modified NAD is biotinylated by a copper‐catalyzed azide‐alkyne cycloaddition. Thirdly, the biotin‐linked RNA is captured on streptavidin beads and processed further for cDNA library preparation and NGS. The NAD‐biotin‐captured sequences are then identified by comparison with the control samples which were not subjected to the first step of chemo‐enzymatic biotinylation [43].

Although we do not have full knowledge on the effects of RNA modification on physiological function, there is increasing evidence that they play critical roles in the regulation of gene expression, cellular functions, and development. Disruptions of RNA modification mechanisms have also been associated with disease. We present here a few examples, which dem-

A sites within 6990 and 250 protein‐coding and non‐coding transcripts, respectively

enriched further within long internal exons and around stop codons, suggesting strong

proximal alternative polyadenylation (APA) sites, which resulted in shorter 3′ untranslated regions, whereas non‐methylated transcripts tended to use distal APA sites [37]. This

cantly shorter RNA half lives and slightly lower translational efficiencies than unmarked

A modifications are commonly found throughout eukaryotes, as dem-

A sites were conserved between both mouse and human transcriptomes and

A peaks were identified in 3376 and 66 protein‐coding and

A‐LAIC‐seq showed that methylated transcripts utilized

A‐seq analyses revealed 12,769 putative

A consensus motif, RRACU, was iden-

A‐modified transcripts had both signifi-

A peaks of 24 nucleotides [26]. Interestingly, the

I). Subsequently, the

‐cyanoethyl

‐CMC‐Ѱ)],

The tRNA T‐loop at position 58 commonly contains a m1 A modification [50], along with position 9 of metazoan mitochondrial tRNAs [51] and eukaryotic rRNAs [52]. Initiator tRNAMet contains fully modified m1 A 58 which stabilizes its tertiary structure. Hypomodification of tRNA m1 A 58 affects the association with polysomes and the subsequent efficiency of translation [53, 54]. m1 A modifications in tRNA function in response to environmental stress [55], whereas m1 A‐modified rRNA regulates ribosome biogenesis [52]. m1 A‐ID‐seq demonstrated that m1 A methylation regulated the dynamic response to stimuli and identified 901 m1 A peaks enriched within the 5ʹ UTR near the start codons of 600 distinct protein‐coding and non‐coding RNAs [39].

m5 C sites have been detected in several eukaryotic tRNA, Rrna, and mRNA. m<sup>5</sup> C marks stabilize the secondary structure of tRNA, alter aminoacylation and codon recognition [56], and regulate translational fidelity [57]. A low level of internal m<sup>5</sup> C was found in mRNA cap structures in mammalian‐ and virus‐infected mammalian cells [58, 59]. BS‐seq identified 10,275 sites in protein‐coding and non‐coding RNAs [41]. m<sup>5</sup> C marks in mRNAs were enriched near argonaute‐binding sites within the 3ʹ UTR [41].

A‐to‐I editing sites are distributed through human mRNA, including exons, introns. and 5ʹ and 3ʹ UTRs [60]. Alu repeat elements contain the highest frequency of A‐to‐I editing sites among the untranslated regions of the genome [61]. Intronic editing mediated by ADAR1 contributes to the maintenance of mature mRNA by protecting it against unfavorable processing of the Alu sequence and by degradation of aberrant transcripts by nonsense‐mediated decay (NMD) [42]. A‐to‐I RNA editing is diminished in brain tissue from patients with Alzheimerʹs disease relative to controls [62]. The reduction occurs predominantly in the hippocampus and to a lesser extent in the temporal and frontal lobes. These alterations result in decreased levels of protein recoding, the process of changing the amino acid sequence by A‐to‐I editing, in Alzheimerʹs disease [62]. The APOBEC3 family of cytidine deaminases has been associated with mutations in cancer genomes in several types of cancer. Accumulated data linking mutations in oncogenes and tumor suppressor genes with APOBEC3B activity are providing evidence that cytidine deaminase‐induced mutagenesis is activated in tumorigenesis, thus providing novel therapeutic targets [63].

identification and characterization of the enzymes responsible for RNA modification since several of these enzymes have been shown to play important roles in development and disease. It is essential to decipher all functions and disease involvements of all RNA modifications. Development of additional technologies to alter RNA modifications, including the engineering of RNA‐modifying enzymes with modified substrate specificity and activity via the CRISPR‐Cas 9 system, will open the door to new types of detection and analysis pipelines. With further technological development, we will be able to elucidate the sequence‐specific signatures in RNA that direct modifications and then better relate these RNA marks to their corresponding biological functions. Finally, the advancement of current approaches, coupled with new technologies, will allow for the development of new therapies and therapeutic targets for human diseases associated with deficient RNA

