Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M501982200 on August 17, 2005 Originally published In Press as doi:10.1074/jbc.M501982200 on August 2, 2005

J. Biol. Chem., Vol. 280, Issue 43, 36429-36441, October 28, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/43/36429    most recent
M501982200v2
M501982200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Santama, N.
Right arrow Articles by Lamond, A. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santama, N.
Right arrow Articles by Lamond, A. I.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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

Niovi Santama{ddagger}1, Stephen C. Ogg§, Anna Malekkou{ddagger}, Spyros E. Zographos{ddagger}, Karsten Weis¶, and Angus I. Lamond§2

From the {ddagger}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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cajal bodies are subnuclear domains that contain newly imported snRNP and snoRNP3 complexes, as well as certain transcription factors. They are thought to be centers for the maturation of these nuclear RNA-protein complexes that are en route to their sites of function in the nucleoplasm and nucleolus, respectively (for review see Ref. 1). Although Cajal bodies do not contain DNA, they have been shown to associate specifically with certain gene loci, including the histone and U snRNA gene clusters (2-4). In the case of the U2 genes, studies on stable cell lines containing arrays of exogenous U2 genes showed that Cajal body association was dependent on the expression of the U2 snRNA transcripts, rather than on the presence of specific DNA sequences in the U2 genes (2). It is possible that Cajal bodies can regulate gene expression from certain loci and/or deliver RNA processing factors or other components required for efficient expression at these sites.

Coilin is a human autoantigen that is widely used as a marker protein for Cajal bodies. It is a specifically nuclear protein that is modified by both serine phosphorylation and symmetrical dimethylation of arginine. Coilin shows a diffuse nucleoplasmic distribution but also concentrates within Cajal bodies, appearing as one or more bright nuclear foci when analyzed by immunofluorescence. Analysis of a mouse knock-out of coilin has revealed that coilin -/- cells are viable in culture, although animals lacking coilin show a decrease in embryonic viability, as judged by the reduced ratios of coilin -/- mice recovered in litters (5). The coilin -/- cells lack normal Cajal bodies but instead have microbodies containing a subset of the usual Cajal body components. Deletion mutants of coilin can disrupt endogenous Cajal bodies when transiently expressed in mammalian cells (6, 7). Coilin is a self-interacting protein, dependent upon sequences in the first 92 amino-terminal residues (8). These data indicate that coilin is likely to play a functional role in the formation of wild type Cajal bodies.

Several proteins have been shown to bind directly to coilin, including the survival of motor neurons (SMN) protein (7), the nucleolar shuttling factor NOPP140 (9), and the {alpha} importins (10), which are adaptors involved in nuclear import. In the case of the SMN protein, its binding to coilin is dependent on the presence in coilin of symmetrical dimethyl arginine residues, a modification shared with the Sm family of snRNP proteins, which also localize in Cajal bodies (11). In the case of NOPP140, it has been shown that deletion mutants removing the amino-terminal portion of NOPP140, which no longer bind to coilin, have a dominant negative effect on Cajal body formation when exogenously expressed in mammalian cells (9). These data support the important role of coilin in Cajal body formation and further indicate that the mechanisms involved likely require the interaction of coilin with other partner proteins.

To search for novel interaction partners for coilin we have conducted a yeast two-hybrid screen using coilin as the bait. Here we report the molecular characterization of hCINAP, a highly conserved nuclear protein that we show binds directly to coilin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Line—HeLa cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (BD Biosciences), 2 mM glutamine, penicillin/streptomycin and maintained at 37 °C in 5% CO2.

Antibodies—Rabbit polyclonal antibodies to full-length, His6-tagged hCINAP were generated at the EMBL animal facility, and by Diagnostics Scotland (Edinburgh, UK). A second rabbit polyclonal antibody, to synthetic peptide DQILKWIEQWIKDHNS, corresponding to amino acid residues 157-175 in hCINAP, was also generated at the EMBL facility. For both antibodies, the specificity of the antiserum from the third bleed was characterized by Western blotting (Fig. 4, and data not shown) and these batches were used for the experiments described here (1:400 dilution for Western blotting and immunofluorescence with the anti-recombinant protein antibody, 1:400 for blotting and 1:200 for immunofluorescence with the anti-peptide antibody).

The following primary antibodies were used: rabit anti-coilin 204/5 (6), mouse anti-coilin monoclonal antibody (mAb) {delta}4 (12), rabit anti-U1A 856 (13), rabbit anti-fibrillarin Fib42, rabbit anti-SMN (Santa Cruz Biotechnology), mouse anti-Sm mAb Y12, rabbit anti-Sp100 (Chemicon), and mouse mAb 9E10 (anti-myc) (14).

