**4. RNA splicing in disease diagnosis**

RNA splicing is a post-transcriptional process necessary to form a mature mRNA [61]. There are two main forms of splicing, that is, constitutive splicing and alternative splicing.

Constitutive splicing involves removal of introns from the pre-mRNA and joining the exons together to form a mature mRNA. Alternative splicing describes how exons can be included or excluded in different combinations to create a

*Recent Progress in Drug Repurposing Using Protein Variants and Amino Acids in Disease… DOI: http://dx.doi.org/10.5772/intechopen.102571*

diverse array of mRNA transcripts from a single pre-mRNA and therefore serves as a process to increase the diversity of the transcriptome. It was initially thought that about 5% of human genes were subjected to alternative splicing [62]. Now, after the implementation of next-generation sequencing technologies, it is now known that the vast majority, >95% of mRNAs, are subjected to alternative splicing [63]. However, the function of a large fraction of these splice isoforms is still unknown.

Splicing is more prevalent in multicellular than in unicellular eukaryotes because of the lower number of intron-containing genes in the latter [64]. As evolution progress, alternative splicing becomes more prevalent in vertebrates than in invertebrates. Skipping of a single exon in the RNA-binding protein (RBP) and polypyrimidine tract binding protein 1 (PTBP1) may be responsible for numerous alternative splicing changes between species, which suggest that one splicing event can augment the varieties observed in transcriptome between species [65].

The hypothesis that alternative splicing largely contributes to organism diversity is fueled by the observation that the total number of protein-coding genes does not differ much between species. And indeed, as we move up the phylogenetic tree, alternative splicing complexity increases, with the highest complexity in primates [66, 67].

#### **4.1 Major and minor spliceosome**

RNA splicing is performed by the spliceosome, a large and dynamic ribonucleoprotein complex composed of proteins and small nuclear RNAs (snRNAs), which assembles on the pre-mRNA (**Figures 1** and **2**).

#### **Figure 1.**

*Two-step splicing reaction. Splicing occurs by a 2-step trans-esterification reaction to remove introns and join exons together. The first step, U1 small nuclear ribonucleoprotein (snRNP) assembles at the 5′ splice site of an exon and U2 snRNP at the branch point sequence (BPS), just upstream of the 3′splice site of the adjacent/ downstream exon. This configuration is known as the pre-spliceosome. Hereafter, U1 and U2 are joined by the snRNPs U5 and U4–U6 complexes to form the pre catalytic spliceosome. Next, U4–U6 complexes unwind, releasing U4 and U1 from the pre-spliceosomal complex. This allows U6 to base pair with the 5′ splice site and the BPS. The 5′ splice site gets cleaved, which leads to a free 3′ OH-group at the upstream exon, and a branched intronic region at the downstream exon called the intron lariat.*

#### **Figure 2.**

*Major and minor splicing. (A) Major, and minor splicing. The major introns are spliced out, and minor introns are either retained (and the mRNA is most often subsequently degraded) or the minor intron is spliced out, and a mature mRNA is formed. (B) The 4 basic splicing signals are the 5′ splice donor site, the 3′ splice acceptor site, the branch point sequence (BPS), and the polypyrimidine tract (PT). Spliceosomal components recognize and bind to these sequences and mediate the splicing reaction. Intronic and exonic splicing enhancers and silencers determine the inclusion rate of exons. The BPS (major, YNYURAY; minor, UCCUUAACU) is located 20–50 bp upstream of the 3′ splice site, and the PT (Y10–12) is located in between the BPS and the 3′ splice site (N*〓*any nucleotide, Y*〓*C or U, R*〓*A, or G and S*〓*C or G). (C) Minor splicing uses different 5′ and 3′ splice sites and BPS, and lacks the PT. ESE indicates exonic splicing enhancers; ESS, exonic splicing silencers; ISE, intronic splicing silencers; and ISS, intronic splicing silencers. Note: The Figures 1 and 2 are a modification from van den Hoogenhof et al. [68].*

During the second step, U5 pairs with sequences in both the 5′ and 3′ splice sites, positioning the 2 ends together. The 3′ OH-group of the upstream (5′) exon fuses with the 3′ intron-exon junction, thereby conjoining the 2 exons and excising the intron in the form of a lasso-shaped intron lariat. Finally, the spliceosome disassembles, and all components are recycled for future splicing reactions.

Recent evidence has shown that splicing does not occur after transcription, but happens during transcription; therefore, the vast majority of human introns are spliced out when transcription is still taking place [69].

*Recent Progress in Drug Repurposing Using Protein Variants and Amino Acids in Disease… DOI: http://dx.doi.org/10.5772/intechopen.102571*

#### **4.2 RNA splicing in cardiomyopathy**

Several mouse models suggest a role for splicing factors in postnatal heart development. One such example is the alternative splicing factor ASF/SF2 (or SFRS1), an SR protein that is ubiquitously expressed and acting as an alternative splicing regulator [70]. ASF/SF2 conditional knockout mice die 6–8 weeks after birth, due to hypercontractile cardiac phenotype caused by a defect in Ca2+ handling. When ASF/SF2 is deleted, it leads to mis-splicing of several genes, including cardiac troponin T (cTnT), LIM-domain binding 3 (LDB3), and Ca2+/calmodulin- dependent protein kinase (CamkIIδ), Atypical alternative splicing of CamkIIδ, cTnT, and LDB3 can present 20 days after birth, even though ASF/SF2 was deleted at the early stages of cardiogenesis.

