INVOLVEMENT OF RAVER1RIBONUCLEOPROTEIN IN SPLICING EVENTSDURING C2C12 MYOBLAST DIFFERENTIATION

Large-scale expressed sequence tag and genome-wide analyses estimate that the majority of human genes undergo alternative splicing with a differential tissue distribution. More than 15% of human genetic diseases are associated to mutations in the consensus splice sites and disruption of splicing regulatory networks contributes to various diseases. Alternative splicing of pre-messenger RNA represents, consequently, an intensive post-transcriptional regulatory activity that involves several RNA binding proteins and splicing regulators. Polypyrimidine tract binding protein (PTB) and its paralog brPTB play a well established role as negative splicing regulators in differentiated tissues. Raver1 and Raver2 are proteins interacting with PTB but their role in splicing events are not understood. During muscle development PTB and brPTB regulate alternative splicing of several tissue specific exons. In the present study it has been investigated the contribution of Raver1 to splicing events occurring during myoblasts differentiation of C2C12 cells. The expression, subcellular localization and functional role of Raver1 in splicing events of selected transcripts during C2C12 myogenic process have been analyzed. The results show that Raver1 protein is expressed in proliferating undifferentiated cells as well as in differentiating myoblast cells. Confocal microscopy analyses show that Raver1 diffuses in the cytoplasm from the nucleus, localizing in polarized cytoplasmic area during myoblasts differentiation. Over-expression of Raver1 during myogenesis affects alternative splicing of regulated exons in protease calpain 3 (CAPN3) and phosphatase myotubularin-related protein 1 (MTMR1). RNA-Immunoprecipitation experiments confirm that Raver1 can bind CAPN3 and MTMR1 pre-mRNAs in regions containing putative PTB recognition sequences. Raver2 expression was also analyzed in human tissues, showing that in contrast to what happens in mouse muscle tissue, Raver2 gene is expressed in human skeletal muscle tissue. These studies may shed new light on the role of ribonucleopreoteins in the posttranscriptional regulation events that occur during muscle development in both nuclear and cytoplasmic compartments.