Epitranscriptomic program promotes skeletal stem cell quiescence and pluripotency by regulating proteostasis via METTL3-FEM1B-GLI1 axis

Skeletal stem cells (SSCs) maintain the hierarchy of skeletal system via pluripotency and differentiation capacity. However, it remains largely unknown how these cells adapt its highly nutrient-deficient niche and precisely regulate their function to keep this skeletal hierarchical organization. Here, we delineate a comprehensive m6A landscape across skeletal hierarchy, and identify a new epitranscriptomic program to promote SSCs quiescence and potency by regulating proteostasis via METTL3-FEM1B-GLI1 axis. We find m6A modifications arise mostly from skeletal stem cells (SSCs) in stem-progenitor population and play critical roles in cell fate maintenance and transformation in skeletal hierarchy. Genetic deletion of Mettl3 in skeletal stem and progenitors impairs bone development with shortened limbs, disturbs growth plate, and decreases bone mass. Moreover, Mettl3 loss induces quiescence loss, and damages the self-renewal ability, and differentiation ability of SSCs. Mechanistically, Mettl3-mediated m6A modification reduces mRNA stability of a Cul2-RING E3 ligase complex substrate-recognition subunit Fem1b, which subsequently influences proteostasis of SSCs and stabilizes Gli1 protein, a key transcription factor of Hedgehog pathway for maintaining SSC identity and function. Thus, our findings present a fascinating portrait of m6A modification of skeletal hierarchy and reveal the essential role of an epitranscriptome-proteostasis axis in maintaining SSC function.