The coupling of mRNA transcription and 3' end formation

mRNA转录和3末端形成的耦合

• 批准号: 7104854
• 负责人: CLAIRE L MOORE
• 金额: $ 56.59万
• 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/7104854
• 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 mRNA 合成的新兴模型是通过不同机器的物理关联在体内精心策划转录和 mRNA 加工事件。例如，RNA聚合酶II影响3'端加工的效率，加工因素影响poly(A)位点下游转录终止的效率。我们对协调这两个事件的精确分子机制感兴趣，并确定了转录和切割/聚腺苷酸化因子之间的几个新的相互作用点。这些发现表明，启动子处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 循环回到启动子进行另一轮转录。这可能作为一种机制，确保加工因子正确加载到转录复合物上，进而确保转录物的后续聚腺苷酸化，这对于 mRNA 的最佳输出、翻译和周转至关重要。为了研究这个问题，我们提出以下具体目标： 1. Ssu72在转录中的活性能否与其在3"末端切割中的作用分开？我们发现，之前被鉴定为影响起始的蛋白质的Ssu72直接参与mRNA 3'末端切割。我们将分析现有的ssu72突变体集合，试图将Ssu72的切割活性与转录起始中的功能分开 并开发新的检测方法来帮助区分这些功能。 2. Sub1和Ssu72与Pta1相互作用的功能意义是什么？这些相互作用是如何调节的？ Ssu72 和 Sub1 最初是基于与 TFIIB 的遗传相互作用而被鉴定的。我们发现这些蛋白质与切割/多聚腺苷酸化因子 (CPF) 的 Ptal 亚基存在遗传相互作用。而且，他们 以互斥的方式物理结合 Pta1。我们将测试以下假设：Pta1 与 Ssu72 和 Sub1 的连续相互作用对于前 mRNA 的有效起始和/或切割非常重要。 3. Swd2是否作为CPF的一部分在mRNA合成中发挥作用？该蛋白与 CPF 和 Set1 组蛋白甲基化酶密切相关。然而，Swd2 缺失对 3' 末端加工没有影响，但会导致转录终止效率低下并降低 mRNA 水平。我们将测试 Swd2 通过将 Set1 招募到转录复合体来影响终止的假设。我们将确定 Swd2 与 CPF 的接触点，并检查这种相互作用的破坏如何影响 mRNA 合成。遗传筛选将用于鉴定与 Swd2 的其他重要功能相互作用。