ASF/SF2: Phosphorylation and RNA Binding

ASF/SF2：磷酸化和 RNA 结合

• 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
• 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

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(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，在结构性和选择性前mRNA剪接中发挥重要作用。遗传学研究表明，ASF/SF2的缺失和过表达都会极大地改变关键基因的剪接模式，从而导致包括癌症在内的多种疾病。这提示了ASF/SF2在细胞生理学中的重要作用。虽然ASF/SF2调控剪接的确切模式尚不清楚，但人们认为它通过N-末端RNA识别基序(RRMS)结合外显子增强序列(ESE)的能力是重要的。此外，在剪接过程中富含精氨酸-丝氨酸(RS)二肽的C-末端区域的磷酸化和去磷酸化的循环也是剪接的关键。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)只对RS结构域的N-末端进行磷酸化，产生低磷酸化的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，用于完全或过度磷酸化(pp-ASF/SF2)。这一建议将检验以下假设，即p-ASF/SF2和pp-ASF/SF2之间的切换是由RS结构域的结构模块决定的，而差异磷酸化对ESE结合从而剪接产生影响。为了验证我们的假设，这一建议集中在以下机制的阐明：i)SRPK1和CLK1对ASF/SF2的顺序磷酸化，ii)ASF/SF2磷酸化在ESE识别中的作用，以及iii)RS结构域磷酸化在剪接中的作用。我们将使用X射线结晶学来研究SRPK1如何将磷酸化限制在RS结构域的N端部分，以及由SRPK1磷酸化的p-ASF/SF2如何成为CLK1的底物。我们将进行生化和结构研究，以测试ASF/SF2如何识别ESES的野生型而不是致癌变体。最后，我们将在体外和体内探索ASF/SF2磷酸化在结构性剪接和选择性剪接中的作用。与公共健康相关：剪接因子的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mRNAs的选择性和结构性剪接，包含大量丝氨酸，这些丝氨酸被SR蛋白激酶连续磷酸化。本研究的重点是了解SR蛋白、ASF/SF2的机制、SRPK1的磷酸化以及磷酸化在识别ESE中的作用。