PCIF1, a novel human WW domain-containing protein, interacts with the phosphorylated RNA polymerase II

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Abstract

Phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAP II) largest subunit has an important role in transcription elongation and in coupling transcription to pre-mRNA processing. To identify proteins that can directly bind to the phosphorylated CTD, we screened a human cDNA expression library using 32P-labeled CTD as a probe. Here we report the cloning and characterization of a novel human WW domain-containing protein, PCIF1 (phosphorylated CTD interacting factor 1). PCIF1 is composed of 704 amino acids. The WW domain of PCIF1 can directly and preferentially bind to the phosphorylated CTD compared to the unphosphorylated CTD. PCIF1 binds to the hyperphosphorylated RNAP II (RNAP IIO) in vitro and in vivo. Double immunofluorescence labeling in HeLa cells demonstrated that PCIF1 and endogenous RNAP IIO are co-localized in the cell nucleus. Thus, PCIF1 may play a role in mRNA synthesis by modulating RNAP IIO activity.

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 32P-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)

  • T. Maniatis et al.

    An extensive network of coupling among gene expression machines

    Nature

    (2002)
  • Cited by (38)

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    The nucleotide sequence of human PCIF1 cDNA reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with Accession No. AB050014.

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    Present address: National Rehabilitation Center for the Disabled, 4-1 Namiki, Tokorozawa, Saitama 359-8555, Japan.

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