Expression, localization and functional role of ribonucleoproteins in myoblasts 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. They belong principally to SR (serine/arginine rich proteins) and hnRNP (heterogeneous nuclear ribonucleoprotein particles) proteins. Both types can act as enhancers or repressors of alternative splicing helping in defining cis-regulatory elements positioned either in the exon or in the intron sequences. Dynamic antagonism between members of the SR and hnRNP protein families is demonstrated to be important for determining a number of alternative splicing patterns. Among the hnRNPs that have been implicated in regulating the alternative splicing events with tissue-specificity, polypyrimidine tract binding protein (PTB) and its paralog nPTB play a well established role as negative splicing regulators. We have previously characterized PTB, its paralog nPTB and Raver1 (a PTB co-repressor factor) gene expression in human tissues and cell lines (1-3). We showed that alternative spliced nPTB transcripts are distributed with a tissue specificity involving brain and skeletal muscle. During muscle development PTB regulates exons splicing in several specific transcribed genes. In the present study we are investigating the contribution of ribonucleoproteins to splicing events occurring during myoblasts differentiation. We are analyzing the expression, subcellular localization and functional role of PTB, Raver1 and Rbm20 (a SR protein which mutations are associated to familial dilated cardiomyopathy) during C2C12 myoblast differentiation. Preliminary results show that Raver1 expression is reduced both at transcriptional and translational levels during differentiation and that its overexpression can affect exon inclusion in calpain 3 (CAPN3) and myotubularin-related protein 1 (MTMR1). Confocal microscopy analyses show that Raver1 diffuses in the cytoplasm from the nucleus, localizing in polarized cytoplasmic area during myoblasts differentiation. These studies may shed new light on the role of ribonucleopreoteins in the post-transcriptional regulation events that occur during muscle development in both nuclear and cytoplasmic compartments. 1. Romanelli et al. Biochim Biophys Acta; 2001, 1520: 85 – 88. 2. Romanelli et al. Gene; 2005, 356:11-18. 3 Romanelli et al. Gene; 2007, 405: 79 – 87