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An Evolutionary Examination of PCIF1 and m6Am in RNA Cap Structure

Researchers continue to uncover the intricacies of RNA methylation patterns. In particular, it has been found that the nucleotide adenosine can be methylated one or multiple times. The location of the adenosine and its methylation status can result in very different biological outcomes. The more commonly known m6A modification occurs on the N6 position of internal adenosines in RNA polymerase II-generated mRNA transcripts. Additionally, the N6 position of adenosines can also be methylated when the adenosine initiates transcription (located at the transcription start site, TSS). Most eukaryotic TSSs have a cap 1 structure, which means that the initial nucleotide is 2’-O-methylated. Therefore, adenosines at the TSS can be dimethylated, denoted as m6Am. The enzyme responsible for the m6Am mark has recently been identified as Phosphorylated CTD-Interacting Factor 1 (PCIF1).

Although the function of PCIF1 has been identified over the past few years, its role in mammalian development and evolution has not been examined. A recent study published in Cell Reports sought to understand the importance of PCIF1 in mammalian development and in other vertebrate or invertebrate systems. Specifically, in Pandey et. al, researchers investigated the role of PCIF1 in mice, drosophila, and trypanosomes.

Pandey et. al included three major experimental sections, each focusing on a different model organism. The first set of experiments investigated the role of PCIF1 in mice. The researchers created a PCIF1 knockout mouse and compared its biology and gene expression to that of its heterozygous littermates and wild-type mice. Physiologically, PCIF1 loss was tolerated during mouse development, with no overt biological effects other than a slightly reduced body weight. PCIF1 loss resulted in altered gene expression (genes both upregulated and downregulated) across the multiple tissues examined, but the tissue with most changes was the testes. Some genes’ expression changes were reflected in an altered translational status, but overall, the loss of PCIF1 did not strongly affect translation.

The second model system studied was Drosophila melanogaster. In flies, there is a PCIF1 ortholog that has high conservation with the human PCIF1. However, Drosophila PCIF1 (dPCIF1) has mutations in a few key residues in the catalytic site, and so was predicted to be inactive as a RNA methylase. Using in vitro methylation assays with m7G-capped RNAs, including a m7G(5)ppp(5)(2’OMeA)pG cap 1 analog (CleanCap® AG) from TriLink Biotechnologies, the scientists found that dPCIF1 was catalytically inactive. When the WW domain was isolated from the rest of the protein however, it bound the phosphorylated C-terminal domain of RNA polymerase. The authors suggest that a conformational change in the protein may be required to permit this binding, and that dPCIF1 has evolved a non-catalytic regulatory role in Drosophila.

The third system these researchers investigated was Trypanosoma cruzi, which causes Chagas Disease. Trypanosomes have a very different cap structure from flies and mice. They typically have a cap 4 structure with six methylation events in the first four nucleotides, including a m6Am on the adenosine in the first position. In these Trypanosomes, isothermal calorimetry experiments showed that PCIF1 can directly bind to m7G(5)ppp(5)(2’OMeA)pG (from TriLink Biotechnologies). In vitro methylation assays demonstrated that Trypanosome PCIF1 can methylate the m7GpppA-RNA that forms the basis of the cap 4 structure. However, they were unable to detect the m6Am by RNA mass spectrometry.

Overall, this paper demonstrates that PCIF1 functions in a variety of catalytic and potentially non-catalytic mechanisms across evolution. Many questions remain in elucidating the details of PCIF1’s biological role in each of the model organisms, but this paper provides a foundation from which to discover more. Surely, a greater understanding of the significance of PCIF1 and the m6Am mark will develop in concert.

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Article Reference: Cell Rep. 2020 Aug 18;32(7):108038. doi: 10.1016/j.celrep.2020.108038.