RBFOX2-dependent alternative splicing of Numb regulates Notch signaling during muscle stem cell activation

Satellite cells (SCs) are somatic stem cells essential for skeletal muscle regeneration. Most SCs remain quiescent in resting muscle, but they rapidly activate in response to stimuli. Although post-transcriptional regulation has been implicated in SC functions, the role of alternative splicing (AS) during SC activation remains unclear. Using in vivo fixation to preserve quiescent SCs, we uncovered rapid and extensive AS changes upon activation, affecting genes involved in fundamental pathways. We identified RBFOX2 as a key AS regulator in SCs; its loss delayed both SC activation and muscle regeneration. Particularly, RBFOX2 promotes the inclusion of exon 6 in Numb, a Notch pathway regulator. This exon is required for SC activation, and its skipping delays activation while upregulating Notch signaling. Altogether, our study provides the AS landscape during SC activation and demonstrates that a single-gene splicing change can significantly influence SC activation and essential pathways such as Notch signaling.