Decoding mAm by simultaneous transcription-start mapping and methylation quantification.

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