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 levels 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 initiation.