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- Title
Antibody cross-reactivity accounts for widespread appearance of m<sup>1</sup>A in 5'UTRs.
- Authors
Grozhik, Anya V.; Olarerin-George, Anthony O.; Sindelar, Miriam; Li, Xing; Gross, Steven S.; Jaffrey, Samie R.
- 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. N1-methyladenosine (m1A) was recently reported as a new mRNA modification but its prevalence has been controversial. Here the authors showed that m1A, if present in mRNA, is at very low stoichiometry, with the notable exception of MT-ND5. Further, they show that the previously reported enrichment of m1A near the start of transcripts are false-positive identifications due to cross-reactivity of the commonly used m1A antibody with mRNA caps.
- Subjects
IMMUNOGLOBULINS; MESSENGER RNA; CARTOGRAPHY; BIOINFORMATICS; NUCLEOTIDES
- Publication
Nature Communications, 2019, Vol 10, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-019-13146-w