Alternative splicing regulation by small RNAs

Alternative pre-nIRNA splicing is emerging as a key mechanism regulating gene expression in higher eukaryotes. Defects in this process cause an increasingly recognized number of human diseases. The applicants demonstrated that nuclear pre-mRNA is packaged in a large macromolecular machine - the supraspliceosome - which is involved in both the catalysis of the splicing reaction and in splice site recognition. Reversible phosphorylations of splicing regulatory factors regulate the selection of numerous alternative exons, often as part of a physiological adaptation mechanism. This proposal will test the hypothesis that dynamic changes in interactions of splicing regulators within the supraspliceosome dictate changes in alternative splicing. To address this question we combined our complementary expertise in: Cell and molecular biology; Structural biology; and Chemistry. The expertise of the Israeli partners in the isolation, biochemical characterization and structural studies by electron microscopy of supraspliceosomes (RS), and their expertise in analysis of protein-nucleic acid complexes by IJV crosslinking and gold chemistry (JS), will be combined with the expertise of the American partner (SS) in cell biology and analysis of signal-dependent alternative splicing. The synergism created by this collaboration will allow to elucidate the regulation of alternative splicing by the supraspliceosome by addressing three specific aims: (i) To determine the distribution of splicing regulatory components within supraspliceosomes; (ii) To characterize supraspliceosomes after cellular stimulation; and (iii) To study the functional consequences of cell stimulation. Our studies, which will for the first time investigate the role of cellular signals on supraspliceosome function, will enhance our understanding of a fundamental aspect of gene expression that is important to influence pathological mechanisms leading to human diseases.