UNDERSTANDING THE MECHANISMS UNDERLAYING 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: 10794651
• 负责人: Frederic Louis Chedin
• 金额: $ 3.35万
• 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/10794651
• 关键词: Address; Affect; Attention; Biochemical; Biogenesis; Cell Line; Cell model; Cell physiology; Cells; Chromatin; Chromosome Segregation; Chromosome abnormality; Complement; CpG Islands; DNA; DNA Damage; DNA Structure; DNA-Directed RNA Polymerase; Data; Defect; Deposition; Developmental Gene; Disease; Enzymes; Functional disorder; Gene Expression; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Health; Histones; Human; Human Genome; Hybrids; Inherited; Knowledge; Length; Life; Light; Link; Maintenance; Mammalian Cell; Mammals; Measures; Mediating; Metabolism; Monitor; Nature; Normal Cell; Nuclear; Nucleosomes; Outcome; Pathologic; Pathway interactions; Pattern; Phenotype; Physiological; Physiological Processes; Planet Earth; Play; Population; Prevalence; Process; RNA; RNA Processing; RNA Splicing; RNA annealing; Resolution; Role; Single-Stranded DNA; Stress; Structure; Technology; Time; Transcription Process; Work; Yeasts; genetic information; human disease; innovative technologies; insight; mammalian genome; nuclease; nucleic acid structure; overexpression; preservation; programs; promoter; recruit; remediation; ribonuclease H1; single molecule; termination factor; transcription termination

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.

项目概要 在转录过程中，新生 RNA 可以与前进 RNA 后面的模板 DNA 链退火 聚合酶并导致称为 R 环的替代 DNA 结构的形成。 R 环分析研究 研究表明，这些结构普遍存在于所有基因组中，并且正常且动态地形成。正常情况下 在某些条件下，R 环发挥着重要的生理作用。然而，在过去十年中，出现了有害的 R 循环 当转录受到干扰时，被认为是从酵母到基因组不稳定的强大触发因素 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”R 环和“有害”R 环的方法仍然大多未知。在本提案中，我们的目标是 剖析转录扰动、R 环代谢和基因组不稳定性之间的联系机制。这将是 通过解决三个核心问题来完成。 (1) 有害 R 环的定义是什么？虽然 R 环有害 许多研究都提出了这一点，但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来表征这些拟议结构的背景 明确的 RNA 加工功能障碍的人类细胞模型。这项工作将定义多样性 由于 RNA 剪接、终止和输出缺陷而导致的 R 环景观改变，这将使我们能够 确定转录扰动如何导致 R 环分布改变，从而增进我们对 R 环的了解 生物发生途径。 (2) 基因组不稳定是由有害的 R 环还是转录改变引起的 本身？虽然人们的注意力集中在有害的 R 环上，但 RNA 加工缺陷的负面影响 转录本身很少被考虑。解开纯 R 环可能产生的影响 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损伤标记物的积累 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“有害”，或者它们是否 只是反映转录异常。 （3）R-loop中核糖核酸酶H1（RNase H1）的作用是什么 代谢？ RNase H1 具有明显的生化能力来解析 R 环及其在细胞中的过度表达 抑制由有害 R 环引起的多种基因组不稳定性表型。然而，直接证据很少 存在表明细胞 RNase H1 表达可以解决核 R 环问题。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发挥作用 本身。为了解决这两种可能性，我们将开发 RNase H1 耗竭和过度的细胞模型 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组织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假定的有害 R 环。核RNase H1的功能和靶点 也将得到澄清，可能以全新的视角揭示这种酶。我们期望这项工作将 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 其特点是基因组不稳定和RNA加工功能障碍。