Role of protein phosphorylation in RNA splicing

描述（由申请人提供）：近年来，细胞核的传统观点发生了重大变化。今天，人们认识到，许多与基因表达相关的过程都定位在一个非常复杂的，动态的核框架内。对于RNA剪接重要的大分子复合物如何在细胞核内组装是生物学中的一个重要焦点领域。剪接错误与一系列人类神经退行性疾病和肿瘤发生有关，这强调了RNA加工事件所需的高水平特异性。最近，人们已经清楚，蛋白质因子在决定剪接体组装和剪接位点选择中起着关键作用。一类剪接因子因其不寻常的丝氨酸-丝氨酸二肽重复序列而被称为SR蛋白，其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，这种修饰对剪接体内的蛋白质功能有深远的影响。尽管磷酸化的生物学意义，很少有人知道SR蛋白是如何磷酸化的，以及特定的磷酸化事件或模式是如何与剪接的控制。该提案的目标将包括在SR蛋白ASF/SF 2，人SRPK 1的底物的丝氨酸磷酸化位点的鉴定。这些丝氨酸是如何磷酸化的将使用位点置换突变体，放射自显影和质谱法进行研究。这些丝氨酸的剪接控制的重要性将在大分子复合物中使用体外剪接测定来解决。启动磷酸化的因素将使用缺失突变体和底物嵌合体进行研究。虽然所有SRPKs都是组成型活性的，但激活的结构机制可能是物种特异性的。这种现象的起源将使用诱变和X射线晶体学来解决。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剂。

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.