RNA m6A modification regulates cell fate transition between pluripotent stem cells and 2-cell-like cells

N⁶-methyladenosine (m⁶A) exerts essential roles in early embryos, especially in the maternal-to-zygotic transition stage. However, the landscape and roles of RNA m⁶A modification during the transition between pluripotent stem cells and 2-cell-like (2C-like) cells remain elusive. Here, we utilised ultralow-input RNA m⁶A immunoprecipitation to depict the dynamic picture of transcriptome-wide m⁶A modifications during 2C-like transitions. We found that RNA m⁶A modification was preferentially enriched in zygotic genome activation (ZGA) transcripts and MERVL with high expression levels in 2C-like cells. During the exit of the 2C-like state, m⁶A facilitated the silencing of ZGA genes and MERVL. Notably, inhibition of m⁶A methyltransferase METTL3 and m⁶A reader protein IGF2BP2 is capable of significantly delaying 2C-like state exit and expanding 2C-like cells population. Together, our study reveals the critical roles of RNA m⁶A modification in the transition between 2C-like and pluripotent states, facilitating the study of totipotency and cell fate decision in the future.