Antibody cross-reactivity accounts for widespread appearance of m 1 A in 5'UTRs

• Published in: Nature communications 2019-11, Vol.10 (1), p.5126
• Main Authors: Grozhik, Anya V, Olarerin-George, Anthony O, Sindelar, Miriam, Li, Xing, Gross, Steven S, Jaffrey, Samie R
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 2041-1723
• DOI: 10.1038/s41467-019-13146-w

N -methyladenosine (m A) was proposed to be a highly prevalent modification in mRNA 5'UTRs based on mapping studies using an m A-binding antibody. We developed a bioinformatic approach to discover m A 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 m A only in one mRNA: the mitochondrial MT-ND5 transcript. As an alternative approach, we also developed an antibody-based m A-mapping approach to detect m A at single-nucleotide resolution, and confirmed that the commonly used m A 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 m G-cap. A different m A antibody that lacks cap-binding cross-reactivity does not show enriched binding in 5'UTRs. These results demonstrate that high-stoichiometry m A sites are exceedingly rare in mRNAs and that previous mappings of m A to 5'UTRs were the result of antibody cross-reactivity to the 5' cap.