Systematic identification of tissue-conserved m⁶A sites reveals a stable epitranscriptomic regulatory layer...

Chemical modifications to RNA are fundamental regulators of cellular identity and function. Among these, N6-methyladenosine (m⁶A) is the most abundant mRNA modification in mammalian cell, governing major post-transcriptional processes. While conditional m⁶A dynamics are well studied, the extent and function of condition-independent, tissue-conserved (TC) m⁶A in humans remain unclear. Here we show that 5,945 TC sites are consistently methylated across 24 human tissues. These sites are enriched near stop codons, evolutionarily conserved, and characterized by distinct sequence signatures. RBM15/B are identified as candidate mediators of TC m⁶A deposition, and YTHDF1-3 and UPF1 are preferentially enriched at TC sites, supporting their role for m⁶A-linked mRNA decay. TC m⁶A sites mark 1,386 genes essential for core cellular processes like autophagy and homeostasis, showing stable expression and evolutionary constraint. Pan-cancer analysis reveals that TC m⁶A genes are disproportionately differentially expressed, alongside with altered RBM15/B expression, suggesting that disruption of this stable m⁶A layer may contribute to transcriptional changes in cancer.