The conserved landscape of RNA modifications and transcript diversity across mammalian evolution

Gene expression programs underpin the development of shared phenotypes, yet the importance of transcript complexity in shaping mammalian evolution remains unclear. Here we present a comprehensive long-read direct RNA sequencing atlas of full-length transcripts and their m6A modifications across six tissues (hippocampus, frontal cortex, cerebellum, testes, skeletal muscle, and liver) in five mammals (human, mouse, rat, dog, and cow) and a non-mammalian out-group (chicken). Our analysis reveals that 29% of genes have multiple mammalian-conserved alternative transcripts, with 31% of these genes showing tissue-specific switching of the major transcript isoforms. We uncover extensive conservation of coordinated splicing events, primarily driven by tissue-specific mutually associated exon splicing, particularly in neural tissues and cytoskeletal genes. At the epitranscriptome level, we find that 14.2% of m6A RNA modifications are conserved across mammals, with 39% of analysed genes containing a conserved m6A RNA modification. Our work provides unprecedented insight into the evolution of transcript complexity and the epitranscriptome, highlighting their potential roles in shaping mammalian phenotypic diversity and providing a valuable resource for understanding post-transcriptional regulation across mammalian evolutionary time.