MECHANISMS OF ENHANCER DEPENDENT SPLICE SITE ACTIVATION

增强子依赖性剪接位点激活机制

• 批准号: 6845708
• 负责人: Klemens J Hertel
• 金额: $ 21.96万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6845708
• 关键词: DNA directed RNA polymerase; RNA binding protein; RNA splicing; enzyme activity; genetic enhancer element; genetic transcription; immunoprecipitation; protein structure function; spliceosomes; tissue /cell culture

DESCRIPTION (from the application): Our long term goals are to understand how splice sites for pre-mRNAs are recognized by the spliceosome and to determine how the coupling of pre-mRNA processing with transcription by RNA polymerase II (pol II) contributes to this decision. The general strategy is to characterize pre-mRNA splicing in vitro, as it allows precise control over the reaction conditions. The central hypothesis is that complexes assembled on exonic splicing enhancers (ESE) assist in the recruitment of the spliceosome to adjacent introns, and that the elongation transcription machinery aides this assembly by interacting with splicing factors. Three specific aims are proposed: Understand the mechanisms involved in the positive control of fru pre-mRNA alternative splicing. It is hypothesized that the fru ESE increases U1 snRNP binding to the regulated 5' splice site. We will carry out an in-depth study to analyze the structure and function of a splicing enhancer that controls the activity of the fru female specific 5' splice site. We will examine the role of the fru ESE in spliceosomal assembly, and analyze the positional effects of the dsx/fru ESE complex in activating a 3' or a 5' splice site. Characterize the architecture and assembly of the dsx splicing enhancer complex. It is hypothesized that the assembly of the dsx ESE complex requires highly cooperative interactions. We propose to carry out a detailed structural analysis of the protein complex formed on the dsx ESE. We will determine the function of Tra, Tra2, and RBP1 in the recruitment of the splicing machinery, define the cooperative assembly and stoichiometry of the dsx ESE heterotrimeric complex, map protein-protein interactions within the complex, and characterize mutants in Tra and Tra2. Determine how transcription of pre-mRNAs by polymerase II affects ESE dependent splicing. We have established an in vitro assay to analyze the efficiency of pre-mRNA splicing when coupled to transcription. Using this assay we will test the hypothesis that the level of C-terminal domain (CTD) phosphorylation influences ESE dependent splice site activation and determine at which step during transcription the splicing machinery associates with nascent pre-mRNA.

描述（来自应用程序）：我们的长期目标是了解如何 前mRNA的剪接位点被剪接体识别， 前体mRNA加工与RNA聚合酶II转录的耦合 (pol（二）促成了这一决定。总的策略是描述 前体mRNA体外剪接，因为它允许精确控制反应 条件中心假设是，复合物组装在外显子上， 剪接增强子（ESE）有助于剪接体的募集， 相邻的内含子，而延伸转录机制有助于这一点， 通过与剪接因子相互作用进行组装。三个具体目标是 提议的： 了解fru前体mRNA阳性调控的机制 选择性剪接据推测，fru ESE增加了U1 snRNP 与受调节的5 ′剪接位点结合。我们会进行深入研究， 分析剪接增强子的结构和功能， FRU雌性特异性5 ′剪接位点的活性。我们将研究 在剪接体组装中的位置效应，并分析了 dsx/fru ESE复合物在激活3'或5'剪接位点中的作用。 描述dsx剪接增强子的结构和组装 复杂.假设dsx ESE复合物的组装需要 高度合作的互动。我们建议进行详细的结构设计， 在dsx ESE上形成的蛋白质复合物的分析。康贝特人将以 Tra、Tra 2和RBP 1在剪接机制募集中的功能， 定义dsx ESE异源三聚体的协同组装和化学计量 复合物，映射复合物内的蛋白质-蛋白质相互作用，并表征 Tra和Tra 2中的突变体。 确定聚合酶II如何转录前mRNA影响ESE依赖性 拼接我们已经建立了一种体外试验来分析 前体mRNA剪接与转录偶联。我们将使用该分析测试 假设C-末端结构域（CTD）磷酸化水平 影响ESE依赖性剪接位点激活并确定在哪个步骤 在转录过程中，剪接机制与新生的前mRNA结合。