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.

项目概要 R 环是非 B DNA 结构，在新生转录物重新退火至 DNA 模板链，产生 RNA:DNA 杂交体和移位的单链 DNA。正常条件下，哺乳动物基因组中数千个保守基因位点动态变化。在与 RNA 加工功能失调相关的条件下，“有害”R 环被认为会出现并导致 DNA 损伤和基因组不稳定表型，从而导致癌症或神经系统疾病。正常和有害 R 环的区别以及有害 R 环如何导致 DNA 损伤仍不清楚我们的研究小组最近发现了两类 R 环： I 类 R 环在 RNA 过程中形成。聚合酶 II (RNAPII) 启动子近端暂停频率升高，同时发生 II 类 R 环重要的是，R 环相关的基因组不稳定表型的频率适中。 RNase H1 的过度表达可以缓解这种情况，RNase H1 是一种专门降解 RNA:DNA 杂交体中 RNA 的酶。观察到 RNase H1 主要结合启动子近端暂停区域，而不是基因体区域，表明 I 类 R 环是基因组不稳定的主要驱动因素。 RNA 加工异常时升高，导致 RNAP 聚合酶 II (RNAPII) 长期暂停复合物、转录复制冲突和启动子处的 DNA 双链断裂 (DSB) 为了检验这个假设，我将通过耗尽 THOC5 建立一个有缺陷的 RNA 输出的细胞模型，众所周知，它会触发 R 环诱导的基因组不稳定，并利用整合和公正的基因组- 广泛的作图方法可直接测量 R 环形成、新生转录和 DSB 中的扰动随着时间的推移，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 环、减少暂停的 RNAPII 复合物并降低 DSB（目标 2）。通过 RNase H1 OE 稳定基因组，我将研究 RNase H1 活性允许通过 XRN2（5'-3' 核糖核酸外切酶 2）“鱼雷”途径终止暂停的 RNAPII 复合物，因此缓解转录复制冲突（目标 3）我预计这项工作将建立相关的 I 类 R 环。将暂停的 RNAPII 复合物作为一类主要的基因组不稳定障碍，澄清了我还希望揭示有害的 R 环及其对基因组稳定性的影响。 RNase H1 稳定基因组的能力，解决了很大程度上被忽视的知识差距和强调 XRN2 在启动子区域基因组维护中的新作用，这将从根本上说。增进我们对异常 RNA 加工、R 环代谢和基因组之间联系的理解疾病相关过程中的维护，例如与疾病相关的 RNA 输出缺陷智力障碍。