Methylation and RNA sequencing to test if m6A is an integral component of RNA:DNA hybrids. N6-methyladenosine regulates cell cycle dynamics of RNA:DNA hybrids.

R-loops are specific nucleic acid structures formed by an RNA:DNA hybrid and an unpaired single stranded DNA1. RNA:DNA hybrids contribute to a number of important biological processes ranging from transcriptional regulation to DNA repair and represent a source of genomic instability in mammalian cells2-4. Notably, the presence of non-canonical bases on the RNA component of R-loops has not been reported to date. Here we show that N6-methyladenosine (m6A), modification involved into the regulation of mRNA stability and translation5, 6, is present on majority of the RNA:DNA hybrids in human pluripotent stem cells (hPSCs). Moreover, we demonstrate that m6A-containing R-loops accumulate in the introns, LINE1 and SINE/Alu repeats during G2/M and getting depleted at G0/G1 phases of the cell cycle in hPSCs. Furthermore, we show that YTHDF2, one of the previously characterized m6A reader proteins regulating mRNA degradation7, migrates to mitoticchromatin in dividing cells where it interacts with genomic regions enriched in RNA:DNA hybrids and another m6A reader, HNRNPA2B18, binds to R-loops-containing intronic regions in interphase nuclei. Correspondingly, siRNA-mediated depletions of YTHDF2 and m6A methyltransferase METTL3 both lead to increase in repeat-specific and intronic RNA:DNA hybrids. Our results provide a new perspective on m6A as an integral component of RNA:DNA hybrids that is involved in the regulation of their cell cycle-specific degradation and imply potential roles for this modification in safeguarding genomic stability, regulating splicing and suppressing retrotransposition in hPSCs.