VIRMA mediates preferential m⁶A mRNA methylation in 3'UTR and near stop codon and associates with alternative...

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.