Coupling of mRNA transcription and 3' end formation

描述(申请人提供)：真核细胞信使核糖核酸合成的新兴模式是转录和信使核糖核酸处理事件在体内通过不同机制的物理联系而被仔细地协调。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确切分子机制很感兴趣，并发现了转录和切割/多聚腺苷化因子之间的几个新的相互作用点。这些发现表明，启动子上加工因子的存在可能会影响转录启动的效率和/或特异性，并促进RNAP II循环回到启动子进行另一轮转录。这可能是一种机制，以确保加工因子适当地加载到转录复合体上，进而确保转录本随后的多腺苷基化，这对于最佳的mRNA输出、翻译和周转是必不可少的。为了研究这一问题，我们提出了以下具体目标：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，Ssu72是一种影响起始的蛋白质，它直接参与了mRNA3‘端的切割。我们将分析现有的Ssu72突变体集合，试图将Ssu72的切割活性从转录启动功能中分离出来，并开发新的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。我们发现这些蛋白在基因上与切割/多聚腺苷化因子(CPF)的PTAL亚基相互作用。此外，它们以相互排斥的方式物理地结合Pta1。我们将检验这样的假设，即Pta1与Ssu72和Sub1的顺序相互作用对于有效地启动和/或切割Pre-mRNA是重要的。 3.作为CPF的一部分，Swd2是否在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‘端加工没有影响，但导致转录终止效率低下和mRNA水平降低。我们将通过将Set1招募到转录复合体来验证Swd2影响终止的假设。我们将确定Swd2与CPF的接触点，并研究这种相互作用的中断如何影响mRNA的合成。基因筛查将用于确定与Swd2的其他重要功能相互作用。

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.