METTL3-METTL14 Heterodimer Assembly Enables Multivalent RNA Engagement and Is Tuned by Interface-Targeting Peptides.
8 affiliations
- 1Department of Biochemical Sciences ″A. Rossi Fanelli″, Sapienza University of Rome, Piazzale Aldo 5, 00185Rome, Italy.
- 2School of Physiology, Pharmacology & Neuroscience, University of Bristol, Biomedical Sciences Building, University Walk, BristolBS8 1TD, U.K.
- 3Department of Biology and Biotechnologies ″Charles Darwin″, Sapienza University of Rome, Piazzale Aldo 5, 00185Rome, Italy.
- 4Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, Texas77030, United States.
- 5Department of Drug Chemistry and Technologies, Sapienza University of Rome, Piazzale Aldo 5, 00185Rome, Italy.
- 6Department of Science, Roma Tre University, Viale Marconi 446, 00146Rome, Italy.
- 7Department of Life Sciences, Health and Health Professions, Link Campus University, Via del Casale di S. Pio V, 44, 00165Roma, Italy.
- 8Biostructures and Biosystems National Institute (INBB), Via dei Carpegna 19, 00165Rome, Italy.
Abstract
RNA N6-methyladenosine (m6A) deposition by the METTL3-METTL14 complex regulates RNA metabolism and thereby influences cell differentiation and stress responses. Dysregulated m6A deposition has been implicated in multiple cancers, where METTL3-METTL14 supports oncogenic programs. Yet the molecular basis of RNA recognition by this complex remains unclear, in part due to the lack of an RNA-bound structure of the full assembly. Here, we combined native mass spectrometry (MS), molecular modeling, biochemical assays, and cell-based studies to define how METTL3-METTL14 engages RNA and how this process can be perturbed through interface targeting. Native MS of the intact heterodimer revealed simultaneous binding of multiple RNA molecules, with RNA-dependent stoichiometries and higher-occupancy states consistent with cooperative interactions. Molecular dynamics simulations identified a structured RNA-bound assembly in which the METTL14 C-terminal RGG-rich region stabilizes two RNA molecules, supporting a pre-engaged substrate configuration. We further developed a focused library of METTL14-derived peptides that bind METTL3 and interfere with protein-protein interactions, identifying the minimal METTL14 epitope for interface engagement. The most potent peptide, R8-14, binds with submicromolar affinity, disrupts both METTL3-METTL14 association and higher-order RNA-bound states, and reduces m6A levels, c-Myc expression, and cancer cell viability. Together, these findings demonstrate that RNA recognition by METTL3-METTL14 is governed by multivalent, higher-order interactions that enhance binding avidity. Notably, this assembly can be perturbed allosterically through protein-protein interface targeting rather than active-site inhibition, highlighting multivalent RNA recognition as a mechanistically and pharmacologically actionable feature of the RNA m6A writer complex.
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BibTeX
@article{fiorentino2026mettl3mettl14,
title = {{METTL3-METTL14 Heterodimer Assembly Enables Multivalent RNA Engagement and Is Tuned by Interface-Targeting Peptides.}},
author = {Francesco Fiorentino and Boyi Xiao and Bianca Cesaro and Sara Venezia and Jujun Zhou and Emanuele Fabbrizi and Fabrizio Casano and Gebremedhin Solomon Hailu and Xiaodong Cheng and Robin A Corey and Alessandro Fatica and Antonello Mai and Stefano Tomassi and Dante Rotili},
journal = {Journal of the American Chemical Society},
year = {2026},
month = oct,
doi = {10.1021/jacs.6c09634},
url = {https://europepmc.org/article/MED/42912293},
note = {CC BY},
}
RIS
TY - JOUR
TI - METTL3-METTL14 Heterodimer Assembly Enables Multivalent RNA Engagement and Is Tuned by Interface-Targeting Peptides.
AU - Francesco Fiorentino
AU - Boyi Xiao
AU - Bianca Cesaro
AU - Sara Venezia
AU - Jujun Zhou
AU - Emanuele Fabbrizi
AU - Fabrizio Casano
AU - Gebremedhin Solomon Hailu
AU - Xiaodong Cheng
AU - Robin A Corey
AU - Alessandro Fatica
AU - Antonello Mai
AU - Stefano Tomassi
AU - Dante Rotili
PY - 2026
DA - 2026/10/01
JO - Journal of the American Chemical Society
DO - 10.1021/jacs.6c09634
UR - https://europepmc.org/article/MED/42912293
AB - RNA N6-methyladenosine (m6A) deposition by the METTL3-METTL14 complex regulates RNA metabolism and thereby influences cell differentiation and stress responses. Dysregulated m6A deposition has been implicated in multiple cancers, where METTL3-METTL14 supports oncogenic programs. Yet the molecular basis of RNA recognition by this complex remains unclear, in part due to the lack of an RNA-bound structure of the full assembly. Here, we combined native mass spectrometry (MS), molecular modeling, biochemical assays, and cell-based studies to define how METTL3-METTL14 engages RNA and how this process can be perturbed through interface targeting. Native MS of the intact heterodimer revealed simultaneous binding of multiple RNA molecules, with RNA-dependent stoichiometries and higher-occupancy states consistent with cooperative interactions. Molecular dynamics simulations identified a structured RNA-bound assembly in which the METTL14 C-terminal RGG-rich region stabilizes two RNA molecules, supporting a pre-engaged substrate configuration. We further developed a focused library of METTL14-derived peptides that bind METTL3 and interfere with protein-protein interactions, identifying the minimal METTL14 epitope for interface engagement. The most potent peptide, R8-14, binds with submicromolar affinity, disrupts both METTL3-METTL14 association and higher-order RNA-bound states, and reduces m6A levels, c-Myc expression, and cancer cell viability. Together, these findings demonstrate that RNA recognition by METTL3-METTL14 is governed by multivalent, higher-order interactions that enhance binding avidity. Notably, this assembly can be perturbed allosterically through protein-protein interface targeting rather than active-site inhibition, highlighting multivalent RNA recognition as a mechanistically and pharmacologically actionable feature of the RNA m6A writer complex.
SN - 0002-7863
SN - 1520-5126
ER -