RNA splicing is a tightly regulated process that involves the spliceosome and additional RNA binding proteins that can repress or activate splice sites selection. Recent studies have indicated that mutations in RBM20, a gene encoding a novel ribonucleic acid - binding protein, are associated to human dilated cardiomyopathy (DCM). RBM20 regulates alternative splicing of expressed genes that have a key role in cardiac function, including ion homeostasis, sarcomere biology, and signal transduction. The functional motifs of the RBM20 protein have been poorly investigated.The focus of this study is to characterize the protein domains that contribute to the nuclear function of RBM20. Predictive in silico analysis of the translated RBM20 gene identifies an RNA recognition motif (RRM motif), a serine /arginine (RS) domain and Zn2+ finger domains. We have produced GFP-RBM20 fusion proteins in order to map the functional domains of the protein that contribute to subcellular distribution. We have produced truncated mutants of the RBM20 proteins and analyzed separately in immunofluorescence assays in transfected cells. We identified a region necessary and sufficient to nuclear localization of RBM20 protein that maps between the RRM and the RS domain. Actually we are producing RBM20 mutant proteins in order to characterize the nuclear localization signal (NLS). Further structural and functional characterization of RBM20 may contribute to understand the molecular pathogenesis of familiar DCM.
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