Role of protein phosphorylation in RNA splicing

描述(由申请人提供)：近年来，对核的传统观点发生了很大的变化。今天，人们认识到，与基因表达相关的许多过程都局限在一个非常复杂的、动态的核框架内。如何在核内组装对RNA剪接至关重要的大分子复合体是生物学中的一个重要研究领域。剪接错误与一系列人类神经退行性疾病和肿瘤发生有关，这突显了RNA加工事件所需的高度特异性。最近，蛋白质因素在决定剪接组装和剪接位点选择中的关键作用已经变得清晰起来。一类被称为SR蛋白的剪接因子因其不寻常的精氨酸-丝氨酸二肽重复而在几个阶段调节剪接。这些因子被SR蛋白激酶(SRPKs)多重磷酸化，这种修饰对剪接体中的蛋白质功能有深远的影响。尽管磷酸化具有生物学意义，但人们对SR蛋白如何被磷酸化以及特定的磷酸化事件或模式如何与剪接的控制联系在一起知之甚少。这项提案的目标将包括鉴定SR蛋白ASF/SF2中的丝氨酸磷酸化位点，SR蛋白是人类SRPK1的底物。这些丝氨酸是如何被磷酸化的，将使用位点替换突变体、放射自显影和质谱学来研究。这些丝氨酸对控制剪接的重要性将通过体外剪接试验在大分子复合体中得到解决。启动磷酸化的因素将使用缺失突变体和底物嵌合体来研究。虽然所有SRPKs都是结构性活性的，但激活的结构机制可能是物种特有的。这一现象的起源将用诱变和X射线结晶学来解决。本提案中概述的实验旨在充分表征一类新的剪接因子及其酶调节因子。

DESCRIPTION (provided by applicant): The traditional view of the nucleus has changed substantially in recent years. Today, it is recognized that many of the processes associated with gene expression are localized within a very complex, dynamic nuclear framework. How macromolecular complexes important for RNA splicing are assembled within the nucleus is an important focus area in biology. Errors in splicing have been linked to an array of human neurodegenerative diseases and tumorigenesis underscoring the high level of specificity required for RNA processing events. Recently, it has become clear that protein factors play a key role in determining splicesome assembly and splice site selection. One class of splicing factors known as SR proteins for their unusual arginine-serine dipeptide repeats regulates splicing at several stages. These factors become multiply phosphorylated by SR protein kinases (SRPKs), modifications that have profound effects on protein function within the splicesome. Despite the biological significance of phosphorylation, little is known about how the SR proteins become phosphorylated and how specific phosphorylation events or patterns are linked to the control of splicing. The goals of this proposal will include the identification of the serine phosphorylation sites in the SR protein ASF/SF2, a substrate for human SRPK1. How these serines get phosphorylated will be studied using site replacement mutants, autoradiography and mass spectrometry. The importance of these serines for the control of splicing will be addressed in large macromolecular complexes using an in vitro splicing assay. Factors which initiate phosphorylation will be studied using deletion mutants and substrate chimeras. While all SRPKs are constitutively active, it is likely that the structural mechanism of activation is species specific. The origins of this phenomenon will be addressed using mutagenesis and X-ray crystallography. The experiments outlined in this proposal are designed to fully characterize a novel class of splicing factors and their enzymatic regulators.