Structural and functional investigations on the consequences of Sam68 post-translational modifications on alternative splicing regulation

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The identification of 20-25,000 human genes by the human genome project came as a big surprise since the estimated number of human proteins is around 130,000 and most scientists assumed a similar number of human genes. This discrepancy can only be explained if one single gene can generate many proteins. It then became clear that alternative RNA splicing is a major regulatory event in cells. Alternative splicing allows for the production of many messenger RNAs and consequently proteins from a single gene by splicing the precursor messenger RNA in many different ways. This process greatly increases the diversity of proteins in humans from a limited number of genes. It is highly regulated by RNA binding proteins, called splicing factors, and defects in its regulation lead to a large number of diseases, including genetic disorders and cancer, often due to overexpression or mutations of splicing factors. Alternative splicing regulation is modulated by extracellular signals through cell signalling pathways that typically modify specific amino acids on target proteins to modulate their functions. A typical example is the splicing factor Sam68, which is overexpressed in a large number of cancers and whose function in alternative splicing is strongly modulated by signalling pathways. Many modifications of Sam68 in response to signalling pathways have been identified, such as phosphorylation, methylation and acetylation. However, very little is known about the molecular mechanisms that govern these regulations and modulations. We have recently revealed the structure of Sam68 in complex with its RNA target and proposed a model for Sam68's mechanism of action in alternative splicing regulation.In this proposal, we will further investigate the regulatory network involving Sam68 by analyzing the effects of cell signalling pathways on Sam68 alternative splicing functions. To that aim, we will address three complementary questions:1- Which amino acids of Sam68 are modified in cells?2- Which modifications of Sam68 occur in response to EGFR pathway, a pathway that is often associated with cancer progression?3- What are the consequences of these modifications on Sam68 structure, RNA binding, localization and alternative splicing function?This will allow us to (i) investigate the mechanism of action of each amino acid modifications on Sam68 structure, dynamic, localization, RNA binding and function, and (ii) evaluate the contribution of Sam68 in the EGFR signalling pathways that is often deregulated in diseases such as cancer.