UNDERSTANDING THE MECHANISMS OF UNDERLYING R-LOOP BIOGENESIS AND RESOLUTION IN MAMMALS

项目总结 在转录过程中，新生的rna可以与前进的rna后面的模板dna链一起退火。 聚合酶并导致另一种称为R-环的DNA结构的形成。R-环侧写研究已经 揭示了这些结构普遍存在于所有基因组中，并正常和动态地形成。低于正常 在这种情况下，R环起着重要的生理作用。然而，在过去的十年里，出现了有害的R-环 当转录被干扰时，被认为是从酵母菌到 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。在这项建议中，我们的目标是 剖析转录受扰、R环代谢和基因组不稳定之间的联系机制。这将是 通过解决三个核心问题来实现。(1)什么定义有害的R-环？而有害的R-环 虽然在许多研究中已经提出，但从来没有在基因组水平上直接定义它们。我们会 利用我们在R环分析方面的独特专业知识，在以下背景下描述这些建议的结构 明确定义的RNA处理功能障碍的人类细胞模型。这项工作将定义 由于RNA拼接、终止和输出中的缺陷而导致的改变的R环环境，并将允许我们 识别干扰转录是如何导致R环分布改变的，从而提高我们对R环的知识 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，但有缺陷的RNA处理的负面影响 关于转录本身的问题很少被考虑。将可能的R-loop效应从纯R-loop中分离出来 转录效应，我们将仔细监测RNA细胞模型中的转录扰动。 加工功能障碍。此外，我们还将直接测量DNA损伤标志物在 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 只是反映了异常转录。(3)核糖核酸酶H1(RNaseH1)在R-环中的作用 新陈代谢？RNaseH1具有明显的生化能力来分解R-环及其在细胞中的过度表达 抑制由有害的R-环引起的多种基因组不稳定表型。然而，几乎没有直接的证据 存在的目的是表明细胞RNaseH1的表达可以分解核R-环。此外，最近的研究和 我们的初步数据表明，RNaseH1可以通过减轻转录改变的影响来发挥作用 它本身。为了解决这两种可能性，我们将建立RNaseH1耗尽和过度的细胞模型。 在哺乳动物细胞中的表达，并对由此产生的基因组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生物多样性的形成和作用的关键知识空白 人类细胞基因组不稳定中可能存在有害的R-环。核RNaseH1的功能和靶点 也将被澄清，可能从一个全新的角度揭示这种酶。我们预计这项工作将 持久影响基因组维护领域，并提供对一系列人类疾病的洞察 以基因组不稳定和RNA加工功能障碍为特征。

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.