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 gamma H2AX, 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 gamma H2AX.