METTL3–METTL14 Heterodimer Assembly Enables Multivalent RNA Engagement and Is Tuned by Interface-Targeting Peptides

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.