Comprehensive analysis of pre-mRNA alternative splicing regulated by m6A methylation in pig oxidative and glycolytic skeletal muscles.

ABSTRACT

BACKGROUND: Different types of skeletal myofibers exhibit distinct physiological and metabolic properties that are associated with meat quality traits in livestock. Alternative splicing (AS) of pre-mRNA can generate multiple transcripts from an individual gene by differential selection of splice sites. N6-methyladenosine (m6A) is the most abundant modification in mRNAs, but its regulation for AS in different muscles remains unknown.  RESULTS: We characterized AS events and m6A methylation pattern in pig oxidative and glycolytic muscles. A tota1 of 1294 differential AS events were identified, and differentially spliced genes were significantly enriched in processes related to different phenotypes between oxidative and glycolytic muscles. We constructed the regulatory network between splicing factors and corresponding differential AS events and identified NOVA1 and KHDRBS2 as key splicing factors. AS event was enriched in m6A-modified genes, and the methylation level was positively correlated with the number of AS events in genes. The dynamic change in m6A enrichment was associated with 115 differentially skipping exon (SE-DAS) events within 92 genes involving in various processes, including muscle contraction and myofibril assembly. We obtained 23.4% SE-DAS events (27/115) regulated by METTL3-meditaed m6A and experimentally validated the aberrant splicing of ZNF280D, PHE4DIP, and NEB. The inhibition of m6A methyltransferase METTL3 could induce the conversion of oxidative fiber to glycolytic fiber in PSCs. CONCLUSION: Our study suggested that m6A modification could contribute to significant difference in phenotypes between oxidative and glycolytic muscles by mediating the regulation of AS. These findings would provide novel insights into mechanisms underlying muscle fiber conversion.