An epitranscriptomic program maintains skeletal stem cell quiescence via a METTL3-FEM1B-GLI1 axis

Skeletal stem cells (SSCs) maintain the skeletal system via pluripotency and differentiation capacity. However, it remains largely unknown how these cells precisely regulate their function to maintain skeletal organization. Here, we delineate the RNA m⁶A modification landscape across skeletal cell populations in the mouse epiphysis. Our findings show that m⁶A modifications are prevalent in skeletal stem cell and progenitor populations and play critical roles in cell fate determination. Genetic deletion of Mettl3, the core catalytic subunit of the m⁶A-methyltransferase complex, in murine skeletal stem and progenitors impaired bone development, leading to shortened limbs, disrupted growth plate zonation, and decreased bone mass. Moreover, Mettl3 deficiency induced quiescence exit in SSCs, together with compromised self-renewal capacity and differentiation potential. Mechanistically, Mettl3-mediated m⁶A modification reduced mRNA stability of the Cul2-RING E3 ligase complex subunit Fem1b, which subsequently stabilizes Gli1 protein, a key transcription factor of Hedgehog pathway for maintaining SSC identity and function. Thus, we present a comprehensive RNA m⁶A modification landscape of skeletal cell hierarchy and uncover the essential function of epitranscriptomically-regulated proteostasis in maintaining SSCs quiescence and potency.