Using fission yeast to unravel co-transcriptional 3' end processing and transcription termination

3'末端加工是基因表达的基本步骤，先于RNA多聚腺苷酸化并且与转录终止密切相关。3'末端加工是一个共转录过程，涉及RNA聚合酶II（RNAPII）最大亚基的羧基末端结构域（CTD）募集RNA成熟因子。尽管对参与RNA加工的参与者进行了某种程度上公平的评估，但关于它们与转录周期整合的调控机制仍有很多未知之处。这对于3'末端加工和转录终止尤其如此。事实上，最近的全转录组研究表明，多聚腺苷酸化位点（PAS）在大多数真核基因中使用，如在人类，小鼠，植物，苍蝇和酵母中所证明的。这一过程被称为选择性多聚腺苷酸化（阿帕），正在成为基因调控的一个主要层面，允许包含或排除控制mRNA定位、稳定性和翻译的序列。然而，PAS识别的机制以及PAS选择如何与转录终止协调仍不清楚。因此，我们研究计划的长期目标是阐明共转录3'端加工和转录终止决定细胞RNA命运的分子基础。 我们的研究使用酵母裂殖酵母粟酒裂殖酵母最近在这一领域作出了贡献，显示保守的RNA结合蛋白Seb 1促进聚（A）位点选择和终止RNAPII转录的蛋白质编码和非编码基因。因此，本申请的一般目的是确定Seb 1控制3'末端加工和转录终止的机制。我们的中心假设，这是基于强有力的初步研究结果，是Seb 1的CTD RNAPII的结合影响转录延伸动力学，从而控制聚（A）位点选择的位置。我们将检验我们的假设，并通过追求以下具体目标来实现本提案的目标： 1.确定Seb 1控制多聚腺苷酸化位点选择的机制; 2.建立RNAPII磷酸化与共转录3'端加工之间的联系; 3.阐明了S.粟球。 该提议的起源依赖于研究与RNAPII的CTD和新生转录物结合的蛋白质，以阐明转录如何协调3'端加工和终止。鉴于S. pombe和人类多聚腺苷酸化信号，包括使用典型的AAUAAA六聚体，我们预测，我们在裂殖酵母中的研究所描述的转录和多聚腺苷酸化位点选择之间的联系将适用于所有真核生物。

3' end processing is a fundamental step of gene expression that precedes RNA polyadenylation and that is intimately associated with transcription termination. 3' end processing is a co-transcriptional process that involves the recruitment of RNA maturation factors by the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Despite a somewhat fair assessment of the players implicated in RNA processing, much is still unknown about the regulatory mechanisms underlying their integration with the transcription cycle. This is particularly true for 3' end processing and transcription termination. Indeed, recent transcriptome-wide studies indicate that multiple polyadenylation sites (PAS) are used at most eukaryotic genes, as demonstrated in humans, mouse, plants, flies, and yeast. This process, known as alternative polyadenylation (APA), is emerging as a major layer of gene regulation, allowing the inclusion or exclusion of sequences that control the localization, stability, and translation of mRNAs. However, the mechanism of PAS recognition and how PAS selection is coordinated with transcription termination remains unclear. The long-term goal of our research program is therefore to elucidate the molecular basis by which co-transcriptional 3' end processing and transcription termination determine the fate of cellular RNAs. Our studies using the yeast Schizosaccharomyces pombe have recently contributed to this field by showing that the conserved RNA-binding protein Seb1 promotes poly(A) site selection and termination of RNAPII transcription at protein-coding and noncoding genes. Accordingly, the GENERAL OBJECTIVE of this application is to determine the mechanism by which Seb1 controls 3' end processing and transcription termination. Our central HYPOTHESIS, which is based on strong preliminary findings, is that binding of Seb1 to the CTD of RNAPII influences transcription elongation kinetics, thereby controlling the position of poly(A) site selection. We will test our hypothesis and accomplish the objective of this proposal by pursuing the following SPECIFIC AIMS: 1. Determine the mechanism by which Seb1 controls polyadenylation site selection; 2. Establish the link between RNAPII phosphorylation and co-transcriptional 3' end processing; 3. Define the mechanism of snoRNA 3' end processing and transcription termination in S. pombe. The ORIGINALITY of this proposal relies on studying a protein that binds to both the CTD of RNAPII and to nascent transcripts to elucidate how transcription coordinates 3' end processing and termination. Given the similarities between S. pombe and human polyadenylation signals, including use of the canonical AAUAAA hexamer, we predict that the links between transcription and polyadenylation site selection described by our studies in fission yeast will apply to all eukaryotes.