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

Nat Commun. 2019 Nov 12;10(1):5126. doi: 10.1038/s41467-019-13146-w.

Abstract

N1-methyladenosine (m1A) was proposed to be a highly prevalent modification in mRNA 5'UTRs based on mapping studies using an m1A-binding antibody. We developed a bioinformatic approach to discover m1A and other modifications in mRNA throughout the transcriptome by analyzing preexisting ultra-deep RNA-Seq data for modification-induced misincorporations. Using this approach, we detected appreciable levels of m1A only in one mRNA: the mitochondrial MT-ND5 transcript. As an alternative approach, we also developed an antibody-based m1A-mapping approach to detect m1A at single-nucleotide resolution, and confirmed that the commonly used m1A antibody maps sites to the transcription-start site in mRNA 5'UTRs. However, further analysis revealed that these were false-positives caused by binding of the antibody to the m7G-cap. A different m1A antibody that lacks cap-binding cross-reactivity does not show enriched binding in 5'UTRs. These results demonstrate that high-stoichiometry m1A sites are exceedingly rare in mRNAs and that previous mappings of m1A to 5'UTRs were the result of antibody cross-reactivity to the 5' cap.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions / genetics*
  • Adenosine / analogs & derivatives*
  • Adenosine / metabolism
  • Animals
  • Antibodies / immunology*
  • Base Sequence
  • Cross Reactions / immunology*
  • Female
  • HEK293 Cells
  • Humans
  • Mice, Inbred C57BL
  • Nucleotides / metabolism
  • RNA Caps / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Transcriptome / genetics

Substances

  • 5' Untranslated Regions
  • Antibodies
  • Nucleotides
  • RNA Caps
  • RNA, Messenger
  • 1-methyladenosine
  • Adenosine