Investigating the connection between aberrant R-loop formation and genome instability

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Project Summary R-loops are non-B DNA structures that form co-transcriptionally upon reannealing of the nascent transcript to the DNA template strand, resulting in an RNA:DNA hybrid and a displaced single-strand of DNA. R-loops form dynamically over thousands of conserved genic loci in mammalian genomes under normal conditions. However, under conditions associated with dysfunctional RNA processing, “harmful” R-loops are thought to arise and contribute to DNA damage and genome instability phenotypes, resulting in cancer or neurological diseases. What differentiates normal and harmful R-loops remains unclear, and how harmful R-loops lead to DNA damage is not fully understood. Our group recently identified two classes of R-loops: Class I R-loops form during RNA polymerase II (RNAPII) promoter-proximal pausing at an elevated frequency, while Class II R-loops occur throughout gene bodies at moderate frequencies. Importantly, R-loop-associated genome instability phenotypes can be relieved by overexpression of RNase H1, an enzyme that specifically degrades RNA in RNA:DNA hybrids. The observation that RNase H1 primarily binds to promoter-proximal pause regions, and not gene body regions, implicates Class I R-loops as major drivers of genome instability. I hypothesize that Class I R-loops become elevated upon abnormal RNA processing, resulting in long-lasting paused RNAP polymerase II (RNAPII) complexes, transcription-replication conflicts, and DNA double-stranded breaks (DSBs) at promoter regions. To test this hypothesis, I will build upon a cellular model of defective RNA export by depleting THOC5, which is known to trigger R-loop-induced genomic instability and leverage integrative and unbiased genome- wide mapping approaches to directly measure perturbations in R-loop formation, nascent transcription, and DSB formation over time (Aim 1). I will overexpress (OE) RNase H1 in vivo and determine if it can suppress Class I R-loops, reduce paused RNAPII complexes, and lower DSBs (Aim 2). To further clarify the mechanism of genome stabilization by RNase H1 OE, I will investigate the possibility that RNase H1 activity permits the termination of paused RNAPII complexes via the XRN2 (5’-3’ exoribonuclease 2) “torpedo” pathway, thus relieving transcription-replication conflicts (Aim 3). I expect that this work will establish Class I R-loops associated with paused RNAPII complexes as a major class of genome-destabilizing obstacles, clarifying the identity of harmful R-loops and their impact on genomic stability. I also expect to reveal the molecular mechanism underlying the ability of RNase H1 to stabilize the genome, addressing largely ignored gaps in knowledge and highlighting novel roles for XRN2 in genome maintenance at promoter regions. Overall, this will fundamentally advance our understanding of the links between aberrant RNA processing, R-loop metabolism, and genome maintenance in the context of disease relevant processes, such as defects in RNA export associated with intellectual disabilities.