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

生命依赖于储存在DNA中的信息，这些信息被表达为执行细胞功能的RNA和蛋白质。基因表达的第一步是转录，一种名为RNA聚合酶的分子机器读取DNA来合成RNA。当RNA聚合酶沿着DNA移动时，它可能会被过早终止转录的调控停止信号打断。早期转录停止对基因表达的下调广泛存在于从细菌到人类的各种细胞中，但其潜在的机制和选择性仍不清楚。这项研究项目将研究酵母酿酒酵母的提前转录终止，这是一个研究许多保守生物过程的易于处理的模型。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。将确定新的基因靶点，更广泛的目标是确定共同的监管特征。学员将有机会在国家研究会议和合著者出版物上展示他们的工作。这项研究还将被纳入本科生物实验室的核心课程，动员另外50名学生来验证基因靶标。课程资源将被广泛共享，因此更多的教育社区可能会做出贡献和受益。真核细胞RNA聚合酶II(POL II)的提前转录终止(衰减)比以前认识到的更普遍，但仍然定义不清。研究人员假设，涉及Hrp1 RNA结合蛋白和Sen1解旋酶的混合转录终止途径广泛地有助于酵母Pol II的衰减。这项研究项目将生成10个新发现的衰减器的全面遗传图谱，并确定整个酵母基因组中更大的依赖于Hrp1的衰减器。在目标1中，基因选择将识别破坏基于质粒的报告基因中衰减器功能的顺式作用突变，然后在CRISPR编辑的基因组DNA中确认。基因筛查将探索改变终止因子招募、POL II CTD修饰和POL II暂停的反式作用突变的影响。直接机制将使用Hrp1突变体进行测试，这些突变体在结合RNA或相关蛋白方面存在缺陷。在目标2中，生长素诱导的降解子系统将耗尽Hrp1，随后是精确的核连续测序(PRO-Seq)以监测全基因组的Pol II通读缺陷。这项工作将为Hrp1直系同源HNRNPDL的分析提供信息，Hrp1是一种人类蛋白质，同样能结合富含AU的RNA并调节转录。此外，工程衰减器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智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.