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Peer-reviewedCC BY 4.0PLOS Genetics

Characterization of METTL3/14-mediated m⁶A modification in human transcriptome using Nanopore direct RNA sequencing

  • Emily Kurtyan1
  • Andrew J. Stein2
  • Kelly J. Abdalla3
  • Zhangerjiao Yuan4
  • Miten Jain
  • Fadia Ibrahim
4 affiliations
  1. 1Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
  2. 2Department of Bioengineering, Northeastern University, Boston, Massachusetts, United States of America
  3. 3Department of Physics, Northeastern University, Boston, Massachusetts, United States of America
  4. 4Khoury College of Computer Sciences, Northeastern University, Boston, Massachusetts, United States of America
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Abstract

Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), 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 m6A 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 m6A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an in vitro transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m6A 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 m6A 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. Author summary: N6-methyladenosine (m6A) modifications regulate RNA metabolism by dynamically modulating RNA processing, translation, stability, and decay. This modification is primarily deposited by the METTL3/METTL14 complex, facilitating various cellular processes. Oxford Nanopore Technologies enables direct RNA sequencing (dRNA-Seq) at single-molecule resolution without disrupting its associated modifications. However, despite continued advancements, several challenges remain unresolved. To harness the full potential of this technology and uncover key biological insights into m6A modifications, we evaluated m6Anet and Dorado modification detection models and the two existing dRNA-Seq chemistries (RNA002/RNA004) following METTL3 or METTL14 depletion in human cells. We report genome- and transcriptome-wide changes in m6A sites and stoichiometry, as well as a global reduction of m6A levels across highly modified genes/sites, coupled with poly(A) tail shortening following METTL3/14 depletion. We reveal a functional complexity of METTL3/14-mediated regulation of modified transcripts and lay the foundation for understanding how post-transcriptional RNA modifications govern mRNA fate and contribute to disease pathogenesis.

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Cite

BibTeXRIS
BibTeX
@article{kurtyan2026characterization,
  title = {{Characterization of METTL3/14-mediated m⁶A modification in human transcriptome using Nanopore direct RNA sequencing}},
  author = {Emily Kurtyan and Andrew J. Stein and Kelly J. Abdalla and Zhangerjiao Yuan and Miten Jain and Fadia Ibrahim},
  journal = {PLOS Genetics},
  year = {2026},
  month = sep,
  doi = {10.1371/journal.pgen.1012278},
  url = {https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012278},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Characterization of METTL3/14-mediated m⁶A modification in human transcriptome using Nanopore direct RNA sequencing
AU  - Emily Kurtyan
AU  - Andrew J. Stein
AU  - Kelly J. Abdalla
AU  - Zhangerjiao Yuan
AU  - Miten Jain
AU  - Fadia Ibrahim
PY  - 2026
DA  - 2026/09/01
JO  - PLOS Genetics
DO  - 10.1371/journal.pgen.1012278
UR  - https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012278
AB  - Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), 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 m6A 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 m6A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an in vitro transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m6A 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 m6A 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. Author summary: N6-methyladenosine (m6A) modifications regulate RNA metabolism by dynamically modulating RNA processing, translation, stability, and decay. This modification is primarily deposited by the METTL3/METTL14 complex, facilitating various cellular processes. Oxford Nanopore Technologies enables direct RNA sequencing (dRNA-Seq) at single-molecule resolution without disrupting its associated modifications. However, despite continued advancements, several challenges remain unresolved. To harness the full potential of this technology and uncover key biological insights into m6A modifications, we evaluated m6Anet and Dorado modification detection models and the two existing dRNA-Seq chemistries (RNA002/RNA004) following METTL3 or METTL14 depletion in human cells. We report genome- and transcriptome-wide changes in m6A sites and stoichiometry, as well as a global reduction of m6A levels across highly modified genes/sites, coupled with poly(A) tail shortening following METTL3/14 depletion. We reveal a functional complexity of METTL3/14-mediated regulation of modified transcripts and lay the foundation for understanding how post-transcriptional RNA modifications govern mRNA fate and contribute to disease pathogenesis.
SN  - 1553-7404
ER  -