Comprehensive analysis of m6A-seq data reveals distinct features of conserved and unique m6A sites in mammals.

N6-methyladenine (m6A) stands out as the most prevalent internal chemical modification on mammalian mRNA, playing a vital role in diverse biological processes. However, the characteristics of m6A across different cell lines and tissues remain poorly understood. In this study, we systematically evaluated 193 published m6A-seq data sets using newly established quality metrics, identifying ∼1.5 million high-confidence m6A sites in human and mouse. By categorizing m6A sites into different consistency levels, we observed that high-consistency m6A sites were enriched near mRNA stop codons and lncRNA 5' ends, exhibited stronger interactions with canonical m6A-binding proteins, and contributed to mRNA/lncRNA expression homeostasis. Furthermore, the promoters of genes marked by these consistent sites exhibited higher CpG density, with METTL3 preferentially binding to these regions. Conversely, low-consistency or unique m6A sites were enriched near mRNA start codons and distributed evenly across lncRNA, interacting with newly discovered m6A-binding proteins. These findings enhance our understanding of the diverse characteristics and potential functional roles of m6A in mammals.