Identification of novel mRNA splicing isoforms in human myogenesis

Myogenesis, the process of muscle formation and regeneration, requires significant changes in gene expression program. However, alternative splicing during human myogenesis has not been systematically studied. Here, we examined mRNA splicing changes during myogenesis in two human myoblast cell lines using hybrid transcriptomic approaches by combining short-read (Illumina) and long-read (Nanopore) RNA-seq analyses. We identified 13,853 new significant splicing isoforms (60,582 in total), with RNAs increasing and decreasing in abundance between days 0 and 3 (3,771 and 3,649, respectively), and between days 3 and 5 (1,302 and 1,109, respectively). We identified 1,937 significant differential transcript usage events (DTU), which pathways were involved in muscle regulation. Findings were validated using RT-qPCR and across mouse and human models, including clinical samples from peripheral artery disease patients. Artificial Intelligence algorithms predicted 595 myogenesis-associated high-confidence novel protein-coding splicing isoforms. Our findings underscore the importance of mRNA splicing isoforms in muscle biology.