The cap-specific m6A methyltransferase, PCIF1/CAPAM, is dynamically recruited to the gene promoter in a transcription-dependent manner.

N 6-methyladenosine (m6A), the most abundant modification in eukaryotic mRNAs, plays an important role in mRNA metabolism and functions. When adenosine is transcribed as the first cap-adjacent nucleotide, it is methylated at the ribose 2'-O and N6 positions, thus generating N6, 2'-O-dimethyladenosine (m6Am). Phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1) is a novel cap-specific adenine N6-methyltransferase responsible for m6Am formation. As PCIF1 specifically interacts with the Ser5-phosphorylated CTD of RNA polymerase II (Pol II), which is a marker for the early phase of transcription, PCIF1 is speculated to be recruited to the early elongating Pol II. In this study, subcellular fractionation and immunofluorescence microscopy demonstrated that PCIF1 is mainly localized to the transcriptionally active chromatin regions in HeLa cells. Chromatin immunoprecipitation (ChIP) revealed that PCIF1 was predominantly localized to the promoter of a broad range of Pol II-transcribed genes, including several protein-coding genes and non-coding RNA genes. Moreover, PCIF1 accumulation on these promoters depended entirely on transcriptional activity and Ser5 phosphorylation of the CTD. These results suggest that PCIF1 dynamically localizes to the Pol II early in transcription and may efficiently catalyze N6-methylation of the first adenosine residue of nascent mRNAs cotranscriptionally.

Sugita A ，Kuruma S ，Yanagisawa N ，Ishiguro H ，Kano R ，Ohkuma Y ，Hirose Y ... -  《-》

PCIF1 Catalyzes m6Am mRNA Methylation to Regulate Gene Expression.

mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs. Furthermore, PCIF1 catalyzes only 5' m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.

Sendinc E ，Valle-Garcia D ，Dhall A ，Chen H ，Henriques T ，Navarrete-Perea J ，Sheng W ，Gygi SP ，Adelman K ，Shi Y ... -  《-》

The Mammalian Cap-Specific m(6)Am 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.

Pandey RR ，Delfino E ，Homolka D ，Roithova A ，Chen KM ，Li L ，Franco G ，Vågbø CB ，Taillebourg E ，Fauvarque MO ，Pillai RS ... -  《Cell Reports》

Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs.

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2'O-methyladenosine (Am), generating N6, 2'O-dimethyladenosine (m6Am) when Am is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and Am), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2'O-methylated Am over unmodified A nucleosides is due mainly to increased binding affinity for Am. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5'-Am oligonucleotide with approximately the same affinity as that of a cap analog (KM = 0.4 versus 0.3 μM). In addition, PCIF1 methylates the uncapped 5'-Am with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.

Yu D ，Dai N ，Wolf EJ ，Corrêa IR Jr ，Zhou J ，Wu T ，Blumenthal RM ，Zhang X ，Cheng X ... -  《-》

HIV reprograms host m(6)Am RNA methylome by viral Vpr protein-mediated degradation of PCIF1.

N6,2'-O-dimethyladenosine (m6Am) is an abundant RNA modification located adjacent to the 5'-end of the mRNA 7-methylguanosine (m7G) cap structure. m6A methylation on 2'-O-methylated A at the 5'-ends of mRNAs is catalyzed by the methyltransferase Phosphorylated CTD Interacting Factor 1 (PCIF1). The role of m6Am and the function of PCIF1 in regulating host-pathogens interactions are unknown. Here, we investigate the dynamics and reprogramming of the host m6Am RNA methylome during HIV infection. We show that HIV infection induces a dramatic decrease in m6Am of cellular mRNAs. By using PCIF1 depleted T cells, we identify 2237 m6Am genes and 854 are affected by HIV infection. Strikingly, we find that PCIF1 methyltransferase function restricts HIV replication. Further mechanism studies show that HIV viral protein R (Vpr) interacts with PCIF1 and induces PCIF1 ubiquitination and degradation. Among the m6Am genes, we find that PCIF1 inhibits HIV infection by enhancing a transcription factor ETS1 (ETS Proto-Oncogene 1, transcription factor) stability that binds HIV promoter to regulate viral transcription. Altogether, our study discovers the role of PCIF1 in HIV-host interactions, identifies m6Am modified genes in T cells which are affected by viral infection, and reveals how HIV regulates host RNA epitranscriptomics through PCIF1 degradation.

Zhang Q ，Kang Y ，Wang S ，Gonzalez GM ，Li W ，Hui H ，Wang Y ，Rana TM ... -  《Nature Communications》