The Mammalian Cap-Specific m⁶Am RNA Methyltransferase PCIF1 Regulates Transcript Levels in Mouse Tissues

The 5' end of eukaryotic mRNAs is protected by the m⁷G-cap structure. The transcription start site nucleotide is ribose methylated (Nm) in many eukaryotes, whereas an adenosine at this position is further methylated at the N⁶ position (m⁶A) by the mammalian Phosphorylated C-terminal domain (CTD)-interacting Factor 1 (PCIF1) to generate m⁶Am. Here, we show that although the loss of cap-specific m⁶Am in mice does not affect viability or fertility, the Pcif1 mutants display reduced body weight. Transcriptome analyses of mutant mouse tissues support a role for the cap-specific m⁶Am modification in stabilizing transcripts. In contrast, the Drosophila Pcif1 is catalytically dead, but like its mammalian counterpart, it retains the ability to associate with the Ser5-phosphorylated CTD of RNA polymerase II (RNA Pol II). Finally, we show that the Trypanosoma Pcif1 is an m⁶Am methylase that contributes to the N⁶,N⁶,2'-O-trimethyladenosine (m⁶₂Am) in the hypermethylated cap4 structure of trypanosomatids. Thus, PCIF1 has evolved to function in catalytic and non-catalytic roles.