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.
In all life forms on earth, DNA carries the hereditary genetic information. Transcription copies DNA into RNA, a key process that permits gene expression. On occasion, the nascent RNA can become entangled with the DNA, resulting in an alternative three-stranded nucleic acid structure called an R-loop. R-loops form universally under normal conditions and are thought to play physiological roles in cells, including human cells. Studies have suggested, however, that under conditions where transcription is perturbed, harmful R-loops are created, leading to DNA breaks and chromosome anomalies. What distinguishes ?good? from ?harmful? R-loops remains unknown. Similarly, how harmful R-loops arise as a result of perturbed transcription is unclear. Using innovative technologies developed by my group, we will characterize the formation of so-called harmful R- loops under conditions of perturbed transcription in human cells. We will also determine whether harmful R- loops, or perturbed transcription itself, are the cause of the DNA breaks. Finally, we will determine if an enzyme known to relieve the DNA break problem is acting by resolving harmful R-loops or remediating the altered transcription. This work will benefit our understanding of fundamental cellular processes acting in our genome and will provide insights into a range of human disorders linked to perturbed transcription.
