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

> **NIH NIH F32** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2024 · $76,756

## Abstract

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 as...

## Key facts

- **NIH application ID:** 10915439
- **Project number:** 5F32GM151833-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Talysa C Viera
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $76,756
- **Award type:** 5
- **Project period:** 2023-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10915439

## Citation

> US National Institutes of Health, RePORTER application 10915439, Investigating the connection between aberrant R-loop formation and genome instability (5F32GM151833-02). Retrieved via AI Analytics 2026-07-31 from https://api.ai-analytics.org/grant/nih/10915439. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*