RESUMO
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 cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, 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.
Assuntos
Adenosina/análogos & derivados , DNA/química , Instabilidade Genômica , Células-Tronco Pluripotentes/metabolismo , Estabilidade de RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/fisiologia , RNA/química , Adenosina/farmacologia , Animais , DNA/efeitos dos fármacos , DNA/genética , Dano ao DNA , Humanos , Camundongos , Camundongos Knockout , Mitose , Células-Tronco Pluripotentes/citologia , RNA/efeitos dos fármacos , RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
A scalable and cost-effective synthetic polymer substrate that supports robust expansion and subsequent multilineage differentiation of human pluripotent stem cells (hPSCs) with defined commercial media is presented. This substrate can be applied to common cultureware and used off-the-shelf after long-term storage. Expansion and differentiation of hPSCs are performed entirely on the polymeric surface, enabling the clinical potential of hPSC-derived cells to be realized.