The following secondary antibodies were used (Dianova, Hamburg, Germany): fluorescein isothiocyanate-conjugated, donkey anti-mouse IgG, Texas Red-conjugated, goat anti-mouse IgG+M, fluorescein isothiocyanate-conjugated, donkey anti-rabbit IgG, and rhodamine-conjugated, donkey anti-rabbit IgG.

Oligonucleotides—The following oligonucleotides were used in polymerase chain reaction (PCR) amplifications: oligonucleotide 1, 5'-cggaattcatgttgcttccgaacatcctg-3'; oligonucleotide 2, 5'-ccagactccatgatatccgatgatc-3'; oligonucleotide 3, 5'-accgtcgacttaagtagctagccttataag-3'; oligonucleotide 4, 5'-acattcagcaaggctagattacag-3'; oligonucleotide 5, 5'-acaccaaagtgtcccttac-3'; oligonucleotide 6, 5'-taatgtgggtgatttagctcgagaag-3'; oligonucleotide 7, 5'-cttcattgacctcaactacatggt-3'; and oligonucleotide 8, 5'-tcatggatgaccttggccaggg-3'.

Two-hybrid System Analysis—Vectors and yeast strains for the two-hybrid analysis were used as described by Brent and coworkers (15), and {beta}-galactosidase assays were performed as reported (16, 17). A HeLa cDNA library was used to generate the activation domain fusion constructs. Interacting clones were identified as described (10). For mapping the CINAP interaction domain on coilin, deletion constructs of coilin (as shown in Fig. 7) were subcloned either as restriction- or PCR-amplified fragments in yeast vector pGBKT7 or vector pACT2 for construct C1. They were each tested for interaction by co-transformation in yeast strain AH109 with full-length hCINAP in yeast vector pACT2 (or in pGBKT7 for testing with C1), followed by screening of reporter gene expression on appropriate selective media. First, transformants were selected with double selection on media lacking leucine and tryptophan and then transferred to quadruple selection on media also lacking adenine and histidine. Clones that were viable on quadruple selection were subjected to colony {beta}-galactosidase assay for confirmation of the interaction. For independent confirmation of the interaction, coilin constructs were in vitro transcribed and translated in the presence of [35S]methionine and incubated either with lysate from bacteria expressing glutathione S-transferase (GST)-tagged full-length CINAP after induction with 1 mM IPTG or without induction as negative control. Proteins interacting with GST-CINAP were recovered by glutathione-agarose pull-down and subjected to SDS-PAGE, followed by autoradiography to detect coilin fragments.

RT-PCR, Semiquantitative RT-PCR for the TAF9 Locus Transcripts, and PCR on Genomic DNA—cDNA was synthesized from 1 µg of purified human placenta poly(A)+ RNA (Clontech) and served as a template for the amplification of the complete ORF of hCINAP with oligonucleotides 1 and 3. The amplified product was digested with EcoRI/SalI, cloned into Bluescript, and sequenced.

For semiquantitative RT-PCR, poly(A)+ RNA was extracted from human tissues or human cell lines with the Oligotex mRNA mini kit (Qiagen), according to the manufacturer's instructions. To evaluate the efficacy of RT-PCR conditions with the various tissues, and compare the hybridization efficiency of the two sets of oligonucleotides used for the experiments shown in Fig. 3 (B and E) (one set specific for the hCINAP transcript and two sets specific for the TAFIID32 transcript), control reactions with plasmid DNA were performed. Reactions were carried out in parallel with identical conditions, using 20 ng of plasmid as template with either TAFIID32- or hCINAP-specific oligonucleotides of the same concentration at 18, 23, and 28 cycles. The results indicated that, in the linear range of the reaction (achieved between 18 and 23 cycles), the concentration of two types of products is comparable (supplementary Fig. S2). Semiquantitative RT-PCR was therefore subsequently performed with the same PCR conditions (except for the increased number of cycles that were necessary to detect the amplified product from cDNA samples). Control reactions using glyceraldehyde-3-phosphate dehydrogenase-specific oligonucleotides were run in parallel as internal standards.

