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

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.