, Shamima Islam1

1 Division of Experimental and Translational Genetics, The Children's Mercy Hospital, MO,

2 Department of Pediatrics, Tangdu Hospital, Fourth Military Medical University, Xian, China 3 Division of Pediatric Gastroenterology, University of Missouri Kansas City School of

4 Department of Biomedical and Health Informatics, University of Missouri Kansas City

[1] Djebali S, et al. Landscape of transcription in human cells. Nature. 2012;**489**(7414):101‐108 [2] Maeda N, et al. Transcript annotation in FANTOM3: Mouse gene catalog based on phys-

[3] Kirchner S, Ignatova Z. Emerging roles of tRNA in adaptive translation, signalling

[4] Sato H, Kamiya M. Deleterious effect of prednisolone on the attachment of *Taenia crassiceps* cysticerci to the intestine of gerbils. Nihon Juigaku Zasshi. 1989;**51**(5):1099‐1101 [5] Fatica A, Bozzoni I. Long non‐coding RNAs: New players in cell differentiation and

dynamics and disease. Nature Reviews Genetics. 2015;**16**(2):98‐112

development. Nature Reviews Genetics. 2014;**15**(1):7‐21

, Li Qin Zhang1

, Ding‐You Li<sup>3</sup>

Epitranscriptomics for Biomedical Discovery http://dx.doi.org/10.5772/intechopen.69033

and

287

modification.

Min Xiong1

USA

Shui Q. Ye1,4\*

**Author details**

Medicine, MO, USA

**References**

School of Medicine, MO, USA

, Daniel P. Heruth1

\*Address all correspondence to: sqye@cmh.edu

ical cDNAs. PLoS Genetics. 2006;**2**(4):e62

, Xun Jiang<sup>2</sup>

Pseudo‐seq revealed that mRNA Ψ marks mRNA are regulated in response to stimuli, such as serum starvation in human cells and nutrient deprivation in yeast. The observations indicate that Ψ triggers a rapid regulatory mechanism to rewire the genetic code through inducible mRNA [16]. Pseudouridylation of rRNA and telomerase RNA component (TERC) were also found to be reduced in dyskeratosis congenita patients [17]. Furthermore, missense mutations in pseudouridine synthase 1 (PUS1) may lead to deficient pseudouridylation of mitochondrial tRNAs in mitochondrial myopathy and sideroblastic anemia (MLASA) patients [64].

NAD captureSeq identified NAD as a 5ʹ RNA cap in a subset of regulatory RNAs in bacteria [43] and subsequently proposed that this type of capping may be common across all of life [65]. It is safe to predict that investigation of the roles and mechanisms of 5ʹ NAD caps in eukaryotes will draw increasing attention in the biomedical field. This is due to mainly two reasons. First, the chemical modification of the 5ʹ end of RNA is critical for RNA processing, localization, stability, translational efficiency, and epitranscriptomic regulation of gene expression [66]. Second, NAD is both a co‐substrate for enzymes, such as the sirtuins and poly(adenosine diphosphate‐ribose) polymerases, and a critical electron‐carrying coenzyme for enzymes that catalyze oxidation‐reduction reactions. NAD is involved in nearly all physiological processes. For example, cellular NAD+ levels are modulated during aging, and the use and production of NAD+ usage has been associated with prolonged health and life spans [67]. Regulation of NAD‐mediated RNA capping and hence gene expression will undoubtedly enrich our understanding of NADʹs expanding roles in normal physiology and disease pathogenesis.

#### **5. Perspective**

Although rapid advances have been made in the past few years in epitranscriptomics, more work is needed in this field. To date, more than 140 different RNA modifications have been identified. However, there are only a few reliable high‐throughput techniques available to determine the global occurrence of a particular RNA modification. Thus, there is a need for the development of more high‐throughput techniques to characterize the full spectra of RNA modifications. It is also important to pursue the comprehensive identification and characterization of the enzymes responsible for RNA modification since several of these enzymes have been shown to play important roles in development and disease. It is essential to decipher all functions and disease involvements of all RNA modifications. Development of additional technologies to alter RNA modifications, including the engineering of RNA‐modifying enzymes with modified substrate specificity and activity via the CRISPR‐Cas 9 system, will open the door to new types of detection and analysis pipelines. With further technological development, we will be able to elucidate the sequence‐specific signatures in RNA that direct modifications and then better relate these RNA marks to their corresponding biological functions. Finally, the advancement of current approaches, coupled with new technologies, will allow for the development of new therapies and therapeutic targets for human diseases associated with deficient RNA modification.
