Antibody crossreactivity with mRNA cap accounts for widespread appearance of m1A throughout the transcriptome

N1-methyladenosine (m1A) was recently identified as a new mRNA modification based on its mapping to the 5’UTRs of thousands of mRNAs with an m1A-binding antibody. More recent studies have questioned the prevalence of m1A. To address this discrepancy, we mapped m1A using ultra-deep RNA-Seq datasets based on m1A-induced misincorporations during reverse transcription. Using this approach, we find m1A only in the mitochondrial MT-ND5 transcript. In contrast, m1A mapping using an m1A-binding antibody showed binding to transcription start nucleotides in mRNA 5’UTRs. Biochemical measurements indicate that m1A is not present at these sites. Instead, we find that the m1A antibody exhibits m1A-independent binding to mRNA cap structures. Our data demonstrate that high-stoichiometry m1A sites are rare in the transcriptome and that previous mapping of m1A to mRNA 5’UTRs reflect an unintended binding to mRNA caps. The data for this study contains the following samples: m1A miCLIP of HEK 293T cell poly(A) RNA: 2 biological replicates RNA-Seq of HEK 293T cell poly(A) RNA: 1 biological replicate m1A miCLIP in mouse brain poly(A) RNA: 6 biological replicates (3 control, 3 AlkB-treated) Second 1mA miCLIP of HEK 293T cells poly(A) RNA: 6 samples (2 biological replicates per antibody, 3 different antibodies)