For the amplification of human genomic sequences, purified high molecular weight human genomic DNA (Roche Applied Science) was used as a template and PCR reactions, with a variety of oligonucleotides (see "Oligonucleotides") were carried out with the Expand Long Template PCR System (Roche Applied Science), following the exact reaction and amplification instructions of the manufacturer. PCR products were directly cloned into vector pCR2.1, using the Original T/A cloning kit (Invitrogen). DNA sequencing was performed by the EMBL sequencing service.

Construction of Bacterial Expression and Mammalian Transfection Vectors—The bacterial expression vector pRSETB-hCINAP was constructed by an in-frame EcoRI/SalI insertion of hCINAP, excised from Bluescript (see previous section) to vector pRSETB (Invitrogen). This created a 5' His6-tagged version of hCINAP. The same hCINAP insert was also ligated to expression vector pGEX-4T-1 (Amersham Biosciences) to generate GST-tagged hCINAP. To create YFP-hCINAP, we made a vector fragment derived from pEYFP-C1 (BD biosciences) by digestion with BglII and HindIII. An insert fragment containing hCINAP was derived from pRSETB-hCINAP by digestion with BglII and HindIII.

Bacterial Expression and Purification of His6-hCINAP—Competent BL21-pLysS strain of E. coli was transformed with pRSETBhCINAP. Large cultures were induced with 1 mM IPTG during exponential growth in LB at 37 °C and harvested 3 h post-induction. The bacterial pellet was resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 8.0, 10% glycerol, 5 mM {beta}-mercaptoethanol, 0.5% Triton X-100, 10 mM imidazole, and 1 tablet/50 ml of Complete protease inhibitor (Roche Applied Science), mechanically lysed with a French press and centrifuged at 13,000 rpm for 15 min at 4 °C. The insoluble pellet was resuspended in buffer B (as per lysis buffer but with 100 mM NaH2PO4 instead of NaCl), containing 6 M guanidinium HCl, and the recombinant protein was purified over a nickel-nitrilotriacetic acid column (Qiagen) under denaturing conditions, according to the manufacturer's instructions. For animal immunization, the purified protein was dialyzed extensively against PBS.

The same construct was used for the production of soluble protein, suitable for enzyme kinetic experiments, with culture growth at 30 °C for 5 h after induction with 0.5 mM IPTG. This resulted in the production of 30-40 mg of soluble His6-hCINAP/liter. The recombinant protein was affinity purified over a nickel-nitrilotriacetic acid column, followed by gel filtration through a Superdex-200 column (Amersham Biosciences), equilibrated in 25 mM Hepes, pH 7.5, 0.3 M NaCl, 2 mM MgCl2, and 5 mM {beta}-mercaptoethanol, and desalted by dialysis. It was stored at -20°C in a buffer containing 12.5 mM Hepes, pH 7.5, 12.5 mM KCl, 1 mM MgCl2, 2.5 mM {beta}-mercaptoethanol, and 50% glycerol for use in ATPase assays.

ATPase Assays with His6-hCINAP—The ATP hydrolysis activity of hCINAP was measured by an assay in which ADP production is coupled to the {beta}-NADH oxidation by pyruvate kinase and L-lactic dehydrogenase (18). The rate of {beta}-NADH disappearance was monitored at 340 nm, and all enzymatic reactions were performed at 30 °C. The final assay mixture was 75 mM Tris-HCl, pH 8.0, 65 mM KCl, 0.2 mM {beta}-NADH, 1 mM phenyl enol pyruvate, 5 mM MgCl2, 1mM dithiothreitol, 20 units/ml pyruvate kinase (Sigma), 20 units/ml L-lactic dehydrogenase (Sigma), 3 µg/ml CINAP, and 0.05-1 mM ATP. Control samples, containing reaction mixture without CINAP, were used to subtract background ATP hydrolysis, mainly due to the activity of pyruvate kinase. Kinetic data were analyzed with the use of the nonlinear regression program GraFit. Calculations of molarity were based on a molar extinction coefficient of 6220 M-1 cm-1 for {beta}-NADH at 340 nm.

