{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T18:13:54Z","timestamp":1788286434485,"version":"build-2803163510"},"reference-count":78,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01GM149825, R01GM149825-02S1"],"award-info":[{"award-number":["R01GM149825, R01GM149825-02S1"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01HG013876"],"award-info":[{"award-number":["R01HG013876"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Cancer Institute, Cancer Genomics Core at Sidney Kimmel Cancer Center, Thomas Jefferson University","award":["P30 CA56036"],"award-info":[{"award-number":["P30 CA56036"]}]}],"content-domain":{"domain":["www.plosgenetics.org"],"crossmark-restriction":false},"short-container-title":["PLoS Genet"],"abstract":"<jats:p>\n                    Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N\n                    <jats:sup>6<\/jats:sup>\n                    -methyladenosine (m\n                    <jats:sup>6<\/jats:sup>\n                    A), one of the most abundant internal RNA modifications, is deposited by the core methyltransferase complex, METTL3 and METTL14. Oxford Nanopore Technologies (ONT) platform permits direct, single RNA molecule sequencing while preserving native modifications. However, without rigorous benchmarking, the accuracy and reproducibility of modification detection remain uncertain. Here, we leveraged ONT to comprehensively profile bona fide m\n                    <jats:sup>6<\/jats:sup>\n                    A modifications in cellular RNAs at single-nucleotide resolution by integrating two direct RNA sequencing chemistries (RNA002 and RNA004) with the m6Anet and Dorado modification-detection models. We independently depleted METTL3 and METTL14 in human cells and rigorously validated modification calls through several assays and independent orthogonal methods (GLORI and miCLIP). We find that Dorado detected a higher number of m\n                    <jats:sup>6<\/jats:sup>\n                    A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an\n                    <jats:italic>in vitro<\/jats:italic>\n                    transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m\n                    <jats:sup>6<\/jats:sup>\n                    A sites and stoichiometry within coding sequences and across genes, particularly in highly modified genes and sites, and at consensus DRACH motifs. We report a differential and complex regulation of modified transcripts, accompanied by a global reduction in poly(A) tail length. Notably, METTL3 and METTL14 depletion produced distinct transcript-specific effects, supporting non-redundant roles within the m\n                    <jats:sup>6<\/jats:sup>\n                    A writer complex. Together, our study illustrates a notable advancement of ONT capabilities and establishes a robust transcriptome-wide framework for RNA modification detection, thereby laying the groundwork for exploring the contribution of METTL3\/METTL14 to cellular functions and disease.\n                  <\/jats:p>","DOI":"10.1371\/journal.pgen.1012278","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T17:25:51Z","timestamp":1788283551000},"page":"e1012278","update-policy":"https:\/\/doi.org\/10.1371\/journal.pgen.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Characterization of METTL3\/14-mediated m6A modification in human transcriptome using Nanopore direct RNA sequencing"],"prefix":"10.1371","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-0332-0865","authenticated-orcid":true,"given":"Emily","family":"Kurtyan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-6525-6512","authenticated-orcid":true,"given":"Andrew J.","family":"Stein","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelly J.","family":"Abdalla","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhangerjiao","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miten","family":"Jain","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4344-765X","authenticated-orcid":true,"given":"Fadia","family":"Ibrahim","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"issue":"6","key":"pgen.1012278.ref001","doi-asserted-by":"crossref","first-page":"1388","DOI":"10.1016\/j.cell.2015.05.014","article-title":"N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency","volume":"161","author":"X 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