RUI: Early Endings - Characterizing the Role of Hrp1 in RNA Polymerase II Transcription Attenuation

RUI：早期结局 - 表征 Hrp1 在 RNA 聚合酶 II 转录减弱中的作用

• 批准号: 2152496
• 负责人: Jason Kuehner
• 金额: $ 37.2万
• 依托单位: Emmanuel College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2152496&HistoricalAwards=false
• 关键词: RUI; Early; Endings; Characterizing; Role

Life depends on information stored in DNA, which is expressed into RNA and proteins that perform cellular functions. An initial step of gene expression is transcription, where a molecular machine called RNA polymerase reads DNA to synthesize RNA. As RNA polymerase moves along DNA, it can be interrupted by regulatory stop signals that terminate transcription prematurely. Downregulation of gene expression by early transcription stoppage occurs widely in cells ranging from bacteria to human, but the underlying mechanism and selectivity remains unclear. This research project will investigate premature transcription termination in the yeast S. cerevisiae, a tractable model for studying many conserved biological processes. Undergraduate student researchers will be trained to use classical genetics and modern bioinformatic tools to dissect transcription stop signals and discover mutants that alter recognition. New gene targets will be identified, with a broader goal of identifying shared regulatory features. Student trainees will have opportunities to present their work at national research conferences and coauthor publications. This research will also be incorporated into a core undergraduate biology laboratory course, mobilizing 50 additional students to validate gene targets. Course resources will be shared broadly so additional educational communities may contribute and benefit.Premature transcription termination (attenuation) of eukaryotic RNA Polymerase II (Pol II) is more prevalent than once appreciated but remains ill-defined. The investigators hypothesize that a hybrid transcription termination pathway involving the Hrp1 RNA-binding protein and Sen1 helicase contributes broadly to yeast Pol II attenuation. This research project will generate a comprehensive genetic profile of ten newly identified attenuators and identify the larger repertoire of Hrp1-dependent attenuators across the yeast genome. In Aim 1, a genetic selection will identify cis-acting mutations that disrupt attenuator function in plasmid-based reporter genes, followed by confirmation in CRISPR-edited genomic DNA. A genetic screen will probe the effect of trans-acting mutations that alter termination factor recruitment, Pol II CTD modification, and Pol II pausing. A direct mechanism will be assayed using Hrp1 mutants defective for binding RNA or affiliated proteins. In Aim 2, an auxin-inducible degron system will deplete Hrp1, followed by precision nuclear run-on sequencing (PRO-Seq) to monitor genome-wide Pol II read-through defects. This work will inform analysis of the Hrp1 ortholog HNRNPDL, a human protein that likewise binds AU-rich RNA and regulates transcription. In addition, engineered attenuators may be harnessed for dynamic gene control in biotechnology applications, including yeast expression of industrial enzymes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

生命依赖于储存在DNA中的信息，这些信息被表达成RNA和蛋白质，执行细胞功能。基因表达的第一步是转录，其中称为RNA聚合酶的分子机器读取DNA以合成RNA。当RNA聚合酶沿着DNA移动时，它可以被过早终止转录的调节停止信号中断。早期转录停止导致的基因表达下调广泛存在于从细菌到人类的细胞中，但其潜在机制和选择性仍不清楚。本研究计划将探讨酵母S. cerevisiae是研究许多保守生物过程的一个易处理的模型。本科生研究人员将接受培训，使用经典遗传学和现代生物信息学工具来剖析转录停止信号，并发现改变识别的突变体。将确定新的基因靶点，更广泛的目标是确定共同的调控特征。学员将有机会在国家研究会议和合著出版物上介绍他们的工作。这项研究也将被纳入一个核心的本科生生物学实验室课程，动员50名额外的学生来验证基因靶点。课程资源将被广泛共享，因此其他教育社区可能会做出贡献并从中受益。真核RNA聚合酶II（Pol II）的过早转录终止（衰减）比以前更普遍，但仍然不明确。研究人员假设，一个混合转录终止途径，涉及Hrp 1 RNA结合蛋白和Sen 1解旋酶广泛有助于酵母Pol II衰减。该研究项目将产生10个新鉴定的衰减剂的综合遗传图谱，并确定整个酵母基因组中Hrp 1依赖性衰减剂的更大库。在Aim 1中，遗传选择将识别破坏基于质粒的报告基因中的衰减子功能的顺式作用突变，然后在CRISPR编辑的基因组DNA中进行确认。遗传筛查将探测改变终止因子募集、Pol II CTD修饰和Pol II暂停的反式作用突变的影响。将使用结合RNA或附属蛋白质缺陷的Hrp 1突变体测定直接机制。在Aim 2中，生长素诱导的降解决定子系统将耗尽Hrp 1，然后进行精确的核连续测序（PRO-Seq）以监测全基因组Pol II通读缺陷。这项工作将为Hrp 1直系同源物HNRNPDL的分析提供信息，HNRNPDL是一种同样结合富含AU的RNA并调节转录的人类蛋白质。此外，工程衰减剂可用于生物技术应用中的动态基因控制，包括工业酶的酵母表达。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Mutations in yeast Pcf11, a conserved protein essential for mRNA 3′ end processing and transcription termination, elicit the Environmental Stress Response

• DOI: 10.1093/genetics/iyad199
• 发表时间: 2023-11-15
• 期刊: GENETICS
• 影响因子: 3.3
• 作者: Graber，Joel H.;Hoskinson，Derick;Moore，Claire
• 通讯作者: Moore，Claire

Abstract 2354: RNA Polymerase II transcription attenuation at multiple genes in yeast depends on the mRNA 3ʹ-end processing factor Hrp1 and its RNA recognition motif

摘要 2354：酵母中多个基因的 RNA 聚合酶 II 转录减弱取决于 mRNA 3Ê1 末端加工因子 Hrp1 及其 RNA 识别基序

• DOI: 10.1016/j.jbc.2023.103408
• 发表时间: 2023
• 期刊: Journal of Biological Chemistry
• 影响因子: 4.8
• 作者: Roche, Mackenzie;Edouard, Sidney;Talluto, Justin;Pavan, Vincent;Kuehner, Jason
• 通讯作者: Kuehner, Jason