In Vitro Co-selection Experiments—Plasmid pGEX-4T-1 (Amersham Biosciences) was used for expression of hCINAP as a GST fusion protein and pET32a+ (Novagen) was used for expression of p80-coilin as a His6 and thioredoxin fusion. Bacterial expression was carried out as described in the previous section with culture growth at 25 °C. Two-milliliter aliquots of bacterial cultures were collected 3 h post-induction, bacterial pellets were resuspended in 200 µl of binding buffer (PBS, containing 1% (v/v) Tween 20 and 1 tablet/50 ml Complete protease inhibitor, Roche Applied Science) and lysed by brief sonication. After clarification of bacterial lysates (13,000 rpm at 4 °C for 15 min), GST-hCINAP, and His6-coilin were mixed in the presence of 0, 1, 2, and 3 mM ATP, respectively, and incubated for 30 min at room temperature with constant agitation. An equal volume of pre-equilibrated glutathione-Sepharose 4B beads (Amersham Biosciences) were then added into each protein mix, and the solution was incubated with gentle agitation at room temperature for 30 min. The beads with bound complexes were collected by centrifugation at 500 rpm for 5 min. The supernatant (unbound fraction) was saved for analysis. Beads were washed with ice-cold binding buffer, resuspended in SDS sample buffer, and analyzed, together with the unbound fractions, by SDS-PAGE, and Western blotting.

In Vivo Co-immunoprecipitation Experiments—HeLa cells were transiently transfected with the pEYFP-hCINAP construct (details below) using Effectene (Qiagen) according to the manufacturer's instructions. One microgram of plasmid DNA was used per 10-cm dish, 18 h after transfection the dishes were rinsed 2x with warm PBS, and cells were lysed with 0.5 ml/dish Lysis Buffer (50 mM Tris HCl, pH 7.5, 0.5 M NaCl, 1% v/v Nonidet P-40, 1% w/v deoxycholic acid, 0.1% w/v SDS, 2 mM EDTA, and 1 tablet/10 ml protease inhibitor Complete, Roche Applied Science). The cell lysate was scraped into a 1.5-ml Eppendorf tube, and the DNA was sheared to reduce the viscosity by processing with a QIAshredder (Qiagen) according to the manufacturer's instructions. The lysate was precleared by incubating with protein G-Sepharose for 1 h at 4°C. After brief centrifugation to remove the protein G-Sepharose, the precleared extract was transferred to a new tube, and then 1 µl of anti-green fluorescent protein monoclonal antibody (Roche Applied Science) was added. Incubation at 4 °C for 1 h allowed formation of immune complexes, which were subsequently recovered by adding protein G-Sepharose and rotating overnight at 4 °C. Protein G-Sepharose beads containing the immune complexes were separated from the supernatant by centrifugation and washed. Finally, proteins in both the supernatant and pellet fraction were denatured with SDS sample buffer and separated by SDS-PAGE. After transfer to nitrocellulose, the blot was probed with an antibody against coilin followed by incubation with a second anti-rabbit antibody coupled to HRP (Pierce, 1:5000).

Standard Molecular Biology and Protein Analysis Techniques—All molecular biology techniques (cDNA library screening, plasmid purifications, restriction digests, ligations, blue/white bacterial colony selection, Northern blotting, and others) were performed according to standard procedures (19).

SDS-PAGE on a mini-gel system (Protean, Bio-Rad) or using gradient "Novex" gels from Invitrogen was essentially as described (20). Western blotting was carried out using standard techniques and visualized by the enhanced chemiluminescence system, ECL-Plus (Amersham Biosciences). HeLa nuclear extracts were obtained commercially from the Computer Cell Culture Center (Mons, Belgium)

Transient Transfections of Tagged hCINAP in HeLa Cell Line—Exponentially growing HeLa cultures were harvested and replated onto coverslips in 60-mm diameter dishes, 24 h prior to transfection. Transfections were carried out with the DOTAP reagent (Roche Applied Science) or with Effectene (Qiagen), according to the manufacturer's instructions in each case or with calcium phosphate precipitation (lamondlab.com/f6protocols.htm). Coverslips were retrieved for immunofluorescence 16-20 h after removal of the transfection medium.

Immunolabeling of Cultured Cells—For hCINAP immunofluorescence, using the antiserum to hCINAP recombinant protein or the anti-peptide antiserum, cells grown on glass coverslips were fixed with 3.7% paraformaldehyde in PHEM buffer (30 mM Hepes, 65 mM Pipes, pH 6.9, 10 mM EGTA, and 2 mM MgCl2) for 10 min and permeabilized for 15 min with 0.2% SDS in the same buffer. Transfected cells were fixed with 3.7% paraformaldehyde in PHEM buffer for 10 min. They were quenched with 50 mM NH4Cl in PBS for 10 min, permeabilized for 10 min with 0.5% Triton X-100 in PHEM buffer, and blocked with 2% BSA, 2% fetal calf serum, 0.2% fish skin gelatin in PBS ("blocking mix") for 30 min.

