ASF/SF2: Phosphorylation and RNA Binding

ASF/SF2：磷酸化和 RNA 结合

基本信息

批准号：
7907768
负责人：
GOURISANKAR GHOSH
金额：
$ 32.12万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-10 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7907768
关键词：
Active Sites Address Affect Affinity Alanine Alternative Splicing Arginine Arginine Kinase Binding Biochemical Biological Biological Assay Biological Models C-terminal Catalysis Cell Nucleus Cell physiology Cells Complex Crystallization Crystallography Dipeptides Disease Enhancers Enzymes Equilibrium Event Exons Family Foundations Gene Expression Genes Genetic Glutamic Acid Hereditary Disease Immunoglobulin M In Vitro Introns Malignant Neoplasms Maps Messenger RNA Modeling Molecular Mutate Mutation N-terminal Nuclear Oncogenic Pattern Peptides Phosphorylation Phosphorylation Site Phosphotransferases Play Property Protein Dephosphorylation Protein Family Protein Kinase Proteins Proto-Oncogenes Publishing RNA Binding RNA Recognition Motif RNA Splicing Reaction Regulation Rest Role Serine Site Slide Small Nuclear Ribonucleoproteins Specificity Spliced Genes Spliceosome Assembly Pathway Spliceosomes Structural Biochemistry Structure Testing Variant Work base design genetic regulatory protein in vivo inorganic phosphate insight mRNA Precursor malignant breast neoplasm member mutant novel protein protein interaction public health relevance reconstitution research study survival motor neuron gene

项目摘要

DESCRIPTION (provided by applicant): Alternating splicing factor/splicing factor 2 (ASF/SF2), a member of the serine-arginine (SR) protein family, plays important roles both in constitutive and alternative pre-mRNA splicing. Genetic studies show that both the absence and over-expression of ASF/SF2 dramatically change splicing pattern of key genes leading to several diseases including cancer. This suggests the vital roles of ASF/SF2 in cell physiology. Although the precise mode of how ASF/SF2 regulates splicing is not clear, it is thought that its ability to bind exonic enhancer sequences (ESE) through the N-terminal RNA recognition motifs (RRMs) is important. In addition, cycle of phosphorylation and dephosphorylation of the C-terminal region enriched in arginine-serine (RS) dipeptides during splicing is also critical for splicing. We have discovered that the RS domain of ASF/SF2 is modular in terms of phosphorylation. SR protein kinase 1 (SRPK1) processively phosphorylates only the N-terminal part of the RS domain generating hypo-phosphorylated ASF/SF2 (p-ASF/SF2). This partially phosphorylated ASF/SF2 is the substrate of another SR kinase, CLK1 for full or hyper-phosphorylation (pp-ASF/SF2). This proposal will test the hypothesis that the switch between p-ASF/SF2 and pp-ASF/SF2 is dictated by structural modularity of the RS domain, and differential phosphorylation impacts on ESE binding and hence splicing. To test our hypothesis, this proposal focuses on the elucidation of the mechanisms of i) sequential phosphorylation of ASF/SF2 by SRPK1 and CLK1, ii) the role of ASF/SF2 phosphorylation in ESE recognition and iii) the role of RS domain phosphorylation in splicing. We will use x-ray crystallography to study how SRPK1 limits phosphorylation to the N- terminal part of the RS domain and how p-ASF/SF2 phosphorylated by SRPK1 becomes a substrate of CLK1. We will perform biochemistry and structural studies to test how ASF/SF2 recognizes wild type but not oncogenic variants of ESEs. Finally, we will explore the roles of ASF/SF2 phosphorylation in both constitutive and alternative splicing in vitro, and in vivo. PUBLIC HEALTH RELEVANCE: The SR protein family of splicing factors, which regulates alternative and constitutive splicing of all pre-mRNAs by binding to exonic splicing enhancer (ESE) sequences, contain large number of serines that are processively phosphorylated by the SR protein kinase. The focus of this proposal is to understand the mechanism of SR protein, ASF/SF2, phosphorylation by SRPK1 and the role of phosphorylation in the recognition of ESE.

描述（由申请人提供）：交替剪接因子/剪接因子 2 (ASF/SF2) 是丝氨酸-精氨酸 (SR) 蛋白家族的成员，在组成型和选择性前 mRNA 剪接中发挥着重要作用。遗传学研究表明，ASF/SF2 的缺失和过度表达都会显着改变导致包括癌症在内的多种疾病的关键基因的剪接模式。这表明 ASF/SF2 在细胞生理学中的重要作用。尽管 ASF/SF2 调节剪接的精确模式尚不清楚，但人们认为其通过 N 端 RNA 识别基序 (RRM) 结合外显子增强子序列 (ESE) 的能力很重要。此外，剪接过程中富含精氨酸-丝氨酸（RS）二肽的C端区域的磷酸化和去磷酸化循环对于剪接也至关重要。我们发现 ASF/SF2 的 RS 结构域在磷酸化方面是模块化的。 SR 蛋白激酶 1 (SRPK1) 仅持续磷酸化 RS 结构域的 N 端部分，生成低磷酸化的 ASF/SF2 (p-ASF/SF2)。这种部分磷酸化的 ASF/SF2 是另一种 SR 激酶 CLK1 的底物，可实现完全磷酸化或过度磷酸化 (pp-ASF/SF2)。该提案将测试以下假设：p-ASF/SF2 和 pp-ASF/SF2 之间的转换是由 RS 结构域的结构模块性决定的，以及差异磷酸化对 ESE 结合和剪接的影响。为了检验我们的假设，本提案重点阐明以下机制：i) SRPK1 和 CLK1 连续磷酸化 ASF/SF2，ii) ASF/SF2 磷酸化在 ESE 识别中的作用，以及 iii) RS 结构域磷酸化在 ESE 识别中的作用。拼接。我们将使用X射线晶体学来研究SRPK1如何限制RS结构域N端部分的磷酸化以及SRPK1磷酸化的p-ASF/SF2如何成为CLK1的底物。我们将进行生物化学和结构研究，以测试 ASF/SF2 如何识别 ESE 的野生型而非致癌变体。最后，我们将探讨 ASF/SF2 磷酸化在体外和体内组成型剪接和选择性剪接中的作用。公共健康相关性：剪接因子 SR 蛋白家族通过与外显子剪接增强子 (ESE) 序列结合来调节所有前 mRNA 的选择性和组成型剪接，包含大量被 SR 蛋白激酶进行磷酸化的丝氨酸。本提案的重点是了解SR蛋白ASF/SF2、SRPK1磷酸化的机制以及磷酸化在ESE识别中的作用。