Biochemical and Biophysical Research Communications
PCIF1, a novel human WW domain-containing protein, interacts with the phosphorylated RNA polymerase II☆
Section snippets
Materials and methods
Plasmid construction. For preparing glutathione S-transferase (GST) fused mouse CTD recombinant protein with a protein kinase A recognition site, the entire mouse CTD cDNA fragment was excised from pGCTD [10] (a gift from Dr. Dynan) by BamHI and EcoRI digestion and cloned into pGEX-2TK (Amersham) to create pG2TK-CTD. A pBK-CMV phagemid (Stratagene) clone containing cDNA encoding the entire PCIF1 protein (pBC-PCIF1) was used to create a PCIF1 expression vector. For creating thioredoxin fused
Detection of phosphorylated CTD interacting proteins in HeLa cell nuclear extract
To systematically identify novel human proteins that directly interact with the hyperphosphorylated CTD (pCTD), we screened a human cDNA expression library by blot overlay assay with -labeled GST fused pCTD (GST-pCTD). Recombinant GST mouse CTD fusion protein was purified, hyperphosphorylated by incubation in HeLa cell nuclear extracts (NE), and then repurified by glutathione affinity chromatography. Hyperphosphorylation of GST-CTD was confirmed by the following two observations: (1) an
Discussion
In this study we have cloned and characterized human PCIF1, a novel phosphorylated CTD interacting factor. PCIF1 is composed of 704 amino acids and contains a WW domain near the N-terminus. The PCIF1 WW domain can directly and specifically bind to the phosphorylated CTD. The binding affinity of the PCIF1 WW domain to CTD is significantly increased by phosphorylation of CTD. We also found that the PCIF1 WW domain could stably bind to the endogenous RNAP IIO present in cell extracts and to
Acknowledgements
We are grateful to Toyoko Kikukawa and Kazuyo Terada for technical assistance. We thank Dr. Murakami for advice on the blot overlay assay and Dr. Kuroki for his kind gift of a cDNA library. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas (A) and (C) from the Ministry of Education, Science, Sports and Culture of Japan, The Naito Foundation, and NOVARTIS Foundation (Japan) for the Promotion of Science.
References (22)
- et al.
Integrating mRNA processing with transcription
Cell
(2002) The mRNA assembly line: transcription and processing machines in the same factory
Curr. Opin. Cell Biol.
(2002)Tails of RNA polymerase II
TIBS
(1990)Reversible phosphorylation of the C-terminal domain of RNA polymerase II
J. Biol. Chem.
(1996)- et al.
Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties
Cell
(1992) - et al.
Creatine phosphate, not ATP, is required for 3′ end cleavage of mammalian pre-mRNA in vitro
J. Biol. Chem.
(1997) - et al.
Characterization of a novel protein-binding module: the WW domain
FEBS Lett.
(1995) - et al.
Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3′-end formation
J. Biol. Chem.
(1999) - et al.
Pinning down proline-directed phosphorylation signaling
Trends Cell Biol.
(2002) - et al.
RNA polymerase II and the integration of nuclear events
Genes Dev.
(2000)
An extensive network of coupling among gene expression machines
Nature
Cited by (38)
RNA m6A methylation across the transcriptome
2023, Molecular Cellm<sup>6</sup>Am RNA modification detection by m<sup>6</sup>Am-seq
2022, MethodsCitation Excerpt :Recently, Phosphorylated CTD-Interacting Factor 1 (PCIF1) was independently identified by four groups as the sole cap-specific N6-methyltransferase [8–11]. PCIF1 was originally identified and named due to its ability to interact with the phosphorylated CTD of RNA Pol II by its N-terminal WW domain [12]. However, there are still some controversies surrounding the function of the m6Am in vivo.
Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs
2022, Journal of Biological ChemistryEnzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima
2021, Journal of Biological ChemistryThe Mammalian Cap-Specific m<sup>6</sup>Am RNA Methyltransferase PCIF1 Regulates Transcript Levels in Mouse Tissues
2020, Cell ReportsCitation Excerpt :This work was approved by the Canton of Geneva (GE/16/19). This study concerns the Pcif1 gene (MGI: 2443858; NCBI Gene: 228866) encoding for Phosphorylated CTD-interacting factor 1 (PCIF1) (Fan et al., 2003; Hirose et al., 2008), which was later shown to be a cap-dependent RNA methyltransferase (and termed as CAPAM) (Akichika et al., 2019). The Pcif1 gene studied here is located on mouse chromosome 2 (Chr2: 164879368-164894454 bp, + strand) and consists of 17 exons (Figure S1A).
PCIF1 Catalyzes m6Am mRNA Methylation to Regulate Gene Expression
2019, Molecular CellCitation Excerpt :A putative methyltransferase enzyme that is conserved in zebrafish, mouse, and human but absent in yeast and nematodes is PCIF1 (Figure S2) (Iyer et al., 2016). PCIF1 contains an N-terminal WW domain, which is believed to mediate interactions with RNA polymerase II (Pol II) phospho-CTD, and also a putative C-terminal methyltransferase domain (Ebmeier et al., 2017; Fan et al., 2003). To determine whether PCIF1 is a m6Am methyltransferase, we generated several independent PCIF1 knockout (KO) clonal cell lines in human melanoma MEL624 cells and determined mRNA m6Am levels by LC-MS/MS (Figures 2A and 2B).
- ☆
The nucleotide sequence of human PCIF1 cDNA reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with Accession No. AB050014.
- 1
Present address: National Rehabilitation Center for the Disabled, 4-1 Namiki, Tokorozawa, Saitama 359-8555, Japan.