Local translational program at the muscle-tendon junction endows domain identity in muscle syncytia

Abstract How cells establish specialized subdomains is a fundamental question in cell and tissue biology. Skeletal muscle fibers, among the largest cells in the body, are multinucleated and form distinct regions such as the neuromuscular and myotendinous junctions (MTJ), the latter forming a critical interface between muscle and tendon that transmits contractile force. While transcriptional heterogeneity among myonuclei has been described, whether local translation contributes to domain identity remains unknown, largely due to the lack of tools for domain-specific manipulation. Here, we introduce MTJ-AAV, a viral system that enables selective genetic targeting of MTJ myonuclei. This approach allowed MTJ-specific ribosome tagging and revealed extensive translational regulation underlying MTJ biology and its remodeling during exercise. Interestingly, untranslated regions of these transcripts were sufficient to control regionalized translation. Notably, the KLF-family transcription factors emerged as translationally upregulated targets at the MTJ, where they drive local gene expression. Our findings establish local translation as a key layer of subcellular specialization and provide a versatile toolkit for dissecting spatial molecular regulation within muscle syncytia.