Role of protein phosphorylation in RNA splicing

蛋白质磷酸化在 RNA 剪接中的作用

• 批准号: 6845233
• 负责人: JOSEPH ADAMS
• 金额: $ 24.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845233
• 关键词: RNA splicing; X ray crystallography; cell nucleus; cofactor; enzyme activity; enzyme structure; enzyme substrate; molecular assembly /self assembly; molecular site; phosphorylation; protein kinase; protein structure function; site directed mutagenesis; spliceosomes; RNA splicing; X ray crystallography; cell nucleus; cofactor; enzyme activity; enzyme structure; enzyme substrate; molecular assembly /self assembly; molecular site; phosphorylation; protein kinase; protein structure function; site directed mutagenesis; spliceosomes

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.

描述（由申请人提供）：近年来，核的传统观点发生了很大变化。如今，人们认识到，与基因表达相关的许多过程都定位在一个非常复杂的动态核框架内。大分子复合物对于RNA剪接重要的重要性是在核中组装的是生物学中的重要焦点区域。剪接中的错误与一系列人类神经退行性疾病和肿瘤发生有关，强调了RNA处理事件所需的高水平特异性。最近，很明显，蛋白质因子在确定拼接组装和剪接位点选择方面起着关键作用。一类称为SR蛋白的剪接因子其异常精氨酸二二肽重复调节几个阶段的剪接。这些因素被SR蛋白激酶（SRPKS）磷酸化，对酶线内蛋白质功能具有深远影响的修饰。尽管磷酸化具有生物学意义，但对于SR蛋白如何变得磷酸化以及特定的磷酸化事件或模式与剪接的控制有关。该提案的目标将包括鉴定人类SRPK1的底物SR蛋白ASF/SF2中的丝氨酸磷酸化位点。这些丝氨酸如何使用位点替代突变体，放射自显影和质谱法研究将研究这些丝网。这些丝氨酸对于控制剪接的重要性将在大型大分子复合物中使用体外剪接测定法解决。启动磷酸化的因素将使用缺失突变体和底物嵌合体研究。尽管所有SRPK都是组成型活性的，但激活的结构机理可能是特定于物种的。这种现象的起源将使用诱变和X射线晶体学来解决。该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。