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: 10543443
• 负责人: Frederic Louis Chedin
• 金额: $ 38.34万
• 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/10543443
• 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RNA可以与前进RNA后面的模板DNA链退火， 聚合酶和导致形成替代DNA结构称为R环。R环分析研究 揭示了这些结构普遍存在于所有基因组中，并正常动态地形成。在正常 在某些条件下，R环发挥重要的生理作用。然而，在过去的十年里， 当转录受到干扰时，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“好的”R环和“有害的”R环仍然是未知的。在本建议中，我们的目标是 剖析机制连接干扰转录，R环代谢，和基因组不稳定性。这将是 解决三个核心问题。(1)什么是有害的R环？虽然有害的R环 虽然在许多研究中已经提出，但它们从未在基因组水平上直接定义。我们将 利用我们在R环分析方面的独特专业知识， 明确定义的RNA加工功能障碍的人类细胞模型。这项工作将确定的多样性 RNA剪接、终止和输出缺陷导致的R环景观改变，将使我们能够 确定转录扰动如何导致R环分布改变，增强我们对R环的了解 生物合成途径(2)基因组的不稳定性是由有害的R环还是由转录的改变引起的 本身？虽然注意力集中在有害的R环上，但有缺陷的RNA加工的负面影响 关于转录本身很少被考虑。为了将可能的R环效应从纯 转录的影响，我们将仔细监测转录扰动的细胞模型的RNA 处理功能障碍此外，我们将直接测量DNA损伤标记物在 与R环的关系，使我们能够第一次确定改变的R环是否实际上是“有害的”，或者它们是否 仅仅反映了异常转录。(3)核糖核酸酶H1（RNase H1）在R环中的作用是什么？ 新陈代谢？RNase H1具有清晰的生化能力来解析R环及其在细胞中的过表达 抑制由有害R环引起的多种基因组不稳定性表型。然而，几乎没有直接证据表明 存在表明细胞RNase H1表达解析核R环。此外，最近的研究和 我们的初步数据表明，RNase H1可以通过减轻转录改变的影响来起作用 本身为了解决这两种可能性，我们将开发RNase H1耗尽和过度- 在哺乳动物细胞中表达，并对所得基因组R环模式进行广泛表征 和转录效应。我们的工作将解决有关的形成和作用的关键知识差距， 人类细胞基因组不稳定性中的假定有害R环。核核糖核酸酶H1的功能和作用靶点 也将得到澄清，可能会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护领域，并提供对一系列人类疾病的见解 其特征在于基因组不稳定性和RNA加工功能障碍。