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

## TL;DR

This study identifies an RNA modification pathway that helps maintain skeletal stem cell function and bone development.

## Contribution

The study reveals a novel METTL3-FEM1B-GLI1 axis regulating skeletal stem cell quiescence through RNA m6A modification.

## Key findings

- m6A modifications are prevalent in skeletal stem cells and regulate cell fate.
- Mettl3 deletion disrupts bone development and reduces stem cell potency.
- m6A modification stabilizes GLI1 protein by degrading FEM1B mRNA.

## Abstract

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.

m6A RNA modification has been linked to regulation of osteogenic differentiation. This study defines the RNA m6A methylome of skeletal cell populations and reveals the role of METTL3-dependent regulation of FEM1B-GLI1 module in maintaining the function of skeletal stem cells (SSCs).

Super-low-input m6A sequencing (SLIM-seq) reveals a comprehensive m6A landscape in murine skeletal cell populations.Mettl3 deletion impairs bone development.METTL3 is required for maintaining skeletal stem cell quiescence and multipotency.m6A modification promotes FEM1B mRNA degradation and subsequently increases GLI1 protein stability in SSCs.

Super-low-input m6A sequencing (SLIM-seq) reveals a comprehensive m6A landscape in murine skeletal cell populations.

Mettl3 deletion impairs bone development.

METTL3 is required for maintaining skeletal stem cell quiescence and multipotency.

m6A modification promotes FEM1B mRNA degradation and subsequently increases GLI1 protein stability in SSCs.

The landscape of RNA m6A methylation across murine skeletal cell populations reveals the role of METTL3 in skeletal stem cell maintenance.