Antibody cross-reactivity accounts for widespread appearance of m1A in 5 UTRs

Abstract

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 confirmed the prevalence of m1A, while others have questioned it. 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, when we mapped m1A antibody-binding sites at single-nucleotide resolution, we found binding to transcription start nucleotides in mRNA 5 UTRs. Using different biochemical assays, we find that m1A is not present at these sites. Instead, we find that the m1A antibody exhibits m1A-independent binding to mRNA cap structures. We also tested a new and independently derived m1A antibody. We show that this m1A antibody lacks m7G cap-binding cross-reactivity, and notably does not map to 5 UTRs in the transcriptome. Our data demonstrate that high-stoichiometry m1A sites are rare in the transcriptome and that previous mapping of m1A to mRNA 5 UTRs are due to unintended binding of the m1A antibody to m7G cap structure in mRNA.