Mechanisms of enhancer dependent splice-site activation

增强子依赖性剪接位点激活机制

• 批准号: 7892830
• 负责人: Klemens J Hertel
• 金额: $ 14.03万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-13 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7892830
• 关键词: 3' Splice Site; 5' Splice Site; ATP Hydrolysis; Alternative Splicing; Architecture; Biochemical; Biological Assay; Cell Cycle; Classification; Commit; Complement; Complex; DNA Polymerase II; Defect; Enhancers; Event; Exons; Funding; Gene Expression; Genes; Goals; Growth; Hereditary Disease; Human Genetics; Human Genome; Immunoprecipitation; Introns; Investigation; Kinetics; Lead; Length; Link; Measures; Molecular; Mutation; Pattern; Phase; Positioning Attribute; Probability; Process; Proteins; RNA Interference; RNA Splicing; RNA chemical synthesis; Reaction; Regulation; Relative (related person); Research Proposals; Role; SF3a120; Sequence Analysis; Signal Transduction; Site; Spliceosomes; Staging; Testing; U2 Small Nuclear Ribonucleoprotein; cancer type; cell type; combinatorial; human disease; improved; insight; mRNA Precursor; research study

DESCRIPTION (provided by applicant): Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. Defects in splicing lead to many human genetic diseases, and pre-mRNAs containing multiple introns and exons can be alternatively spliced in a cell type, cell cycle, or developmentally regulated manner by joining different pairs of 5' and 3' splice sites. Insights into the basic mechanisms of pre-mRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. The overall goal of this research proposal is to understand the mechanisms involved in splice-site recognition and pairing of pre-mRNAs. During the previous funding period, we have developed quantitative assays to provide new insights into the mechanisms of splice-site pairing. In the next phase of investigation, we propose to determine the ?molecular events that lock splice sites into a pairing position and to analyze how the combinatorial contribution of multiple splicing signals influence exon inclusion. Specifically, we will determine the biochemical steps that lead to splice-site pairing in A complex (Aim 1). We will test the hypothesis that ATP hydrolysis during A complex formation drives the irreversible juxtaposition of alternative splice sites or exons. In Aim 2 we will determine how the spliceosome executes commitment to splice-site pairing. We will use immuno-depletion and RNAi approaches to test the hypothesis that a subset of U2 snRNP components and associated proteins (CUS2/Tat-SF-1, Prp5, SF3a120, and UAP56) is necessary for irreversible splice-site pairing. Aim 3 describes a systematic and quantitative approach to determine how the probability of exon definition and inclusion is influenced by the combinatorial contributions of variable splice sites, enhancers, silencers, and the exon/intron architecture. We will test the hypothesis that measures of exon inclusion can be quantitated and used to improve the predictability of constitutive and alternative splicing within the human genome. These experiments are important because 1) the commitment to splice-site pairing constitutes arguably the most crucial step during the splicing reaction because it determines the splicing patterns of pre- mRNAs, and because 2) a quantitative framework of combinatorial exon recognition will elucidate mechanisms of splicing regulation and allow to predict the intrinsic pattern of splicing from sequence analysis.

描述（由申请人提供）：MRNA剪接是表达大多数后生基因所需的基本过程。剪接中的缺陷导致许多人类遗传疾病，并且可以通过连接不同的5'和3'剪接位点，以细胞类型，细胞周期或发育调节的方式替代含有多个内含子和外显子的MRNA。因此，对MRNA剪接和剪接位点识别的基本机制的见解是理解受调节的基因表达和人类疾病的基础。该研究建议的总体目标是了解与剪接站点识别和MRNA配对有关的机制。在上一个资金期间，我们开发了定量测定，以提供有关剪接站点配对机制的新见解。在下一阶段调查中，我们建议确定将剪接位点锁定在配对位置的分子事件，并分析多个剪接信号的组合贡献如何影响外显子包容性。具体而言，我们将确定在复合物中导致剪接位点配对的生化步骤（AIM 1）。我们将测试以下假设：复杂形成过程中ATP水解驱动替代剪接位点或外显子的不可逆并置。在AIM 2中，我们将确定剪接体如何执行剪接站点配对的承诺。我们将使用免疫缺乏和RNAI方法来检验以下假设：U2 SNRNP成分和相关蛋白的子集（CUS2/TAT-SF-1，PRP5，SF3A120和UAP56）对于不可逆的Splice Splice Spite配对是必需的。 AIM 3描述了一种系统和定量的方法，以确定外显子定义和包容的概率如何受到可变剪接位点，增强剂，消音器和外显子/内含子体系结构的组合贡献的影响。我们将检验以下假设：外显子包容的测量可以进行定量，并用于改善人类基因组中构型和替代剪接的可预测性。这些实验很重要，因为1）对剪接位点配对的承诺可以说是剪接反应过程中最关键的一步，因为它决定了前mRNA的剪接模式，并且2）组合外显子识别的定量框架将阐明拼接调控机制和允许从序列分析中进行启动分析。