Antibody cross-reactivity accounts for widespread appearance of m1A in 5’UTRs
Year: 2019
Type: article
Abstract: N 1 -methyladenosine (m 1 A) was proposed to be a highly prevalent modification in mRNA 5’UTRs based on mapping studies using an m 1 A-binding antibody. We developed a bioinformatic approach to discover m 1 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 1 A only in one mRNA: the mitochondrial MT-ND5 transcript. As an alternative approach, we also developed an antibody-based m 1 A-mapping approach to detect m 1 A at single-nucleotide resolution, and confirmed that the commonly used m 1 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 7 G-cap. A different m 1 A antibody that lacks cap-binding cross-reactivity does not show enriched binding in 5’UTRs. These results demonstrate that high-stoichiometry m 1 A sites are exceedingly rare in mRNAs and that previous mappings of m 1 A to 5’UTRs were the result of antibody cross-reactivity to the 5’ cap.
Source: Nature Communications
Institution: Weill Cornell Medicine
Language: English
Topic: RNA modifications and cancer
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $5,200
Funders: Burroughs Wellcome Fund, National Institutes of Health, National Heart, Lung, and Blood Institute, National Institute on Drug Abuse, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Neurological Disorders and Stroke, National Center for Advancing Translational Sciences, Eunice Kennedy Shriver National Institute of Child Health and Human Development
Awards: Risk Genes and Environmental Interactions in Neural Tube Defects, Training Program in Developmental and Stem Cell Biology, Pathways controlling mRNA translation pathways in dopaminergic neurons, Clinical and Translational Science Center, Cancer Pharmacology, HIV+ Alveolar Macrophage Oxidant-mediated Apoptosis of Pulmonary Endothelium, Characterizing the transcriptome-wide location and function of 8oxoG, G9000004251, G9000015526, Clinical and Translational Science Center, New mechanisms and technologies for understanding post-transcriptional gene regulation in neurons, I. Institutional Career Development Core
OpenAlex ID: W2986901350