Single-base resolution atlas reveals moderate conservation and regulatory diversity of m6A modifications across mammals.

RNA methylation, notably m6A modification, is a predominant epitranscriptomic alteration in mRNA, yet its evolutionary properties and the selective constraints acting on it across mammals remain poorly understood. Here, we generated a single-base-resolution m6A modification atlas in liver, kidney, and brain tissues across 21 non-model mammals using Nanopore direct RNA sequencing. We found that 25.54-35.70% of orthologous transcripts across examined species harbor m6A modifications, with m6A-modified sites exhibiting significantly greater conservation than nearby unmodified regions, probably under purifying selection. While m6A sites were preferentially enriched in RNA loops rather than stems, an inverse correlation between overall m6A levels and RNA splicing complexity was observed, a pattern which is compatible with a model in which exon junction complex (EJC)-associated, splice-junction-proximal mechanisms contribute to suppression of nearby m6A deposition. Further analyses of m6A-modified genes and life history traits uncovered that genes with higher m6A modification ratios in long-lived mammals were mainly characterized by relaxed selection. This study explores the evolutionary landscape of m6A modifications in non-model organisms, underscoring their diverse regulatory roles and evolutionary significance across mammalian lineages.