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

3‘末端加工是RNA多聚腺苷化之前基因表达的基本步骤，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)招募RNA成熟因子。尽管对RNA加工中涉及的参与者进行了某种程度上的公平评估，但关于它们与转录周期整合的调控机制仍有许多未知之处。对于3‘末端处理和转录终止尤其如此。事实上，最近的转录组研究表明，在大多数真核基因中都使用了多个多聚腺苷酸化位点(PA)，如在人类、小鼠、植物、苍蝇和酵母中证明的那样。这一过程被称为选择性多聚腺苷酸化(APA)，正在成为基因调控的一个主要层面，允许包含或排除控制mRNAs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相协调仍不清楚。因此，我们研究计划的长期目标是阐明共转录3‘端处理和转录终止决定细胞RNA命运的分子基础。 我们使用酵母裂殖酵母的研究最近对这一领域做出了贡献，表明保守的RNA结合蛋白Seb1促进蛋白质编码和非编码基因的聚(A)位点选择和RNAPII转录的终止。因此，本申请的总体目标是确定Seb1控制3‘端处理和转录终止的机制。我们的中心假设基于强有力的初步发现，即Seb1与RNAPII的CTD结合影响转录延伸动力学，从而控制Poly(A)位点的选择位置。我们将通过追求以下具体目标来检验我们的假设并实现本提案的目标： 1.确定Seb1控制多聚腺苷化位点选择的机制； 2.建立RNAPII磷酸化与共转录3‘端加工之间的联系； 3.确定了snoRNA3‘端加工和转录终止的机制。 这一建议的原创性依赖于研究一种蛋白质，该蛋白质既能与RNAPII的CTD结合，又能与新生转录本结合，以阐明转录如何协调3‘端的处理和终止。鉴于S.pombe和人类多腺化信号之间的相似之处，包括使用典型的AAUAAA六角体，我们预测我们在分裂酵母中的研究描述的转录和多腺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.