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

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• R-loops (RNA/DNA hybrids) are linked to genome instability and human diseases.
• Current methods for R-loop detection in yeast often require fixed cells and lack real-time visualization.
• There is a need for advanced cytological tools to study R-loops in live cells.

Purpose of the Study:

• To develop and validate a simplified cytological method for detecting R-loops in live Saccharomyces cerevisiae cells.
• To establish a reporter system for sensitive and specific R-loop visualization and quantification.
• To enable high-throughput screening and deeper understanding of R-loop regulatory mechanisms.

Main Methods:

• Development of a catalytically inactive RNase H1 protein fused to GFP (dRnh1-GFP) as a live-cell reporter.
• Utilizing yeast cells deficient in endogenous RNase H1 for reporter expression.
• Colocalization studies with the S9.6 antibody for R-loop validation.
• Tracking R-loop level changes induced by genetic mutations and small molecules.

Main Results:

• The dRnh1-GFP reporter forms bright nuclear foci in live yeast cells lacking endogenous RNase H1.
• Reporter foci colocalize with R-loops identified by S9.6 immunocytology.
• The reporter system sensitively detects R-loop alterations induced by known R-loop-increasing factors.
• dRnh1-GFP demonstrates superior specificity compared to the S9.6 antibody.

Conclusions:

• The dRnh1-GFP reporter provides a robust and simplified method for live-cell R-loop detection in yeast.
• This system facilitates sensitive monitoring of R-loop dynamics and responses to various stimuli.
• The reporter is suitable for high-throughput applications, advancing R-loop research and its implications in disease.