N⁶-methyladenosine (m⁶A) is enriched in 3′untranslated region (3′UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m⁶A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3′UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m⁶A enrichment in 3′UTR. Depletions of VIRMA and METTL3 induce 3′UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3′UTR of over 2800 mRNAs, 84% of which are modified with m⁶A and have increased m⁶A peak density in 3′UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m⁶A deposition specificity in 3′UTR and its correlation with alternative polyadenylation.

N6-Methyladenosine (m⁶A) modification has been implicated in the progression of several cancers. We reveal that during epithelial-mesenchymal transition (EMT), one important step for cancer cell metastasis, m⁶A modification of mRNAs increases in cancer cells. Deletion of methyltransferase-like 3 (METTL3) down-regulates m⁶A, impairs the migration, invasion and EMT of cancer cells both in vitro and in vivo. m⁶A-sequencing and functional studies confirm that Snail, a key transcription factor of EMT, is involved in m⁶A-regulated EMT. m⁶A in Snail CDS, but not 3’UTR, triggers polysome-mediated translation of Snail mRNA in cancer cells. Loss and gain functional studies confirm that YTHDF1 mediates m⁶A-increased translation of Snail mRNA. Moreover, the upregulation of METTL3 and YTHDF1 act as adverse prognosis factors for overall survival (OS) rate of liver cancer patients. Our study highlights the critical roles of m⁶A on regulation of EMT in cancer cells and translation of Snail during this process.

N⁶-methyladenosine (m⁶A) is the most abundant ribonucleotide modification among eukaryotic messenger RNAs. The m⁶A “writer” consists of the catalytic subunit m⁶A-METTL complex (MAC) and the regulatory subunit m⁶A-METTL-associated complex (MACOM), the latter being essential for enzymatic activity. Here, we report the cryo-electron microscopy (cryo-EM) structures of MACOM at a 3.0-Å resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and that ZC3H13 stretches the conformation by binding VIRMA. Furthermore, the 4.4-Å resolution cryo-EM map of the MACOM–MAC complex, combined with crosslinking mass spectrometry and GST pull-down analysis, elucidates a plausible model of the m⁶A writer complex, in which MACOM binds to MAC mainly through WTAP and METTL3 interactions. In combination with in vitro RNA substrate binding and m⁶A methyltransferase activity assays, our results illustrate the molecular basis of how MACOM assembles and interacts with MAC to form an active m⁶A writer complex.