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

Transcriptome-wide mapping of N-6-methyladenosine (m(6)A) at base resolution remains an issue, impeding our understanding of m(6)A roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA m(6)A transcriptome-wide at base resolution, called 'm(6)A-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 N-6-allyladenosine (a(6)A) at supposed m(6)A-generating adenosine sites. We pinpointed the mRNA a(6)A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA m(6)A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m(6)A-label-seq with available m(6)A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m(6)A sites and holds promise to locate nuclear nascent RNA m(6)A modifications.