Mechanisms of enhancer dependent splice-site activation

描述（由申请人提供）：Pre-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，含有多个内含子和外显子的pre- mrna可以通过连接不同对的5‘和3’剪接位点，以细胞类型、细胞周期或发育调节的方式进行选择性剪接。因此，了解pre-mRNA剪接和剪接位点识别的基本机制是理解受调控基因表达和人类疾病的基础。本研究计划的总体目标是了解pre- mrna的剪接位点识别和配对机制。在之前的资助期间，我们开发了定量分析，为剪接位点配对机制提供了新的见解。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复合体中剪接位点配对的生化步骤（目的1）。我们将测试在A复合物形成过程中ATP水解驱动替代剪接位点或外显子的不可逆并置的假设。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。我们将使用免疫消耗和RNAi方法来验证U2 snRNP成分和相关蛋白（CUS2/Tat-SF-1, Prp5， SF3a120和UAP56）对不可逆剪接位点配对所必需的假设。目的3描述了一种系统和定量的方法，以确定外显子定义和包含的概率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。我们将测试外显子包含的测量可以量化并用于提高人类基因组内组成剪接和替代剪接的可预测性的假设。这些实验很重要，因为1)剪接-位点配对是剪接反应中最关键的一步，因为它决定了pre- mrna的剪接模式；2)组合外显子识别的定量框架将阐明剪接调节机制，并允许从序列分析中预测剪接的内在模式。

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.