N6-methyladenosine (m⁶A) depletion regulates pluripotency exit by activating signaling pathways in embryonic stem cells.

N6-methyladenosine (m6A) deposition on messenger RNA (mRNA) controls embryonic stem cell (ESC) fate by regulating the mRNA stabilities of pluripotency and lineage transcription factors (TFs) [P. J. Batista et al., Cell Stem Cell 15, 707-719 (2014); Y. Wang et al., Nat. Cell Biol. 16, 191-198 (2014); and S. Geula et al., Science 347, 1002-1006 (2015)]. If the mRNAs of these two TF groups become stabilized, it remains unclear how the pluripotency or lineage commitment decision is implemented. We performed noninvasive quantification of Nanog and Oct4 TF protein levels in reporter ESCs to define cell-state dynamics at single-cell resolution. Long-term single-cell tracking shows that immediate m6A depletion by Mettl3 knock-down in serum/leukemia inhibitory factor supports both pluripotency maintenance and its departure. This is mediated by differential and opposing signaling pathways. Increased FGF5 mRNA stability activates pErk, leading to Nanog down-regulation. FGF5-mediated coactivation of pAkt reenforces Nanog expression. In formative stem cells poised toward differentiation, m6A depletion activates both pErk and pAkt, increasing the propensity for mesendodermal lineage induction. Stable m6A depletion by Mettl3 knock-out also promotes pErk activation. Higher pErk counteracts the pluripotency exit delay exhibited by stably m6A-depleted cells upon differentiation. At single-cell resolution, we illustrate that decreasing m6A abundances activates pErk and pAkt-signaling, regulating pluripotency departure