MECHANISMS OF ENHANCER DEPENDENT SPLICE SITE ACTIVATION

描述(来自应用程序)：我们的长期目标是了解如何 剪接体识别前mRNAs的剪接位点并确定 RNA聚合酶II对前mRNA的加工与转录的耦合 (POL II)促成了这一决定。一般的策略是将 体外剪接前信使核糖核酸，因为它允许精确控制反应 条件。中心假设是在外显子上组装的复合体 剪接增强剂(ESE)有助于招募剪接体以 相邻的内含子，伸长转录机制帮助了这一点 通过与剪接因子相互作用进行组装。三个具体目标是 建议： 了解FRU-Pre-mRNA正向调控的机制 另一种拼接。假设FRU ESE增加了U1 SNRNP 结合到受调控的5‘剪接位点。我们将深入研究 分析控制剪接增强剂的结构和功能 FRU雌性特异的5‘剪接位点的活性。我们将研究这一角色 在剪接体组装中的位置效应，并分析位置效应。 激活3‘或5’剪接位点中的DsX/FRU ESE复合体。 描述DSX剪接增强器的体系结构和组装 很复杂。据推测，组装DSX ESE综合体需要 高度合作的互动。我们建议进行一项详细的结构设计 DSSX ESE上形成的蛋白质复合体的分析。我们将确定 TrA、Tra2和RBP1在剪接机械招募中的作用， 确定DSX ESE杂三聚体的协同组装和化学计量比 复合体，绘制复合体内的蛋白质-蛋白质相互作用图，并表征 TrA和Tra2的突变体。 确定聚合酶II对前-mRNAs转录如何影响ESE依赖性 拼接。我们已经建立了一种体外试验来分析 当与转录结合时，前信使核糖核酸剪接。使用这一化验方法，我们将测试 假设C末端结构域(CTD)的磷酸化水平 影响ESE依赖的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Pre-mRNA相关联。

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.