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，这是一个很大的惊喜，因为人类蛋白质的估计数量约为130，000，大多数科学家假设人类基因的数量相似。这种差异只有在一个基因可以产生许多蛋白质的情况下才能解释。然后很明显，RNA剪接是细胞中的一个主要调控事件。选择性剪接允许通过以许多不同的方式剪接前体信使RNA来从单个基因产生许多信使RNA并因此产生蛋白质。这个过程大大增加了人类蛋白质的多样性，来自有限数量的基因。它受到RNA结合蛋白（称为剪接因子）的高度调节，其调节的缺陷导致大量疾病，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。选择性剪接调节由细胞外信号通过细胞信号传导途径调节，所述细胞信号传导途径通常修饰靶蛋白上的特定氨基酸以调节其功能。一个典型的例子是剪接因子Sam 68，其在大量癌症中过表达，并且其在选择性剪接中的功能受到信号传导途径的强烈调节。已经鉴定了响应于信号传导途径的Sam 68的许多修饰，例如磷酸化、甲基化和乙酰化。然而，很少有人知道的分子机制，管理这些法规和调制。我们最近揭示了Sam 68与其RNA靶标复合的结构，并提出了Sam 68在选择性剪接调控中的作用机制模型。在本研究中，我们将通过分析细胞信号通路对Sam 68选择性剪接功能的影响，进一步研究涉及Sam 68的调控网络。为了这个目的，我们将解决三个互补的问题：1-哪些氨基酸的Sam 68在细胞中被修改？2-Sam 68的哪些修饰发生在对EGFR通路的反应中，EGFR通路通常与癌症进展相关？3-这些修饰对Sam 68结构、RNA结合、定位和可变剪接功能有什么影响？这将使我们能够（i）研究每种氨基酸修饰对Sam 68结构、动力学、定位、RNA结合和功能的作用机制，以及（ii）评估Sam 68在EGFR信号通路中的作用，该通路通常在疾病如癌症中失调。

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.