**2. Natural antisense transcripts that overlap with mRNAs**

### **2.1 Structures of natural antisense transcripts**

The AS transcript is frequently transcribed from inducible genes [14]. Our previous studies showed that an AS transcript harbors an overlapping sequence with 3′UTR of an mRNA [14]. Such a 3′UTR possesses a few AU-rich elements (AREs), which may be involved in mRNA stability because AREs may be the targets of miRNAs and RNAbinding proteins [15, 16]. Interestingly, the location of AREs in the 3′UTR of *iNOS* mRNA is conserved among species (rat, mouse, and human) [17, 18].

The sizes of AS transcripts are variable and show a smear pattern or discrete bands in Northern blot analysis. The former example is AS transcripts that are transcribed from rat inducible nitric oxide synthase (*iNOS*, *NOS2*) gene, and the size is ranging from 600 to 1000 nt (**Figure 1**) [19]. iNOS catalyzes the production of the inflammatory mediator nitric oxide (NO). iNOS is induced by various inflammatory stimuli; interleukin (IL)- 1beta to hepatocytes and bacterial lipopolysaccharide (LPS) to macrophages [17, 18].

*The Natural Antisense Transcript-Targeted Regulation Technology Using Sense Oligonucleotides… DOI: http://dx.doi.org/10.5772/intechopen.108281*

#### **Figure 1.**

*mRNA and AS transcripts transcribed from the inducible nitric oxide synthase (iNOS) gene. The rat iNOS gene is schematically depicted. The iNOS gene consists of 27 exons, and the AS transcript overlaps with the exon 27, which includes the 3*′*UTR (white box) of the mRNA. The iNOS mRNA harbors AREs in its 3*′*UTR. The iNOS AS transcript starts at the end of exon 27 and stops at various sites, resulting in various sizes of the transcripts, which do not harbor a poly(A) tail. Figure reproduced with modification from [19] with permission.*

#### **Figure 2.**

*mRNA and AS transcripts transcribed from interferon alpha1 (IFN-A1) gene. The human IFN-A1 gene consists of a single exon, which encodes IFN-alpha1 [20]. The coding sequence (from ATG to TAA) gives conserved stemloop structures in the IFN-A1 mRNA, i.e., stem-loops (SL) and bulged-stem loop (BSL) [21]. These structures are responsible for chromosome region maintenance 1 (CRM1)-dependent nuclear export of IFN-A1 mRNA [21]. The IFN-A1 mRNA harbors AREs in its 3*′*UTR. Two AS transcripts are shown, both of which are spliced and harbor poly(A) tails. Nucleotides are numbered from the transcription initiation site. Figure reproduced with modification from [20] with permission.*

The latter examples are about 4-kilobase (kb) AS transcript that is transcribed from the human interferon-alpha1 (*IFN-A1*) gene (**Figure 2**) [20]. *IFN-A1* gene encodes the cytokine IFN-alpha1 and is one subset of human *IFN-A* genes, which consist of 13 subsets [22]. Viral infection induces IFN-alpha1, a member of the type I interferon family, which is a main innate immunity response. AS transcripts are also transcribed from other subtypes of *IFN-A* genes [23].

Another example is the AS transcript from the rat tumor necrosis factor-alpha (*Tnf*) gene, which is about 2.5-kb long [24]. AS transcripts are transcribed from many genes that are involved in inflammation, such as mRNAs that encode alpha subunit (p19) of IL-23 (IL-23A), chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-X3-C) motif ligand 1 (CX3CL1), p65 and p50 subunits of nuclear factor kappaB (NF-kappaB) [14]. Additionally, AS transcript is transcribed from the human gene encoding ephrin type A receptor 2 (EPHA2), which is a receptor tyrosine kinase whose over-expression is observed in various cancers [25, 26].

#### **2.2 mRNA-AS transcript interactions and mRNA stability**

#### *2.2.1 iNOS mRNA-AS transcript interaction*

When the AS transcript overlaps with the relevant mRNA, the interaction between the AS transcript and mRNA is expected. Indeed, the AS transcript is transcribed from the rat *iNOS* gene and interacts with and stabilizes the *iNOS* mRNA [19].

#### **Figure 3.**

*A putative mechanism of the mRNA-AS transcript interaction and mRNA degradation by the introduction of a sense oligonucleotide (iNOS gene). (A) Stable complex on the iNOS mRNA. When the iNOS AS transcript is expressed, it partially hybridizes with a single-stranded loop in the iNOS mRNA that harbors a cap structure (open circle) and a poly(A) tail. By recruiting an RNA-binding protein, it forms an mRNA-AS transcriptprotein complex to stabilize the iNOS mRNA in the cytoplasm. (B) Interference with the mRNA-AS transcript interaction. Because a sense oligonucleotide to the iNOS mRNA harbors the same sequence as the mRNA, it competitively inhibits the hybridization of iNOS mRNA with the iNOS AS transcript, leading to interference with the mRNA-AS transcript interaction and then the mRNA degradation. This mechanism confers to the basis of the NATRE technology.*

Further analyses demonstrated that the *iNOS* AS transcript interacts with the *iNOS* mRNA at the single-stranded loop or bulge of the overlapping region between the mRNA and AS transcript (**Figure 3**) [19].