Cells were incubated with primary antibodies, in PBS containing 5% blocking mix, for 1 h at room temperature. For double labeling, incubation was carried out sequentially for each of the primary antibodies and secondary antibodies were applied as a mixture. Coverslips were mounted with Mowiol (Merck, Germany), containing 100 mg/ml DABCO (Sigma, Germany) as an anti-fading agent.

Confocal and Epifluorescence Microscopy—Immunofluorescent preparations were analyzed on the EMBL Compact Confocal Microscope (21) or a Zeiss Axiophot light microscope. Excitation wavelengths of 476 nm (for fluorescein isothiocyanate-coupled antibodies) or 594 nm (Texas Red-coupled antibodies) were selected.

Some images presented in Figs. 5 and 8A were recorded on a Zeiss Axiovert S100 2TV DeltaVision Restoration microscope (Applied Precision) using either a Zeiss Plan-apochromat (100x, 1.40 numerical aperture objective) or a Zeiss Plan-neofluar (40x, 1.30 numerical aperture objective) and a CCD-1300-Y/HS camera (Roper Scientific). Images were captured and processed by constrained iterative deconvolution using SoftWorx (Applied Precision) and prepared as illustrations using Adobe Photoshop. Two-dimensional images presented here are maximal intensity projections of three-dimensional volumes along the optical axis.



View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 1.
A, identification of hCINAP, a protein that interacts specifically with p80-coilin in the yeast two-hybrid assay. Two-hybrid interactions between LexA-p80 coilin (lanes 1 and 3) and either a hCINAP-B42 hybrid protein (lane 1) or the B42 transcriptional activation domain alone (lane 3) were analyzed by liquid {beta}-galactosidase assays. As a negative control, the interaction between the hCINAP-B42 hybrid and the unrelated LexA-bcd fusion protein was included (lane 2). For each experiment, the {beta}-galactosidase activity was determined two times from individual cultures, and {beta}-galactosidase units were calculated as described under "Materials and Methods." B, cloning by RT-PCR and sequencing of hCINAP from human placenta: 1, {lambda}/HindIII MW markers; 2, amplified product from human placenta cDNA using oligonucleotides 1 and 3; 3, identical reaction with no cDNA template; 4, identical reaction with 15 ng of pRSETB-hCINAPHeLa as template. The sequence derived from the cloned product in lane 2 was identical to the sequence from the HeLa cDNA library in lane 4. C, alignment of CINAP protein from different organisms. Sequences were aligned using ClustalX and are represented by the single letter amino acid code. Sequences from human (H. sapiens, accession numbers AJ878880 [GenBank] and AJ878881 [GenBank] ), nematode (C. elegans, NP496065), fruit fly (D. melanogaster, NP610797), thale cress (A. thaliana, BAB10972 [GenBank] ), and baker's yeast (S. cerevisiae, AAS56894 [GenBank] ) are presented. Boxed residues are identical among these five sequences, residues in bold represent conservative amino acid substitutions, and the shaded sequence represents a conserved domain found in many ATP/GTP binding proteins (P-loop motif).

 



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 2.
Enzymatic activity of CINAP with respect to ATP. Plot of Initial ATP hydrolysis velocities versus various ATP concentrations (0.05 to 1 mM). Best-fit curves were computer-generated by non-linear regression according to the Michaelis-Menten equation (GraFit). The apparent kinetic parameters determined by this method are as follows: the Km = 75.3 ± 5 µM and Vmax = 1.27 ± 0.02 µmol of ADP formed. min-1·mg-1. Inset, double reciprocal plot of initial reaction velocities versus ATP concentration.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To identify novel coilin interacting proteins, a yeast two-hybrid screen was performed with a HeLa cDNA library, using human coilin as the bait. Two million colonies were screened and ninety positive clones identified, as previously described (10, 22). Among the ninety positive clones four overlapping cDNAs, encoding the same open reading frame, were isolated. An apparently full-length cDNA clone encoding this protein was used to quantify its interaction with coilin using a liquid {beta}-galactosidase yeast two-hybrid assay (Fig. 1A). This showed a strong interaction of the hCINAP protein with coilin relative to the negative controls.

