Decoding m(6)Am by simultaneous transcription-start mapping and methylation quantification.

N (6),2'-O-dimethyladenosine (m(6)Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m(6)Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5' isoforms. Thus, gene-level annotations cannot capture the diversity of m(6)Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m(6)Am stoichiometry for each 5' isoform that initiates with adenosine. Using CROWN-seq, we map the m(6)Am landscape in nine human cell lines. Our findings reveal that m(6)Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m(6)Am stoichiometry. We find that m(6)Am is associated with increased transcript expression and provide evidence that m(6)Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m(6)Am in influencing transcription.