MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE RIBONUCLEOPROTEIN RBM20: EVIDENCE OF ITS INVOLVEMENT IN REGULATING ALTERNATIVE SPLICING OF FORMIN FHOD3

Alternative splicing is an extremely regulated post-transcriptional process that enhances the genome expression complexity. Very few human tissues specific regulators of alternative splicing have been identified and functionally characterized. Genetic linkage and gene-mapping studies have demonstrated that mutations in an heart specific ribonucleoprotein known as RNA Binding protein 20 (RBM20) cause dilated cardiomyopathy. In the present thesis the molecular and functional characterization of RBM20 is presented and discussed. RBM20 open reading frame from human cardiac tissue has been cloned in expressing vectors. Domains that are necessary for nuclear distribution have been identified by dissection of the RBM20 functional domains through the production and analyses of the sub-cellular distribution of GFP- RBM20 truncated peptides. RBM20 tissue specificity was analyzed in human tissues and cell lines. The involvement of RBM20 in alternative splicing events of tissues regulated exons was analyzed, selecting the FHOD3 regulator of actin filaments as RBM20 specific transcript target. Alternative splicing switch in FHOD3 transcripts was observed in presence of RBM20 over-expression in transfected cells. FHOD3 interaction with RBM20 was demonstrated by RIP experiment. The results have produced evidences that RBM20 is a key factor in determining the issue- splicing signature of FHOD3. This study contributes to the knowledge of the extensive mechanisms that allow to the heart development and, when altered, may lead to molecular defects in dilated cardiomyopathy.