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）：前体mRNA剪接是大多数后生动物基因表达所需的基本过程。剪接缺陷导致许多人类遗传疾病，并且含有多个内含子和外显子的前mRNA可以通过连接不同的5'和3'剪接位点对以细胞类型、细胞周期或发育调节的方式进行选择性剪接。因此，了解前体mRNA剪接和剪接位点识别的基本机制对于理解受调控的基因表达和人类疾病至关重要。这项研究的总体目标是了解剪接位点识别和配对的前体mRNA的机制。在上一个资助期间，我们开发了定量分析，为剪接位点配对的机制提供新的见解。在下一阶段的调查中，我们建议确定？将剪接位点锁定到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含。具体来说，我们将确定导致A复合物中剪接位点配对的生化步骤（目的1）。我们将测试的假设，ATP水解过程中A复合物的形成驱动器的不可逆并列的选择性剪接位点或外显子。在目标2中，我们将确定剪接体如何执行剪接位点配对的承诺。我们将使用免疫耗竭和RNAi方法来检验U2 snRNP组分和相关蛋白（U2/Tat-SF-1，Prp 5，SF 3a 120和UAP 56）的子集对于不可逆的剪接位点配对是必要的这一假设。目的3描述了一个系统的和定量的方法来确定外显子定义和包含的概率是如何受到可变剪接位点，增强子，沉默子和外显子/内含子结构的组合贡献的影响。我们将测试的假设，外显子包含的措施可以量化，并用于提高人类基因组内的组成性和选择性剪接的可预测性。这些实验是重要的，因为1）对剪接位点配对的承诺可以说是剪接反应期间最关键的步骤，因为它决定了前mRNA的剪接模式，并且因为2）组合外显子识别的定量框架将阐明剪接调控的机制并允许从序列分析预测剪接的内在模式。