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

人类基因组计划确定了20-25,000个人类基因，这令人大吃一惊，因为估计人类蛋白质的数量约为130,000个，而大多数科学家假设人类基因的数量相似。只有当一个基因可以产生许多蛋白质时，这种差异才能得到解释。然后，事情变得很清楚，替代的RNA剪接是细胞中的一个主要调节事件。选择性剪接允许通过许多不同的方式剪接前体信使RNA来生产许多信使RNA，从而从单个基因中产生蛋白质。这一过程极大地增加了人类有限基因中蛋白质的多样性。它受被称为剪接因子的RNA结合蛋白的高度调控，其调控缺陷会导致大量的疾病，包括遗传疾病和癌症，通常是由于剪接因子的过度表达或突变。选择性剪接调控是由细胞外信号通过细胞信号通路调节的，这些信号通路通常会修改靶蛋白上的特定氨基酸来调节其功能。一个典型的例子是剪接因子Sam68，它在大量癌症中过度表达，其在选择性剪接中的功能受到信号通路的强烈调控。Sam68的许多修饰已被确定为响应信号通路，如磷酸化、甲基化和乙酰化。然而，人们对控制这些调节和调节的分子机制知之甚少。我们最近揭示了Sam68与其核糖核酸靶标的复合体结构，并提出了Sam68的S选择性剪接调控机制模型。在该模型中，我们将通过分析细胞信号通路对Sam68选择性剪接功能的影响来进一步研究涉及Sam68的调控网络。为此，我们将解决三个互补的问题：1-Sam68的哪些氨基酸在细胞中被修饰？2-Sam68的哪些修饰发生在经常与癌症进展相关的EGFR途径中？3-这些修饰对Sam68的结构、RNA结合、定位和选择性剪接功能有什么影响？这将使我们能够(I)研究每个氨基酸修饰对Sam68的结构、动态、定位、RNA结合和功能的作用机制，以及(Ii)评估Sam68在通常在癌症等疾病中被解除调控的EGFR信号通路中的作用。

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.