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Decoding m6Am by simultaneous transcription-start mapping and methylation quantification.

Jianheng Fox Liu | Ben R Hawley | Luke S Nicholson | Samie R Jaffrey
eLife | 2025

N 6,2'-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am 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 m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5' isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.

Pubmed ID: 40162895

Associated grants

  • Agency: NIMH NIH HHS, United States
    Id: R01 MH121072
  • Agency: NIMH NIH HHS, United States
    Id: MH121072
  • Agency: NHGRI NIH HHS, United States
    Id: RM1 HG011563
  • Agency: ODCDC CDC HHS, United States
    Id: S10 OD030335
  • Agency: NIH HHS, United States
    Id: S10 OD030335
  • Agency: NIDA NIH HHS, United States
    Id: R01 DA059544
  • Agency: NINDS NIH HHS, United States
    Id: R35 NS111631

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