To ensure that the positive clones from the HeLa library encode a bona fide human protein, the transcript was amplified by PCR from human placental cDNA (Fig. 1B). This yielded a cDNA amplification product of identical size to the HeLa cDNA clone (Fig. 1B, compare lanes 2 and 4). DNA sequence analysis showed that both the original HeLa and the placental cDNA clones were identical. The sequences were deposited in the GenBankTM/EBI data base as accession numbers AJ878880 [GenBank] and AJ878881 [GenBank] . We have termed the resulting gene product hCINAP (human coilin interacting nuclear ATPase protein), which is a 172-amino acid protein of molecular mass 20,048 Da. Fig. 1C shows the full protein sequence of hCINAP aligned with putative orthologues from other species spanning the major eukaryotic divide, including plants, flies, worms, and yeast. This shows that hCINAP is highly conserved from human to yeast across its entire length. Thus there is a 22% identity and ~46% overall similarity in amino acid sequence conserved between all orthologues in human, fly, nematode, plants, and yeast. A direct comparison of the human and Saccharomyces cerevisiae CINAP protein sequences shows that they are 41% identical and ~62% similar. The hCINAP gene is apparently unique and includes a consensus nucleotide (ATP or GTP) binding motif (GX4GKT). Sequence comparison shows that hCINAP is not highly homologous to any other known proteins. It does however contain an amino-terminal P-loop motif characteristic of ATP/GTP binding proteins. This consensus motif is strictly conserved in all CINAP orthologues examined and is similar to the adenylate kinase motif found in members of the adenylate kinase family. This similarity with adenylate kinase and in particular the conservation in the P-loop region, suggests that hCINAP likely binds ATP rather than GTP. Consistent with this prediction, we found that recombinant hCINAP shows ATP hydrolysis activity. A kinetic analysis of ATP hydrolysis with recombinant hCINAP was performed (Fig. 2). The Km value for ATP was calculated to be 75.3 ± 5 µM, whereas the Vmax value was 1.27 ± 0.2 µmol of ADP formed·min-1·mg-1 (Fig. 2). These results showed that hCINAP exhibits a significant ATPase activity and an apparent high affinity for ATP as a substrate.

The high primary sequence conservation of hCINAP, extending over the length of the protein, strongly suggests that it is functionally important. To test this we knocked out the corresponding orthologue of hCINAP in the budding yeast, S. cerevisiae and observed that it is essential (data not shown). This essential phenotype has also been independently described (23). These workers isolated the budding yeast hCINAP orthologue, which they termed Fap7p, as a factor required for transcriptional regulation of yeast genes upon oxidative stress. Based on its interaction with human coilin in the yeast two-hybrid assay, its essential phenotype in yeast, and its high sequence conservation, we therefore selected hCINAP for further analysis.

During our characterization of hCINAP, we noticed that the first 141 nucleotides of hCINAP mRNA, which encode the first 41 amino acids of hCINAP protein, were identical with the 5'-untranslated region of another human mRNA encoding subunit 9 of the transcription factor TAFIID (TAFIID32 (24)). However, despite this common nucleotide sequence, hCINAP does not share any amino acid similarity with TAFIID32. This observation prompted us to investigate the genomic organization of hCINAP.

