MECHANISMS OF ENHANCER DEPENDENT SPLICE SITE ACTIVATION

增强子依赖性剪接位点激活机制

• 批准号: 6628936
• 负责人: Klemens J Hertel
• 金额: $ 22.05万
• 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 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6628936
• 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 前体 mRNA 的剪接位点被剪接体识别并确定 RNA 聚合酶 II 的前 mRNA 加工与转录如何耦合 (pol II) 促成了这一决定。总体策略是表征 体外前 mRNA 剪接，因为它可以精确控制反应 状况。中心假设是复合物在外显子上组装 剪接增强子（ESE）有助于招募剪接体 相邻的内含子，并且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 建议的： 了解 fru mRNA 前体阳性控制所涉及的机制 选择性拼接。假设 fru ESE 增加 U1 snRNP 与受调控的 5' 剪接位点结合。我们将深入研究 分析控制剪接增强子的结构和功能 fru 女性特异性 5' 剪接位点的活性。我们将研究的角色 fru ESE在剪接体组装中的作用，并分析其位置效应 dsx/fru ESE 复合物激活 3' 或 5' 剪接位点。 表征 dsx 拼接增强器的架构和组装 复杂的。假设 dsx ESE 复合体的组装需要 高度合作互动。我们建议进行详细的结构 分析 dsx ESE 上形成的蛋白质复合物。我们将确定 Tra、Tra2 和 RBP1 在剪接机器募集中的功能， 定义 dsx ESE 异三聚体的协同组装和化学计量 复合物，绘制复合物内蛋白质-蛋白质相互作用的图谱，并表征 Tra 和 Tra2 的突变体。 确定聚合酶 II 的前 mRNA 转录如何影响 ESE 依赖性 拼接。我们建立了体外测定来分析效率 与转录结合时发生前 mRNA 剪接。我们将使用该测定来测试 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一步 在转录过程中，剪接机制与新生的前 mRNA 相关联。