Mis-splicing of CamkIIδ in ASF/SF2 knockout hearts can lead to perturbation of Ca2+ handling and severe excitation-contraction coupling defects, which in turn leads to dilated cardiomyopathy (DCM).

Embryonic lethality may occur in systemic deletion of SC35 in mice, even before the onset of cardiogenesis [71]. Attempt to bypass this problem by generating a heart-specific knockout of SC35 uncovered the role of SC35 in the heart, as cardiac hypertrophy and DCM developed in these mice at 5–6 weeks of age [71].

In conclusion, ablation of SC35 in the heart shows that proper expression of this splice factor during postnatal heart development is essential to maintain cardiac form and function.

Severe and lethal DCM has been reported to occur 2 weeks after birth in mice with deletion of hnRNP U in the mouse heart [72]. The importance of alternative splicing of Ca2+-handling genes in early postnatal heart development can be observed in the role of heterogeneous nuclear ribonucleoprotein U (HnRNP U) in splicing of calcium/calmodulin-dependent protein kinase IIδ (CamkIIδ).

#### **4.3 Role of alternative splicing in disease phenotype**

Atypical alternative splicing has been documented to contribute to disease severity and susceptibility [73]; as observed in retinitis pigmentosa, Prader-Willi syndrome, and spinal muscular atrophy [74, 75]. Spinal muscular atrophy, for example, is caused by the loss of the survivor of the motor neuron-1 (SMN1) gene, which is required for proper assembly and transport of snRNP [74].

Kong et al. [76] used a genome-wide approach to study alternative splicing changes in the diseased heart. The splicing of 4 key sarcomeric genes, troponin T (TNNT)-2, TNNI3, MYH7, and FLNC, were significantly altered in human ischemic cardiomyopathy, DCM, and aortic stenosis.

### **5. Epigenetic DNA modifiers**

Epigenetics and its attendant markers influence the proliferation of diseases and their phenotypes. Outside, DNA canonical structure, DNA folds into alternative structures including DNA hairpins, cruciforms, triplexes or G-quadruplexes (G4), and holiday junctions [77, 78]. Besides these DNA structural changes, epigenetics processes, using DNA methylation and histone modification as the driver, are another primary vehicle for changes in DNA. Changes due to epigenetics modification with time can alter our phenotypes profoundly. Known facts are that everything from what we eat, drink, and smoke to other factors within our immediate environment including, stress can interfere with the way our genes express themselves up and down the line with the finest totality [79]. The primary

vehicles for epigenetic changes are DNA methylation and histone modification; there are many known enzymes that act on histone modifications by either adding or removing the covalent modifications. Such changes influence the degree of interaction between DNA and histone, which have some profound effects on the ability of that DNA to be transcribed. Histone modifications are subject to rapid changes (in seconds/minutes), giving room for the cell to respond to external stimuli. Furthermore, many of the known enzymes responsible for modifying histone residues have numbers of non-histone substrates such as transcription factors [80, 81].

Some mechanisms for the function of histone modifications have been characterized including; the compression of chromatin, and the recruitment of nonhistone proteins [82]. There are different types of modification and these determine the amino acid residue produced. The modifications of histone lead to either gene activation or repression, and the addition of acetyl groups, to the tail of histone H3, neutralizes the basic charge of the lysine, leading to the unfolding of the chromatin, allowing transcription to occur. Conversely, the removal of these acetyl groups results in chromatin compression, which prevents transcription [82]. These kinds of changes in chromatin structure help to prevent access by other proteins that can further modify the chromatin (e.g., remodeling ATPases).

Understanding the etiology of some of these diseases, from PPI, protein DNA/RNA interaction is important as it will herald in more robust drug treatments for patients

#### **Figure 3.**

*Factors influencing epigenetics (modified from [84]): complex interplay is required for a wholesome gene expression understanding; such complexes will further enhance the use of phytomedicine in disease management.*

*Recent Progress in Drug Repurposing Using Protein Variants and Amino Acids in Disease… DOI: http://dx.doi.org/10.5772/intechopen.102571*

with specific disease phenotypes. Along this line, Okoh et al. [81] recently, using available data, espoused the need for the combination of herbal medicine to target some epigenetic markers by way of epigenetic engineering (site-specific DNA binding module fusions with DNA demethylating enzymes for epigenetic induction of for instance; fetal hemoglobin (HbF) for therapy of sickle cell disease (SCD)). This is in consonance with earlier postulation [83, 84], implying such technique may provide a better way to activate/or repress inherent gene expression, bearing transient modification of DNA and histones should remain stable over many cell divisions helping in delaying HbF switching [83, 84].

Moreover, Okoh et al. [81], suggested that the de-methylation of DNA at the CpGs site on both DNA strands may be possible using the combination of herbal medicine, foods rich in flavonoids could be vital in tweaking histone acetylation, which can modulate gene expression. The figure below postulates the complex interplay between, epigenetics and phyto-compound modifiers towards enabling gene transcription for proper protein translation (**Figure 3**).
