Investigating the connection between aberrant R-loop formation and genome instability

研究异常 R 环形成与基因组不稳定性之间的联系

• 批准号: 10750839
• 负责人: Talysa C Viera
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750839
• 关键词: Address; Binding; Bioinformatics; Body Regions; Cell model; Cells; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Structure; Defect; Disease; Enzymes; Excision; Frequencies; Future; Gene Expression; Gene Expression Process; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomic approach; Genomics; Goals; Hybrids; Institution; Intellectual functioning disability; Knowledge; Lead; Link; Maintenance; Malignant Neoplasms; Maps; Measures; Mediating; Metabolism; Molecular; Nuclear Export; Outcome; Pancreatic ribonuclease; Pathway interactions; Pattern; Phenotype; Polymerase; Process; Promoter Regions; RNA; RNA Polymerase II; RNA Processing; Research Personnel; Research Proposals; Resolution; Role; Single-Stranded DNA; Source; Structure; Testing; Time; Torpedo; Transcript; Work; career; gene conservation; genome-wide; genomic locus; in vivo; mRNA Precursor; mammalian genome; mutant; nervous system disorder; novel; overexpression; poly A specific exoribonuclease; post-doctoral training; promoter; ribonuclease H1; skills; transcription termination

Project Summary R-loops are non-B DNA structures that form co-transcriptionally upon reannealing of the nascent transcript to the DNA template strand, resulting in an RNA:DNA hybrid and a displaced single-strand of DNA. R-loops form dynamically over thousands of conserved genic loci in mammalian genomes under normal conditions. However, under conditions associated with dysfunctional RNA processing, “harmful” R-loops are thought to arise and contribute to DNA damage and genome instability phenotypes, resulting in cancer or neurological diseases. What differentiates normal and harmful R-loops remains unclear, and how harmful R-loops lead to DNA damage is not fully understood. Our group recently identified two classes of R-loops: Class I R-loops form during RNA polymerase II (RNAPII) promoter-proximal pausing at an elevated frequency, while Class II R-loops occur throughout gene bodies at moderate frequencies. Importantly, R-loop-associated genome instability phenotypes can be relieved by overexpression of RNase H1, an enzyme that specifically degrades RNA in RNA:DNA hybrids. The observation that RNase H1 primarily binds to promoter-proximal pause regions, and not gene body regions, implicates Class I R-loops as major drivers of genome instability. I hypothesize that Class I R-loops become elevated upon abnormal RNA processing, resulting in long-lasting paused RNAP polymerase II (RNAPII) complexes, transcription-replication conflicts, and DNA double-stranded breaks (DSBs) at promoter regions. To test this hypothesis, I will build upon a cellular model of defective RNA export by depleting THOC5, which is known to trigger R-loop-induced genomic instability and leverage integrative and unbiased genome- wide mapping approaches to directly measure perturbations in R-loop formation, nascent transcription, and DSB formation over time (Aim 1). I will overexpress (OE) RNase H1 in vivo and determine if it can suppress Class I R-loops, reduce paused RNAPII complexes, and lower DSBs (Aim 2). To further clarify the mechanism of genome stabilization by RNase H1 OE, I will investigate the possibility that RNase H1 activity permits the termination of paused RNAPII complexes via the XRN2 (5’-3’ exoribonuclease 2) “torpedo” pathway, thus relieving transcription-replication conflicts (Aim 3). I expect that this work will establish Class I R-loops associated with paused RNAPII complexes as a major class of genome-destabilizing obstacles, clarifying the identity of harmful R-loops and their impact on genomic stability. I also expect to reveal the molecular mechanism underlying the ability of RNase H1 to stabilize the genome, addressing largely ignored gaps in knowledge and highlighting novel roles for XRN2 in genome maintenance at promoter regions. Overall, this will fundamentally advance our understanding of the links between aberrant RNA processing, R-loop metabolism, and genome maintenance in the context of disease relevant processes, such as defects in RNA export associated with intellectual disabilities.

项目摘要 R-环是一种非B型DNA结构，在新生转录本重新退火时形成共转录 DNA模板链，导致RNA：DNA杂交体和置换的单链DNA。R-环形式 在正常情况下，在哺乳动物基因组中动态地超过数千个保守的基因位点。然而， 在与RNA加工功能障碍相关的条件下，“有害的”R-环被认为会出现 导致DNA损伤和基因组不稳定表型，导致癌症或神经系统疾病。 目前尚不清楚如何区分正常和有害的R-环，以及有害的R-环如何导致DNA损伤 还没有完全被理解。我们的团队最近发现了两类R-环：在RNA过程中形成的I类R-环 聚合酶II(RNAPII)启动子-近端停顿频率升高，同时发生II类R-环 以中等频率贯穿整个基因体。重要的是，与R环相关的基因组不稳定表型 可以通过过度表达RNaseH1来缓解，RNaseH1是一种在RNA：DNA杂交物中特异性降解RNA的酶。 观察到RNaseH1主要与启动子近端的停止区结合，而不是基因体区， 暗示I类R-环是基因组不稳定的主要驱动因素。我假设I类R-循环变成 异常RNA处理时升高，导致长时间暂停的RNAP聚合酶II(RNAPII) 复合体、转录-复制冲突和DNA双链断裂(DSB) 地区。为了验证这一假设，我将建立一个通过耗尽THOC5来实现缺陷RNA输出的细胞模型， 这是已知的触发R环诱导的基因组不稳定，并利用整合和无偏见的基因组- 直接测量R-环形成、新生转录和DSB中扰动的广泛作图方法 随时间形成(目标1)。我将在体内过表达(OE)RNaseH1，并确定它是否能抑制I类 R-环，减少暂停的RNAPII复合体，降低DSB(目标2)。为进一步阐明这一机制 通过RNase H1 OE稳定基因组，我将调查RNase H1活性允许 通过XRN_2(5‘-3’外切核糖核酸酶2)“鱼雷”途径终止暂停的RNAPII复合体，从而 缓解转录-复制冲突(目标3)。我希望这项工作将建立与I类R-环相关的 暂停的RNAPII复合体是一类主要的破坏基因组稳定的障碍，澄清了 有害的R-环及其对基因组稳定性的影响。我还希望揭示分子机制 RNaseH1稳定基因组的能力，解决了在很大程度上被忽视的知识差距和 突出XRN2在启动子区域基因组维护中的新角色。总体而言，这将从根本上 促进我们对异常RNA加工、R环代谢和基因组之间的联系的理解 在疾病相关过程中的维护，例如与以下相关的核糖核酸出口缺陷 智力障碍。