Examination of the Ensembl data base (www.ensembl.org) showed that the sequences of both the hCINAP and TAFIID32 transcripts arise from map position 68.7 Mb on chromosome 5 (annotated as the TAF9 locus, Ensembl Gene ID ENSG00000085231). The hCINAP and TAFIID32 mRNAs are generated as alternatively spliced transcripts that share exons 1 and 2, but diverge downstream of the XhoI site at nucleotide 141 (Fig. 3A). Translation of hCINAP initiates at the first ATG codon in the shared 5'-terminal region of the mRNA. In contrast, initiation of TAFIID32 starts at an internal ATG, which is located 18 nucleotides downstream of the shared 141 nucleotide 5' sequence and is therefore unique to TAFIID32 (Fig. 3A, open triangles indicate translation start sites). Although the TAFIID32 mRNA also has the same upstream ATG used by hCINAP, the downstream ATG actually used to initiate translation of TAFIID32 is in a different reading frame. Thus, it appears that, at the TAF9 locus on chromosome 5, alternative splicing results in two transcripts that encode proteins with zero shared amino acid sequence, despite the common 5' region of the mRNA. This was a sufficiently unusual situation that we decided to verify the genomic organization and transcription pattern at the TAF9 locus directly by PCR analysis and DNA sequencing using human genomic DNA (supplementary Fig. S1). The data obtained were fully consistent with the transcript structures reported in the Ensembl annotation, although we also detected multiple apparent pseudogenes dispersed on different chromosomes (data not shown), again consistent with Ensembl annotations. Parallel RT-PCR data using probes either specific for hCINAP, or TAFIID32 ORF sequences, identified two cDNAs of the sizes expected for hCINAP and TAFIID32 (Fig. 3B). A combination of Northern blotting and semiquantitative RT-PCR analyses showed that both transcripts were expressed in all tissues and cell lines tested (Fig. 3, C-E). Preliminary phylogenetic analysis (data not shown) reveals that this unusual gene arrangement for hCINAP and TAFIID32 is conserved in mammals, including specifically chimp, rat, mouse, and dog. However, it is not conserved in other vertebrates examined, including fish and amphibians. Outside of mammals, protein orthologues of both CINAP and TAFIID32 are conserved, but they are encoded at separate loci. We thus conclude that the hCINAP protein is genetically but not physically related to TAFIID32.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 3.
A, model of alternative splicing of two transcripts from the TAF9 locus. Two transcripts, sharing two exons at their 5'-end are generated, the first encoding hCINAP and the second encoding TAFIID32. Partial characterization of this locus, as shown in this schematic, was achieved with genomic PCR using exon- and intron-spanning oligonucleotides generating overlapping products for sequencing (oligonucleotides 1, 2, 5, and 6, see "Materials and Methods"; data not shown but available as supplemental Fig. S1). The positions of complementary sequences to oligonucleotides used for RT-PCR experiments (shown in B and D) are marked as well as the translation start sites for the two transcripts (open triangles). B, confirmation of the predicted transcripts for the TAF9 locus by amplification with RT-PCR of cDNAs containing full-length ORFs. Simultaneous co-amplification of TAFIID32(expected size 950 bp, EMBL accession number U21858 [GenBank] ) and hCINAP ORF (expected size 550 bp) utilizing a common upstream oligonucleotide (oligo 1, see "Materials and Methods") and downstream oligonucleotides 3 (specific for hCINAP) and 4 (specific for TAFIID32)(lane a) and for comparison single amplification of hCINAP (lane b) and single amplification of TAFIID32 (lane c). C, Northern blot of HeLa poly(A)+ RNA probed with a 3', hCINAP-specific, probe (nucleotides 118-550 of hCINAP transcript) reveals hybridization to a band below the 1.35-kb size marker (arrow), which is very likely to correspond to the hCINAP transcript. D, multitissue Northern blot using the whole of hCINAP cDNA as a probe, showing ubiquitous expression of hCINAP. E, semiquantitative RT-PCR with equivalent amounts of cDNA from different human tissues, using hCINAP- and TAFIID32-specific oligonucleotides (oligonucleotides 1 and 3 and oligonucleotides 1 and 2, respectively, see "Materials and Methods") to generate diagnostic full-length or partial cDNA products. Results indicate that the two cDNAs appear to be expressed in nearly equimolar concentrations in each tissue type but display a differential pattern of tissue-to-tissue expression. Control reactions using glyceraldehyde-3-phosphate dehydrogenase-specific oligonucleotides (oligonucleotides 7 and 8, see "Materials and Methods") were run in parallel and are shown in the bottom panel. PCR conditions and oligonucleotide hybridization were tested to ascertain that RT-PCR results for the two DNAs are comparable (data not shown but included in supplemental Fig. S2).

 



View larger version (64K):
[in this window]
[in a new window]
  		FIGURE 4.
Recombinant and native expression and affinity purification of hCINAP. A, SDS-PAGE showing production of His6-tagged hCINAP (arrow) in E. coli upon induction with IPTG: 1, total lysate from uninduced culture; 2, total culture lysate 3 h post-induction; 3, corresponding supernatant, following high speed centrifugation of lysate; 4, corresponding insoluble pellet. B, purification of His6-tagged recombinant hCINAP on a Ni2+-agarose column (1) total culture lysate with overexpressed recombinant hCINAP; (2) flow-through fraction; (3) one of the eluted serial fractions (peak) containing purified hCINAP (arrow). Peak fractions were combined, dialyzed against PBS and used for rabbit immunization. C, Western blotting probed with hCINAP anti-peptide anti-serum, showing: a total bacterial lysate from an uninduced culture (1) and a total bacterial lysate from an induced culture (2). D, detection of endogenous hCINAP, and YFP-hCINAP by Western blot. Whole cell lysate from HeLa cells was separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with antibodies specific for hCINAP (lanes 3 and 4), its cognate pre-immune serum (lanes 1 and 2), or green fluorescent protein (lanes 5 and 6). The lysate in lanes 1, 3, and 5 was from control cells, whereas the lysate in lanes 2, 4, and 6 was derived from HeLa cells transiently transfected with pEYFP-hCINAP. Arrow indicates the exogenously expressed YFP-hCINAP and breakdown products, whereas the arrowhead indicates endogenous hCINAP.

