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J. Biol. Chem., Vol. 280, Issue 43, 36429-36441, October 28, 2005

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Characterization of hCINAP, a Novel Coilin-interacting Protein Encoded by a Transcript from the Transcription Factor TAFIID₃₂ Locus^*

Niovi Santama1, Stephen C. Ogg, Anna Malekkou, Spyros E. Zographos, Karsten Weis¶, and Angus I. Lamond2

From the Department of Biological Sciences, University of Cyprus and Cyprus Institute of Neurology and Genetics, P.O. Box 20537, 1678 Nicosia, Cyprus, the Division of Gene Regulation and Expression, University of Dundee, MSI/WTB Complex, Dundee DD1 5EH, Scotland, United Kingdom, and the ^¶Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200

Coilin is a marker protein for the Cajal body, a subnuclear domain acting as a site for assembly and maturation of nuclear RNA-protein complexes. Using a yeast two-hybrid screen to identify coilin-interacting proteins, we have identified hCINAP (human coilin interacting nuclear ATPase protein), a nuclear factor of 172 amino acids with a P-loop nucleotide binding motif and ATPase activity. The hCINAP protein sequence is highly conserved across its full-length from human to plants and yeast and is ubiquitously expressed in all human tissues and cell lines tested. The yeast orthologue of CINAP is a single copy, essential gene. Tagged hCINAP is present in complexes containing coilin in mammalian cells and recombinant, Escherichia coli expressed hCINAP binds directly to coilin in vitro. The 214 carboxyl-terminal residues of coilin appear essential for the interaction with hCINAP. Both immunofluorescence and fluorescent protein tagging show that hCINAP is specifically nuclear and distributed in a widespread, diffuse nucleoplasmic pattern, excluding nucleoli, with some concentration also in Cajal bodies. Overexpression of hCINAP in HeLa cells results in a decrease in the average number of Cajal bodies per nucleus, consistent with it affecting either the stability of Cajal bodies and/or their rate of assembly. The hCINAP mRNA is an alternatively spliced transcript from the TAF9 locus, which encodes the basal transcription factor subunit TAFIID₃₂. However, hCINAP and TAFIID₃₂ mRNAs are translated from different ATG codons and use distinct reading frames, resulting in them having no identity in their respective protein sequences.

Received for publication, February 22, 2005 , and in revised form, July 5, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ87888 and AJ878881.

^* This work was funded in part by the Biotechnology and Biological Sciences Research Council (Project Grant 94/C12944), in part by Research Training Network Grant HPRNCT-2000-00079 from the European Union and in part by grant ENISX0603/03 from the Research Promotion Foundation (Cyprus). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ To whom correspondence may be addressed. Tel.: 357-22-392-757; Fax: 357-22-358-237; E-mail: santama{at}ucy.ac.cy.

² Supported by a Wellcome Trust Principal Research Fellowship. To whom correspondence may be addressed. Tel.: 44-1382-345-473; Fax: 44-1382-345-695; E-mail: a.i.lamond{at}lifesci.dundee.ac.uk.

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