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

项目摘要 R环是非B DNA结构，其在新生转录物再退火时共转录形成， DNA模板链，产生RNA：DNA杂交体和置换的单链DNA。R-环形式 在正常条件下，在哺乳动物基因组中的数千个保守基因位点上动态地进行。然而，在这方面， 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。 目前还不清楚正常和有害的R环之间的区别，以及有害的R环如何导致DNA损伤 还没有被完全理解。我们的研究小组最近发现了两类R环：第一类R环在RNA合成过程中形成， 聚合酶II（RNAPII）启动子近端暂停的频率升高，而II类R环发生 在基因体中以中等频率传播。重要的是，R环相关基因组不稳定性表型 可以通过过量表达RNase H1来缓解，RNase H1是一种特异性降解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， 暗示I类R环是基因组不稳定性的主要驱动因素。我假设I类R环 在RNA加工异常时升高，导致长期暂停的RNAP聚合酶II（RNAPII） 复合物、转录-复制冲突和启动子处的DNA双链断裂（DSB） 地区为了验证这一假设，我将建立一个细胞模型的缺陷RNA输出，通过耗尽THOC 5， 已知其触发R环诱导的基因组不稳定性，并利用整合和无偏倚的基因组- 直接测量R环形成、新生转录和DSB中扰动的广泛作图方法 随着时间的推移形成（目标1）。我将在体内过表达（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）。为进一步阐明 通过RNase H1 OE的基因组稳定，我将研究RNase H1活性允许 通过XRN 2（5 '-3'核糖核酸外切酶2）“鱼雷”途径终止暂停的RNAPII复合物， 缓解转录-复制冲突（目标3）。我希望这项工作将建立I类R环相关 暂停RNAPII复合物作为一个主要类别的基因组不稳定的障碍，澄清的身份， 有害的R环及其对基因组稳定性的影响。我还期望揭示分子机制 作为RNase H1稳定基因组的能力的基础，解决了在很大程度上被忽视的知识差距， 突出了XRN 2在启动子区域基因组维持中的新作用。总的来说，这将从根本上 推进我们对异常RNA加工，R环代谢和基因组之间联系的理解 在疾病相关过程的背景下，例如与疾病相关的RNA输出缺陷， 智力残疾。

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.