Decoding m6Am 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.