The molecular chaperone ALYREF promotes R-loop resolution and maintains genome stability.

Unscheduled R-loops usually cause DNA damage and replication stress, and are therefore a major threat to genome stability. Several RNA processing factors, including the conserved THO complex and its associated RNA and DNA-RNA helicase UAP56, prevent R-loop accumulation in cells. Here we investigate the function of ALYREF, an RNA export adapter associated with UAP56 and the THO complex, in R-loop regulation. We demonstrate that purified ALYREF promotes UAP56-mediated R-loop dissociation in vitro, and this stimulation is dependent on its interaction with UAP56 and R-loops. Importantly, we show that ALYREF binds DNA-RNA hybrids and R-loops. Consistently, ALYREF depletion causes R-loop accumulation and R-loop-mediated genome instability in cells. We propose that ALYREF, apart from its known role in RNA metabolism and export, is a key cellular R-loop co-regulator, which binds R-loops and stimulates UAP56-driven resolution of unscheduled R-loops during transcription.