The coupling of mRNA transcription and 3' end formation

描述（由申请人提供）：真核生物mRNA合成的新模型是，转录和mRNA加工事件通过不同机制的物理关联在体内精心安排。例如，RNA聚合酶II影响3'端加工效率，加工因素影响poly(A)位点下游转录终止效率。我们对参与这两个事件协调的精确分子机制很感兴趣，并且已经确定了转录和切割/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起始的效率和/或特异性，并促进RNAP II再循环回启动子进行另一轮转录。这可能是一种机制，以确保加工因子正确装载到转录复合体上，反过来，转录本随后的聚腺苷化，这对于mRNA的最佳输出、翻译和转换至关重要。为了研究这一问题，我们提出以下具体目标：1。能否将Ssu72的转录活性与其在3 ‘ ’端切割中的作用分离开来？我们发现Ssu72，先前被鉴定为影响起始的蛋白质，直接参与mRNA 3'端切割。我们将分析现有的ssu72突变体，试图将ssu72的切割活性与转录起始的功能分离开来，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最初是根据与TFIIB的遗传相互作用确定的。我们发现这些蛋白在遗传上与切割/聚腺苷化因子（CPF）的Ptal亚基相互作用。此外，它们在物理上以互斥的方式结合Pta1。我们将验证Pta1与Ssu72和Sub1的顺序相互作用对于前mrna的有效起始和/或切割很重要的假设。

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.