3'UTR shortening alleviates miRNA repression of mRNAs critical for muscle stem cell differentiation.

Alternative polyadenylation (APA) modulates gene expression by altering 3' untranslated region (3'UTR) length. Although 3'UTR lengthening typically accompanies cell differentiation, we unexpectedly observed preferential APA-mediated 3'UTR shortening events during muscle stem cell (satellite cell, SC) differentiation, coinciding with increased muscle-specific miRNAs (myomiRs) targeting at alternative 3'UTRs. Mechanistically, this shortening primarily results from reduced cleavage factor I (CFI) expression and allows transcripts to escape repression by differentiation-induced myomiRs. Interestingly, perturbation of mRNA 3'UTR shortening of multiple genes impairs myogenic differentiation. Focusing on Matr3-a gene linked to muscle disorders-we demonstrate that its APA-miRNA regulatory balance is critical for efficient SC differentiation in vitro. Genetically mutating Matr3 proximal polyadenylation site (pA site) impaired mouse muscle regeneration in vivo. Together, our findings reveal that APA-mediated 3'UTR shortening counteracts miRNA repression to orchestrate the gene expression program essential for robust muscle regeneration.