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

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.