The coupling of mRNA transcription and 3' end formation

在过去的十年中，人们已经清楚地看到，前mRNA的加工，包括5‘封端、剪接、3’ 末端多聚腺苷化，甚至核输出，与RNA聚合酶II(RNAP II)的转录结合在一起。 然而，将转录与加工联系起来以及加工如何调节转录的机制是 很大程度上是未知的。这一建议的前提是招聘和交换转录和加工 因子通过Ser2和Ser5的磷酸化和去磷酸化与转录周期相协调 羧基末端重复结构域(CTD)是存在于最大的RNAP II亚基上的一种独特结构。我们会 重点研究CPF加工因子中的一种成分Ssu72 CTD Ser5-P磷酸酶在反式- 转录和3‘端加工因子如何影响转录终止。这项研究涉及三个目标 第一个目标是确定Ssu72在转录周期中的作用，并确定特定的因素 其与RNAP II的结合受Ser5-P去磷酸化的影响。染色质免疫沉淀 (CHIP)将用于评估Ssu72如何影响RNAP II、转录因子和 体内转录基因的加工因子。我们将使用体外转录系统来测试 Ssu72促进了起始-延伸的转变。我们的第二个目标，在两个目标中得到解决 拟议的研究是为了了解Ssu72是如何受到RNAP II转录机制和3‘ 结束加工因素。我们将确定与Ssu72基因相互作用的转录因子 影响CTD Ser5-P水平和RNAP II Ser5-P的跨基因分布。一种强大的酵母组合 遗传方法将被用来识别与Ssu72相互作用的新因素。我们还将检验这一假设 Ssu72与CPF的相互作用以及3‘端加工复合体与CTD的相互作用对 将Ssu72活性直接作用于磷酸化的CTD。我们的第三个目标是确定 Poly(A)依赖的终止中的切割/多聚腺苷化机制。关于这一点有很多争论 导致终止的分子事件，包括加工因子是否必须与RNAP II接触， 新生转录本的切割是否重要，以及转录本下游的降解是否重要 需要裂解位点的位置。我们将使用芯片分析，一种改进的转录运行分析，以及一种策略 这使得对致命突变的检查能够解决这些问题。 拟议的实验建立在我们使用生物化学和遗传学成功的悠久历史基础上。 研究转录和mRNA3‘末端加工。我们期待这些新的研究将促进我们的洞察力 深入了解转录周期的动态，并识别和澄清早期和晚期的关键检查点 这一周期的各个阶段。此外，RNAP II转录和RNA加工是高度保守的过程， 由系统发育上相似的蛋白质编排。因此，我们希望从我们的 拟议的研究将直接适用于人类生物学和医学。

During the past decade, it has become clear that processing of pre-mRNA, including 5' capping, splicing, 3' end polyadenylation, and even nuclear export, is coupled with transcription by RNA polymerase II (RNAP II). However, the mechanisms linking transcription to processing and how processing regulates transcription are largely unknown. The premise of this proposal is that recruitment and exchange of transcription and processing factors is coordinated with the transcription cycle by phosphorylation and dephosphorylation of Ser2 and Ser5 of the carboxy-terminal repeat domain (CTD), a unique structure present on the largest RNAP II subunit. We will focus on the role of the Ssu72 CTD Ser5-P phosphatase, a component of the CPF processing factor, in trans- cription and how 3' end processing factors affect transcription termination. The research addresses 3 objectives The first objective is to determine where in the transcription cycle Ssu72 acts and to identify specificfactors whose associationwith RNAP II is affected by Ser5-P dephosphorylation. Chromatin immunoprecipitation (ChIP) will be used to evaluate how Ssu72 affects the distribution of RNAP II, transcription factors, and processing factors across transcribed genes in vivo. We will use an in vitro transcription system to test whether Ssu72 facilitates the initiation-elongation transition. Our second objective,which is addressed in two aims of the proposed research, is to understand how Ssu72 is regulated by the RNAP II transcription machinery and by 3' end processing factors. We will determine whether transcription factors that genetically interact with Ssu72 affect CTD Ser5-P levels and the distribution of RNAP II Ser5-P across genes. A powerful combination of yeast genetic methods will be used to identify novel factors that interact with Ssu72. We will also test the hypothesis that the interactions of Ssu72 with CPF, and of the 3' end processing complex with the CTD, are important to direct Ssu72 activity to the phosphorylated CTD. Our third objective is to determine the role of the cleavage/polyadenylation machinery in poly(A)-dependent termination. There is much debate over the molecular events that lead to termination, including whether the processing factors must contact RNAP II, whether cleavage of the nascent transcript is important, and whether degradation of the transcript downstream of the cleavage site is needed. We will use ChIP analysis, a modified transcription run-on assay, and a strategy that allows the examination of lethal mutations to address these issues. The proposed experiments build upon our long history of using biochemistry and genetics to successfully study transcription and mRNA 3' end processing. We anticipate that these new studies will advance our insight into the dynamics of the transcription cycle and identify and clarify critical checkpoints in the early and late phases of this cycle. Moreover, RNAP II transcription and RNA processing are highly conservedprocesses, orchestrated by phylogenetically similar proteins. Consequently, we expect that information gained from our proposed studies will be directly applicable to human biology and medicine.