Understanding the mechanisms underlying R-loop biogenesis and resolution in mammals

了解哺乳动物 R 环生物发生和分解的机制

• 批准号: 10725028
• 负责人: Frederic Louis Chedin
• 金额: $ 8.16万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725028
• 关键词: Address; Attention; Biochemical; Biogenesis; Cell model; Cell physiology; Cells; Chromosome abnormality; DNA; DNA Damage; DNA Structure; DNA-Directed RNA Polymerase; Data; Defect; Disease; Enzymes; Functional disorder; Gene Expression; Genetic Transcription; Genome; Genomic Instability; Genomics; Human; Inherited; Knowledge; Life; Light; Link; Maintenance; Mammalian Cell; Mammals; Measures; Metabolism; Monitor; Nuclear; Pathway interactions; Pattern; Phenotype; Physiological; Planet Earth; Play; Process; RNA; RNA Processing; RNA Splicing; Resolution; Role; Structure; Time; Work; Yeasts; genetic information; innovative technologies; insight; nucleic acid structure; overexpression; remediation; ribonuclease H1

Project summary During transcription, the nascent RNA can anneal with the template DNA strand behind the advancing RNA polymerase and cause the formation of alternative DNA structures called R-loops. R-loop profiling studies have revealed that these structures are prevalent in all genomes and form normally and dynamically. Under normal conditions, R-loops serve important physiological roles. Yet, over the last decade, harmful R-loops that arise when transcription is perturbed have been implicated as powerful triggers of genome instability from yeast to humans. Harmful R-loops have also been linked to an increasing number of human disorders. What distinguishes “good” R-loops from “harmful” R-loops remains mostly unknown. In this proposal, we aim to dissect the mechanisms linking perturbed transcription, R-loop metabolism, and genome instability. This will be accomplished by addressing three central questions. (1) What defines harmful R-loops? While harmful R-loops have been proposed in many studies, they have never been directly defined at the genomic level. We will leverage our unique expertise in R-loop profiling to characterize these proposed structures in the context of well-defined human cellular models of RNA processing dysfunction. This work will define the diversity of altered R-loop landscapes that result from defects in RNA splicing, termination, and export and will allow us to identify how perturbed transcription results in altered R-loop distributions, boosting our knowledge of R-loop biogenesis pathways. (2) Does genome instability result from harmful R-loops or from altered transcription itself? While attention has been focused on harmful R-loops, the negative impacts of defective RNA processing on transcription itself have seldom been considered. To disentangle possible R-loop effects from pure transcriptional effects, we will carefully monitor transcriptional perturbations in cellular models of RNA processing dysfunction. In addition, we will directly measure the accumulation of DNA damage markers in relation to R-loops, allowing us to determine for the first time if altered R-loops are actually “harmful” or if they simply reflect abnormal transcription. (3) What is the role of Ribonuclease H1 (RNase H1) in R-loop metabolism? RNase H1 has a clear biochemical ability to resolve R-loops and its over-expression in cells suppresses a variety of genome instability phenotypes attributed to harmful R-loops. Yet, little direct evidence exists to show that cellular RNase H1 expression resolves nuclear R-loops. Furthermore, recent studies and our preliminary data suggest that RNase H1 could instead work by mitigating the impact of altered transcription itself. To address these two possibilities, we will develop cellular models of RNase H1 depletion and over- expression in mammalian cells and conduct a broad characterization of the resulting genomic R-loop patterns and transcriptional effects. Our work will resolve crucial knowledge gaps concerning the formation and roles of putative harmful R-loops in genome instability in human cells. The function and targets of nuclear RNase H1 will also be clarified, possibly revealing this enzyme in a fundamentally new light. We expect that this work will durably impact the field of genome maintenance and provide insights into a range of human disorders characterized by genome instability and RNA processing dysfunction.

项目摘要 在转录过程中，新生的RNA可以用模板DNA链在前进的RNA后面退火 聚合酶并引起称为R环的替代DNA结构的形成。 R环分析研究具有 揭示了这些结构在所有基因组中都普遍存在，并且正常形成。在正常情况下 条件，R环起着重要的身体作用。然而，在过去的十年中，出现有害的R环 当转录被扰动时，已作为基因组不稳定性的强大触发器从酵母到 人类。有害的R环也与越来越多的人类疾病有关。什么 区分“良好”的R环与“有害”的R环仍然未知。在此提案中，我们的目标是 解剖连接扰动转录，R环代谢和基因组不稳定性的机制。这将是 通过解决三个中心问题来完成。 （1）什么定义有害的R环？而有害的R弹 已经在许多研究中提出过，从未在基因组水平上直接定义它们。我们将 利用我们独特的专业知识在R环分析中，以表征这些提议的结构 RNA加工功能障碍的明确定义的人类细胞模型。这项工作将定义多样性 RNA剪接，终止和出口缺陷导致的R-loop景观改变了，将使我们能够 确定扰动的转录如何导致R-loop分布改变，从而增强我们对R环的了解 生物发生途径。 （2）基因组不稳定性是由于有害的R环或转录改变而导致的 本身？尽管注意力集中在有害的R环上，但RNA处理有缺陷的负面影响 很少考虑转录本身。解散可能的R环效应 转录效应，我们将仔细监测RNA细胞模型中的转录扰动 处理功能障碍。此外，我们将直接测量DNA损伤标记的积累 与R环有关，允许我们首次确定R-loops实际上是“有害的”，或者他们是否是否 只需反映异常转录即可。 （3）核糖核酸酶H1（RNase H1）在R环中的作用是什么 代谢？ RNase H1具有清晰的生化能力，可以解决R-loops及其在细胞中的过表达 抑制了归因于有害的R环的多种基因组不稳定性表型。但是，几乎没有直接证据 存在以表明细胞RNase H1表达能够解析核R环。此外，最近的研究和 我们的初步数据表明，RNase H1可以通过减轻转录的影响来起作用 本身。为了解决这两种可能性，我们将开发RNase H1部署和过度的蜂窝模型 在哺乳动物细胞中的表达，并对所得的基因组R环模式进行广泛的表征 和转录效应。我们的工作将解决有关形成和角色的重要知识差距 人类细胞中基因组不稳定性中假定的有害R环。核RNase H1的功能和目标 还将澄清，可能在从根本上揭示这种酶。我们希望这项工作将 持久地影响基因组维持领域，并为一系列人类疾病提供见解 以基因组不稳定性和RNA处理功能障碍为特征。