Isoform-specific m6A deposition and coordinated splicing shape mammalian transcriptome evolution

Alternative isoform usage and RNA modifications are fundamental to transcriptome evolution. Among these, N6-methyladenosine (m6A), the most abundant internal mRNA modification, plays a key role in gene regulation. However, due to the limitations of short-read technology, the evolutionary conservation and phenotypic impact of transcript isoforms and m6A modifications remain incomplete. Here we present a comparative evolutionary analysis using direct-RNA long-read sequencing of six tissues from species representing three major clades within Mammalia, along with an avian outgroup. We find that although 71% of transcript isoforms are species-specific, they contribute less than 3% to total mRNA gene expression, whereas 18% of mammalian-conserved isoforms account for the majority of mRNA gene expression. We also identify that 14.2% of m6A modification sites, present in 39% of genes, are conserved across mammals, with enrichment in 3’-untranslated regions and stop codon-proximal regions. Notably, 27.3% of conserved m6A sites display isoform-specific deposition, supporting a role for epitranscriptomic regulation in maintaining functional transcript diversity. Finally, we uncover widespread conservation of coordinate splicing, in which exon co-regulation compensates for frameshift-inducing changes in individual exons, suggesting a buffering mechanism in isoform regulation. Together, these findings provide insight into how post-transcriptional regulation shapes phenotypic diversity and evolutionary adaptation in mammals. How RNA isoforms and chemical marks evolve across species remains poorly understood. This study uses direct RNA long-read sequencing across mammals to show that conserved isoforms account for most expression, while isoform-specific conserved m6A and coordinated splicing reveal conserved regulatory mechanisms.