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 N 6 -methyladenosine (m 6 A) 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 m 6 A-containing R-loops accumulate during G 2 /M and are depleted at G 0 /G 1 phases of the cell cycle, and that the m 6 A 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 m 6 A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability. N 6 -methyladenosine (m 6 A) is prevalent at RNA:DNA hybrids in human pluripotent stem cells. The m 6 A reader YTHDF2 interacts with R-loop-enriched loci in dividing cells, and YTHDF2 loss leads to increased R-loop levels and accumulation of γH2AX.