Cap-specific, terminal N6-methylation by a mammalian m6Am methyltransferase

Identification of N6,2′-O-dimethyladenosine methyltransferase. a Chemical structure of m6Am, which is adjacent to the m7G mRNA cap. b Cartoon view of the predicted domain structure of PCIF1, with the conserved “NPPF” motif in the zoom-in view. A sequence alignment is shown below to highlight the high conservation of the key residues for PCIF1 orthologues. Residue 43–77 (blue segment) represents the WW domain, and the red segment denotes the putative catalytic methyltransferase domain. c LC-MS/MS quantification of the m6Am/A ratios of HEK293T polyA？+？RNA treated with control or PCIF1 siRNA (n？=？3). d LC-MS/MS quantification of the m6A/A ratios of HEK293T polyA+ RNA treated with control or PCIF1 siRNA (n？=？3). e Quantification of the m6Am/(Am？+？m6Am) ratios in RNA probes starting with different cap structure (n？=？3). f Quantification of the methylation activity of WT and mutant PCIF1 proteins (n？=？3). g Distribution of enriched m6A/m6Am peak density across mRNA segments of control and PCIF1 knockdown samples using an m6A-seq protocol with random priming. Each segment was normalized according to its average length in Ref-seq annotation. h One representative transcript harboring m6Am and m6A peaks. The m6Am peak at the 5′-terminal is significantly decreased upon PCIF1 knockdown, while the m6A peak at the 3′-UTR stays the same. The grey line denotes “Input”, and the red line denotes “IP”. i Boxplot of log2fold change of peak score in PCIF1 knockdown and control mRNA. Enriched peaks are classified into three groups: m6Am peak (near TSS and without GGACH motif), m6A peak (not in TSS and with GGACH motif), and the rest (potentially m6Am？+？m6A). j A proposed model for mammalian mRNA m6Am modification mediated by PCIF1. *P？<？0.05; **P？<？0.01; ***P？<？0.001; ns, not significan