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

基本信息

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
项目类别：
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

项目摘要

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环是非B DNA结构，它们在新生转录本重新进行后共转录形成 DNA模板链，导致RNA：DNA杂交和一个位移的DNA单链。 R环形在正常条件下，在哺乳动物基因组中动态超过数千个保守的基因座。然而，在与功能失调的RNA处理相关的条件下，“有害”的R环被认为是Arye和导致DNA损伤和基因组不稳定性表型，导致癌症或神经系统疾病。哪些区别正常和有害的R环仍不清楚，以及有害的R环如何导致DNA损伤不完全理解。我们的小组最近确定了两类的R环：RNA期间的I类R-Loops形式聚合酶II（RNAPII）启动子 - 抗频率以较高的频率暂停，而II类R环发生通过中等频率的基因体。重要的是，与R环相关的基因组不稳定性表型 RNase H1的过表达可以缓解，RNase H1是一种特异性降解RNA中RNA的酶：DNA杂交。 RNASE H1主要与启动子 - 抗氧化暂停区域而不是基因体区域结合的观察结果，暗示I类R-loops是基因组不稳定性的主要驱动因素。我假设我的r-loops成为在异常的RNA处理后提升，导致持续暂停的RNAP聚合酶II（RNAPII）启动子处的复合物，转录复制冲突和DNA双链断裂（DSB）地区。为了检验这一假设，我将通过耗尽THOC5的细胞模型来建立在有缺陷的RNA导出的细胞模型上，已知会引发R环诱导的基因组不稳定性，并利用综合和无偏基因组 - 广泛的映射方法直接测量R环形成，新生转录和DSB的扰动随着时间的推移形成（目标1）。我将在体内过度表达（OE）RNase H1，并确定它是否可以抑制I类 R-loops，减少暂停的RNAPII复合物和下降DSB（AIM 2）。进一步阐明 RNase H1 OE的基因组稳定，我将研究RNase H1活性允许的可能性通过XRN2（5'-3'Exoribonclelease 2）终止暂停的RNAPII复合物2）“鱼雷”途径，因此缓解转录复制冲突（AIM 3）。我希望这项工作将建立与R-loops相关的类暂停的RNAPII复合物是一类主要的基因组障碍障碍，使人的身份确定有害的R环及其对基因组稳定性的影响。我还希望揭示分子机制 RNase H1稳定基因组的能力的基础，在很大程度上忽略了知识和强调XRN2在启动子区域维持基因组维持中的新作用。总体而言，这将从根本上促进我们对异常RNA处理，R环代谢和基因组之间的联系的理解在疾病相关过程的背景下进行维护，例如与RNA导出的缺陷智力残疾。