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

Dan Yu, Nan Dai, Eric J. Wolf, Ivan R. Corrêa, Jujun Zhou, Tao Wu, Robert M. Blumenthal, Xing Zhang, Xiaodong Cheng

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number101751
JournalJournal of Biological Chemistry
Issue number4
StatePublished - Apr 1 2022

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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