MOLECULAR CLONING, CHARACTERIZATION AND EXPRESSION ANALYSIS OF THE RBM20, A NOVEL RIBONUCLEOPROTEIN GENE ASSOCIATED TO FAMILIAL DILATED CARDIOMYOPATHY

Alternative splicing of pre-messenger RNA is a tightly regulated process that involves the spliceosome and additional RNA binding proteins that can repress or activate splice site selection. More than 15% of human genetic diseases have been associated to mutations in cis-acting splice elements, but very few of them are attributable to trans-factors that control alternative splicing. The effects of these mutations that involve the basal splicing machinery and regulators of alternative splicing have been associated with autism spectrum disorders, amyotrophic lateral sclerosis and cancer. In heart and skeletal muscle, alternative splicing plays a critical role in muscle function. In both systems, splicing transition occurs in tightly grouped temporal ways, partially involving the same proteins. A direct involvement of splicing regulators to cardiac diseases has been proven recently for two RNA binding motif (RBM) protein, the RBM25 and the RBM20. This experimental study is focused on the RBM20 protein. RNA binding motif protein type 20 (RBM20) is a trans-acting factor expressed preferentially in heart tissue, which regulates alternative splicing of gene that have a key role in cardiac function, including ion homeostasis, sarcomere biology and signal transduction. Mutations in the RBM20 gene are linked to familial dilated cardiomyopathy and the most of them alters residues in the arginine-serine domain of the protein. The functional motifs of the RBM20 protein have been predicted by sequence homology, but poorly functionally characterized. In the present study by cloning the cardiac isoform of human RBM20 and the mouse RBM20 full length cDNA I provided functional annotations of structural domains required for the RBM20 nuclear localization. Confocal microscopy analysis showed that RBM20 had a punctuate localization in nuclear speckles, which is typical of RS splicing factors. By producing expressing vectors for truncated proteins and comparing their subcellular distribution we identified the sequences necessary for RBM20 full nuclear retention. The region overlaps both RNA binding motif and arginine-serine domain. The sequence is conserved in many species but belongs only to RBM20 protein orthologs. These studies demonstrate a specific evolutionary selection for post-transcriptional regulation in heart, highlighting RBM20 as a key factor for regulation of splicing events required for cardiac function.