[Nanopore sequencing for RNA modification and splicing analysis in skeletal muscle]

Alternative splicing and chemical modifications of RNA are key mechanisms of post-transcriptional regulation. By modulating RNA structure, localization, stability, translation, and protein expression levels and diversity, they significantly influence cell fate and biological processes. In muscle tissue, these mechanisms are particularly important for myogenesis, muscle fiber type differentiation, energy metabolism regulation, and adaptation to environmental stress. In recent years, increasing attention has been paid to aberrant alternative splicing events and the functions of epitranscriptomic modifications in muscle development and diseases such as muscle atrophy, metabolic disorders, and stress-induced injury. Accurately identifying RNA modifications and alternative splicing changes is crucial for deciphering the intricate regulatory mechanisms in muscle biology and for improving muscle-related production traits in farmed animals. Third-generation sequencing technologies, particularly Nanopore sequencing technology, are advancing the study of RNA splicing and modifications by offering long-read capabilities and the ability to directly detect modification signals from raw data. With the support of bioinformatics tools such as Bonito, Nanopolish, DeepSignal, and m⁶ATM, which continuously optimize base calling, modification detection, and isoform resolution, the resolution and accuracy of data analysis have been greatly enhanced. This review summarizes the fundamental mechanisms and research progress in RNA splicing and modifications, with a focus on the application prospects of Nanopore sequencing in epitranscriptomics, particularly its potential value in the research on muscle development and the diagnosis of muscle-related diseases. The advantages and challenges of nanopore sequencing technology and its associated bioinformatics methods in epigenomic studies are systematically summarized, providing guidance for subsequent research in technical selection, methodological optimization, and functional mechanism exploration. This review also lays a theoretical foundation for advancing studies on muscle development and muscle-related diseases.