A metabolic labeling method detects m⁶A transcriptome-wide at single base resolution

Transcriptome-wide mapping of N⁶-methyladenosine (m⁶A) at base resolution remains an issue, impeding our understanding of m⁶A roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA m⁶A transcriptome-wide at base resolution, called 'm⁶A-label-seq'. Human and mouse cells could be fed with a methionine analog, Se-allyl-L-selenohomocysteine, which substitutes the methyl group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N⁶-allyladenosine (a⁶A) at supposed m⁶A-generating adenosine sites. We pinpointed the mRNA a⁶A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA m⁶A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m⁶A-label-seq with available m⁶A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m⁶A sites and holds promise to locate nuclear nascent RNA m⁶A modifications.