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UNDERSTANDING THE MECHANISMS OF 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

基本信息

批准号：
10389339
负责人：
Frederic Louis Chedin
金额：
$ 6.12万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
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/10389339
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 Genes 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 replication stress ribonuclease H1 single molecule termination factor transcription termination

项目摘要

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.

项目总结