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.

• PMID: 40205716
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Journal Article

Authors

Sanders J, Hakeem Z, Schwacha A

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