A study to find sequence motifs that affect splicing of exons regulated by RBM20

Recent studies showed that RNA binding motif protein 20 (RBM20) is involved in the regulation of the alternative splicing of sevaral genes both in rats and humans. RBM20 affects the expression level of the target genes, through activation (rarely) or repression (more often) of the regulated exons. Mutations in RBM20 have been shown to cause human dilated cardiomyopathy, a major source of mortality in the developed world. RBM20 RNA-binding site maps in the intronic sequence of the target genes in close proximity to differentially spliced exons and a significant overrepresentation of RBM20 binding sites in these regions was noticed. Nevertheless, a rule based on the number and the location of the sites flanking RBM20 regulated exons, and other possible still unknown sequence features, that allow to characterize an exon as regulated by RBM20, has not been found yet. The aim of this study is to find genes, especially exons, whose alternative splicing is affected by RBM20, through a differential analysis of existing RNA sequencing data of human and rat cardiomyocytes in two different conditions: presence and absence of RBM20. Then, we aim to define in greater detail the characteristics of the regions flanking RBM20 regulated exons. RNA sequencing data of human and rat cardiomyocytes used by Guo et al. were downloaded and used. Rat samples presented three different conditions: 3 wild type (RBM20+/+), 3 heterozygosity (RBM20+/-) and 3 deletion (RBM20-/-) samples. Human samples were in the two different conditions: 2 control individuals (RBM20+/+) and an individual with mutated RBM20 (RBM20-/-). Rat reads were mapped against the reference rat genome RGSC3.4, human reads were mapped against the reference human genome GRCh37. I used DEXSeq program, locally installed, to perform the exons differential analysis. The differential analysis between RBM20+/+ and RBM20-/- rat samples found 96 differentially expressed exons (p. adj < 0.05) belonging to 34 different genes. The differential analysis between RBM20+/- and RBM20-/- rat samples found 50 differentially expressed exons (p. adj < 0.05) belonging to 12 different genes. The differential analysis between RBM20+/+ and RBM20+/- rat samples found 6 differentially expressed exons (p. adj < 0.05) belonging to 3 different genes. All the exons, in all the three analyses, are up-regulated (log2foldchange > 1). The differential analysis between RBM20+/+ and RBM20-/- human samples found 1816 differentially expressed exons (p. adj < 0.05) which belongs to 657 different genes. 1744 exons are up-regulated (log2foldchange > 1). Only 8 genes differentially expressed were found common between the two species. After having produced the catalogue of human and rat exons affected by RBM20, we are now proceeding by evaluating if there are sequence differences in the regions flanking the exons affected by RBM20 to identify the characteristics (eg. the density of sites recognized by RBM20) that allow us to mark an exon as RBM20 affected or not affected. Moreover, functional studies are undergoing for transcripts expressed by one of the genes affected by RBM20.