Cotranslational assembly directs the biogenesis of the m⁶A methyltransferase complex

The chemical modification N⁶-methyladenosine (m⁶A) is catalyzed by the m⁶A methyltransferase complex (MTC) comprising METTL3 and METTL14 in the nucleus. Structural evidence reveals that METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding scaffold. However, the mechanism directing the complex assembly in vivo remains enigmatic. Here, we demonstrate that MTC is formed by a cotranslational mechanism in which nascent METTL3 interacts with METTL14 polypeptide chain exposed from the ribosome exit tunnel. The methyltransferase domains in the subunits determine the specificity of their cotranslational interaction. In contrast, WTAP, the regulatory subunit of MTC, is recruited to the complex posttranslationally. We further identify CCT4, the key subunit of cytosolic chaperonin TCP-1, as an essential facilitator of the endogenous MTC assembly process. Depletion of CCT4 results in dramatic reduction of METTL3-METTL14 heterodimer formation. Remarkably, we engineer a cell-permeable peptide M14P1, which could disrupt cotranslational assembly of MTC, thereby impairing m⁶A deposition and significantly attenuating the proliferation of acute myeloid leukemia (AML) cells and promoting apoptosis. Collectively, our findings unravel intrinsic mechanisms governing the in vivo assembly of MTC, and provide a potential therapeutic strategy to disrupt oncogenic m⁶A pathways and impede AML progression.