N⁶-methyladenosine regulates the stability of RNA:DNA hybrids in human cells

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells^1-4. Here we show that N⁶-methyladenosine (m⁶A) modification, contributing to different aspects of messenger RNA metabolism^5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m⁶A-containing R-loops accumulate during G₂/M and are depleted at G₀/G₁ phases of the cell cycle, and that the m⁶A reader promoting mRNA degradation, YTHDF2 (ref. ⁷), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m⁶A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.