Coupling of mRNA transcription and 3' end formation

mRNA 转录和 3 末端形成的偶联

• 批准号: 6677386
• 负责人: CLAIRE L MOORE
• 金额: $ 32.78万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6677386
• 关键词: DNA directed RNA polymerase; RNA binding protein; RNA biosynthesis; genetic screening; genetic transcription; messenger RNA; molecular genetics; polyadenylate; posttranscriptional RNA processing; protein protein interaction; protein structure function; transcription factor; transport proteins

DESCRIPTION (provided by applicant): The emerging model of eukaryotic mRNA synthesis is that transcription and mRNA processing events are carefully orchestrated in vivo by a physical association of the different machineries. For example, RNA polymerase II affects the efficiency of 3' end processing, and processing factors affect the efficiency of transcription termination downstream of poly(A) sites. We are interested in the precise molecular mechanisms involved in the coordination of these two events and have identified several new points of interaction between transcription and cleavage/polyadenylation factors. These findings suggest that the presence of processing factors at the promoter might affect the efficiency and/or specificity of transcription initiation and facilitate recycling of RNAP II back to the promoter for another round of transcription. This may serve as a mechanism to insure the proper loading of processing factors onto the transcriptional complex, and in turn, the subsequent polyadenylation of the transcript, which is essential for optimal export, translation, and turnover of mRNA. To investigate this issue, we propose the following specific aims: 1. Can the activity of Ssu72 in transcription be separated from its role in 3" end cleavage? We have found that Ssu72, previously identified as a protein affecting initiation, is directly involved in mRNA 3'end cleavage. We will analyze an existing collection of ssu72 mutants to try to separate the cleavage activity of Ssu72 from a function in transcription initiation and develop new assays to help discriminate these functions. 2. What is the functional significance of the interactions of Sub1 and Ssu72 with Pta1, and how are these interactions regulated? Ssu72 and Sub1 were initially identified based on genetic interactions with TFIIB. We have found that these proteins genetically interact with the Ptal subunit of Cleavage/Polyadenylation Factor (CPF). Moreover, they physically bind Pta1 in a mutually exclusive manner. We will test the hypothesis that sequential interactions of Pta1 with Ssu72 and Sub1 are important for efficient initiation and/or cleavage of pre-mRNA. 3. Does Swd2 function in mRNA synthesis as part of CPF? This protein is intimately associated with CPF and the Set1 histone methylase. However, Swd2 depletion has no effect on 3' end processing, but causes inefficient transcription termination and reduced mRNA levels. We will test the hypothesis that Swd2 affects termination by recruiting Set1 to the transcription complex. We will identify the contact point of Swd2 with CPF and examine how disruption of this interaction affects mRNA synthesis. A genetic screen will be used to identify other important functional interactions with Swd2.

描述（由申请人提供）：真核生物mRNA合成的新兴模型是转录和mRNA加工事件在体内通过不同机制的物理关联精心编排。例如，RNA聚合酶II影响3'末端加工的效率，加工因子影响poly（A）位点下游转录终止的效率。我们感兴趣的精确的分子机制参与协调这两个事件，并确定了几个新的点之间的相互作用的转录和切割/多聚腺苷酸化因子。这些发现表明，在启动子处的加工因子的存在可能会影响转录起始的效率和/或特异性，并促进RNAP II再循环回到启动子进行另一轮转录。这可以作为一种机制，以确保加工因子的适当加载到转录复合物上，并反过来，随后的多聚腺苷酸化的转录，这是必要的最佳输出，翻译和周转的mRNA。为了研究这个问题，我们提出了以下具体目标：1。Ssu 72在转录中的活性能否与其在3”末端切割中的作用分开？我们已经发现，Ssu 72，以前确定为一个蛋白质的影响启动，是直接参与mRNA的3 '端切割。我们将分析现有的收集的Ssu 72突变体，试图分离Ssu 72的切割活性从转录起始的功能，并开发新的检测方法，以帮助区分这些功能。2. Sub1和Ssu 72与Pta 1相互作用的功能意义是什么，这些相互作用是如何调节的？Ssu 72和Sub1最初是基于与TFIIB的遗传相互作用而鉴定的。我们已经发现，这些蛋白质在遗传上与切割/多聚腺苷酸化因子（CPF）的Ptal亚基相互作用。此外，它们以相互排斥的方式物理结合Pta 1。我们将检验Pta 1与Ssu 72和Sub1的顺序相互作用对于前体mRNA的有效起始和/或切割是重要的这一假设。 3. Swd 2作为CPF的一部分在mRNA合成中起作用吗？这种蛋白质与CPF和Set 1组蛋白甲基化酶密切相关。然而，Swd 2缺失对3'端加工没有影响，但导致低效的转录终止和mRNA水平降低。我们将测试的假设，Swd 2影响终止募集Set 1的转录复合物。我们将确定SWD 2与CPF的接触点，并研究这种相互作用的破坏如何影响mRNA的合成。遗传筛选将用于鉴定与Swd 2的其他重要功能相互作用。