N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA, conserved across species and typically enriched near stop codons at the DRACH consensus motif. While deposition is catalyzed by the METTL3/14 catalytic core, how site specificity is achieved in vivo remains unclear.

Here, we used hepatocyte-specific conditional knockouts to dissect the roles of m6A-METTL- associated complex (MACOM) subunits in mouse liver. Transcriptome-wide mapping revealed that VIRMA, ZC3H13, and WTAP are essential for maintaining bulk methylation levels and enforcing canonical DRACH targeting within coding sequence (CDS) and 3’ untranslated regions (3’ UTRs). Loss of these proteins led to widespread redistribution of m6A, with depletion at canonical sites and emergence of m6A at noncanonical GA-rich motifs such as GAGGA. In parallel, RNA-seq demonstrated that transcripts with multiple m6A depleted sites were stabilized in a dose-dependent manner, whereas m6A increase had limited impact on RNA abundance. Mechanistically, VIRMA, WTAP, and ZC3H13 form a mutually stabilizing scaffold that anchors the METTL3/14 core in the nucleus and enables formation of the full ~ 1,000 kDa writer complex. Disruption of any one subunit prevented assembly of the intact complex.

Together, these findings establish MACOM subunits as central determinants for m6A deposition by maintaining complex integrity and nuclear localization of METTL3/14 in mouse liver.