# An improved cytological assay for R-loop detection in Saccharomyces cerevisiae utilizing a catalytically inactive RNase H

## TL;DR

This paper introduces a new method to detect R-loops in live yeast cells using a modified protein linked to a fluorescent tag, offering a more sensitive and practical approach than existing techniques.

## Contribution

The novel contribution is a live-cell R-loop detection method using a catalytically inactive RNase H1-GFP reporter in yeast.

## Key findings

- The dRnh1-GFP reporter generates bright nuclear foci that colocalize with R-loops as detected by S9.6.
- The reporter system can sensitively track R-loop changes caused by mutations and small molecules.
- The method is suitable for high-throughput experiments due to its ease of use and specificity.

## Abstract

R-loops (RNA/DNA hybrids) are caused by defects in RNA transcription or processing, and their level heavily correlates with genome instability and human disease. Most current yeast methods for R-loop analysis use fixed or disrupted cells probed with an R-loop-specific antibody (S9.6), and relatively few cytological methods are available to visualize R-loops in living cells. Here, we present a simplified cytological method for R-loop detection in live cells of the yeast Saccharomyces cerevisiae using a catalytically inactive RNase H1 protein coupled to GFP (dRnh1-GFP reporter). In cells lacking the endogenous RNase H1 gene, reporter expression generates bright nuclear foci that colocalize with R-loops as defined by S9.6 immunocytology. We find that our dRnh1-GFP reporter system can sensitively identify and track changes in R-loop levels induced by various mutations and small molecules known to increase R-loops. Given its ease of use and superior R-loop specificity relative to S9.6, the dRnh1-GFP reporter is suitable for use in high-throughput experiments and presents an exciting opportunity to deepen our understanding of R-loops and their regulatory mechanisms.