Because the orientation of RNA is 5′-to-3′, base complementarity indicates that the secondary structure of AS transcript is a mirror image of that of mRNA. This means that stem-loop structures in the AS transcript are formed at the complementary sites in the corresponding mRNA, leading to loop-loop hybridization between the mRNA and AS transcript [1, 2]. This loop-loop hybridization forms a short RNA:RNA duplex (usually <10 base pairs), which is thermodynamically unstable due to the low melting temperature of the duplex. Then, RNA-binding proteins (e.g., HuR) bind to the *iNOS* mRNA and/or AS transcript to stabilize the mRNA-AS transcript-protein complex, which protects from the degradation of RNAs and facilitates translation in the cell [19].

#### *2.2.2 INF-A1 mRNA-AS transcript interaction*

As another putative mechanism, the human *IFN-A1* AS transcript interacts with and stabilizes *IFN-A1* mRNA by blocking the microRNA binding to *IFN-A1* mRNA [20]. *IFN-A1* AS transcript is expressed at a low level. After the AS transcript transiently interacts with *IFN-A1* mRNA, it moves on and targets the next mRNA molecule in a 'hit-and-run' fashion [1, 20].

Sense oligonucleotides to BSL of *IFN-A1* mRNA (see **Figure 2**) decreased *IFN-A1* mRNA levels. Because a potent binding site of a microRNA (miR-1270) is present in BSL, miR-1270 may bind to BSL of *IFN-A1* mRNA. A microRNA-binding site is also called a microRNA-responsive element (MRE). Next, a short AS oligoribonucleotide (asORN), which is the part of *IFN-A1* AS transcript corresponding to BSL, was

*The Natural Antisense Transcript-Targeted Regulation Technology Using Sense Oligonucleotides… DOI: http://dx.doi.org/10.5772/intechopen.108281*

#### **Figure 4.**

*A model of interactions among transcripts to regulate mRNA levels. mRNA1 has a site of interaction (loop) with AS transcript1 and an MRE for microRNA3. AS transcript1 stabilizes mRNA1, whereas microRNA destroys mRNA1 through its MRE. AS transcript2 transcribed from another gene has common MREs. AS transcript2 sponges microRNA3, resulting in the sequestration of microRNA3. Therefore, AS transcript2 competes with microRNA3 and functions as a ceRNA. A typical example is seen among IFN-A1 mRNA and AS transcripts from the specific subsets of IFN-A gene family. See details in the text.*

introduced into the cells (This is not NATRE technology.). *IFN-A1* asORN increased *IFN-A1* mRNA levels, but it did not alter *IFN-A1* AS transcript levels [20]. These data imply that *IFN-A1* asORN stabilizes *IFN-A1* mRNA by simulating the AS transcript.

When the levels of transcripts were measured, miR-1270 was much more excess to *IFN-A1* AS transcript [23]. Although *IFN-A1* AS transcripts include several MREs, they are stoichiometrically unable to sponge all miR-1270 molecules. Further study indicated another mechanism.

When microRNAs are shared by mRNAs and AS transcripts, the transcripts function as competing endogenous RNAs (ceRNAs). Several AS transcripts are transcribed from the specific subsets of *IFN-A* gene family (*IFN-A1*, *A7*, *A8*, *A10*, and *A14* genes) and harbor common MREs for miR-1270 [23]. MiR-1270 can bind to both mRNA and AS transcript from *IFN-A* gene, as well as mRNAs from the specific subsets of *IFN-A* gene family (*IFN-A8*, *A10*, *A14*, and *A17* genes). Because the mRNAs and AS transcripts share MREs for miR-1270, they sponge and sequester the miR-1270 molecules. Collectively, *IFN-A* mRNAs and AS transcripts from *IFN-A* gene family form a ceRNA network to antagonize miR-1270 [23, 27]. This network finely tunes *IFN-A1* mRNA levels by common MREs that are present in the mRNAs and AS transcripts at the post-transcriptional level. Possible interactions among transcripts are depicted in **Figure 4**. See detailed discussion in [28].

#### *2.2.3 Tnf mRNA-AS transcript interaction*

Different from the *iNOS* and *IFN-A1* mRNA cases, AS transcripts sometimes downregulate gene expression. The stability of *Tnf* mRNA is modulated by RNAbinding proteins that bind to the AREs in its 3′UTR: human homolog R of embryonic lethal-abnormal visual protein (HuR), which stabilizes the mRNA; and tristetraprolin

#### **Figure 5.**

*A putative mechanism of the increase in mRNA levels by the introduction of a sense oligonucleotide (Tnf gene). (A) The Tnf mRNA that forms with a destabilizing RNA-binding protein. The Tnf AS transcript partially hybridizes with a single-stranded loop in the Tnf mRNA. An RNA-binding protein forms an mRNA-AS transcript-protein complex. (B) Interference with the mRNA-AS transcript interaction. A sense oligonucleotide to the Tnf mRNA competitively inhibits the hybridization of Tnf mRNA with the Tnf AS transcript, releasing a destabilizing RNA-binding protein. Finally, the Tnf mRNA becomes stable in the cytoplasm.*

(TTP), which destabilizes the mRNA [24]. A putative mechanism of mRNA destabilization is schematically shown in **Figure 5** and discussed in [1, 2]. Possible involvement of microRNA in the *Tnf* mRNA-AS transcript interaction is also mentioned [24].

Other than these mechanisms, there are several AS transcript-mediated mechanisms that regulate gene expression [7]. For example, AS transcripts may epigenetically repress transcription at the chromatin level.