 



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 5.
Immunolocalization of native and YFP-tagged hCINAP in HeLa cells. A1, YFP-hCINAP fluorescence; A2, the same cells stained with 204/5 anti-coilin; A3, overlay of images A1 and A2; B1, anti-hCINAP staining using the antiserum to the whole recombinant protein; B2, anti-coilin; B3, overlay of images B1 and B2; C1, anti-hCINAP staining, using anti-peptide antiserum; C2, the same cell stained with anti-coilin antiserum; C3, overlay of images C1 and C2; Scale bar in all images: 5 µm.

 
We next conducted a biochemical characterization of the hCINAP protein. First, recombinant hCINAP was expressed in Escherichia coli fused at the amino terminus to a hexahistidine tag (Fig. 4, lanes 1 and 2). The E. coli-expressed protein was largely insoluble and co-fractionated with the bacterial pellet after cell lysis (lanes 3 and 4, arrow). Due to its insolubility, recombinant hCINAP was affinity-purified over a nickel-nitrilotriacetic acid-agarose column under denaturing conditions, and the resulting purified protein (Fig. 4B, lane 3, arrow) used for antibody production in rabbits (see "Materials and Methods"). Protein blotting analysis using the resulting polyclonal rabbit antiserum showed that it recognized specifically both recombinant hCINAP and endogenous hCINAP in HeLa cell extracts and similar results were obtained using an independent antibody raised against the synthetic peptide DQILKWIEQWIKDHNS, corresponding to carboxyl-terminal amino acid residues 157-175 in hCINAP (Fig. 4, C and D, and other data not shown). Both antibodies show that the endogenous HeLa hCINAP co-migrated with the bacterial recombinant protein, indicating that hCINAP is likely not extensively post-translationally modified (data not shown).



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 6.
Co-selection and immunoprecipitation experiments indicating an interaction between hCINAP and coilin. A, recombinant GST-hCINAP and His6-coilin were expressed in bacteria and probed for interaction in crude bacterial lysates, as described under "Materials and Methods." Complexes were immobilized on glutathione beads and analyzed by SDS-PAGE, followed by visualizing GST-hCINAP and His6-coilin with Coomassie Blue. The molecular mass of recombinant coilin (native form is 66 kDa), expressed from vector pET2+, which bears in addition to the hexahistidine tag (0.66 kDa) a thioredoxin peptide (12 kDa), totals 78.66kDa. B, immunoblotting of an identical gel, using anti-coilin antiserum for the detection of coilin. Reactions were carried out either in the absence or presence of ATP (1-3 mM). The results show that coilin is detectable on the glutathione beads only in the presence of hCINAP and hCINAP-coilin interaction appears to be ATP-independent. Arrows point to the band corresponding to His6-coilin, and an arrowhead to the band corresponding to GST-hCINAP. C, detection of hCINAP-coilin interaction in vivo. HeLa extract from control cells (lanes 2 and 3) or cells transfected with YFP-hCINAP (lanes 4 and 5) was subjected to immunoprecipitation under native conditions using an anti-green fluorescent protein antibody to recover YFP-hCINAP and associated proteins. Bound (lanes 3 and 5) or unbound (lanes 2 and 4) fractions were separated by SDS-PAGE, transferred to nitrocellulose, and native coilin was detected by immunoblotting using a rabbit anti-coilin antibody. As a marker for coilin, 5 µg of a nuclear extract prepared for in vitro splicing was also included on the gel (lane 1). The arrow indicates coilin band. Molecular weight markers are indicated.

 



View larger version (8K):
[in this window]
[in a new window]
  		FIGURE 7.
Mapping of the CINAP-interaction domain of coilin within the carboxyl-terminal 214 amino acid residues of coilin. A, yeast strain AH109 was simultaneously transformed with one of the six coilin bait constructs shown in conjunction with full-length hCINAP. Interaction was defined as the ability of >95% of resulting clones, containing both bait and prey constructs, to grow on quadruple selective media (-Leu, -Trp, -His, and -Ade) and to also display {beta}-galactosidase activity upon filter assay. In addition to the interaction observed between full-length coilin and full-length CINAP (positive control), an additional positive result was obtained between coilin construct C1 and full-length CINAP. For validation of the later, negative controls included co-transformation of C1 and p53 (negative control 1) and of full-length CINAP and AD vector pACT2 (negative control 2), which failed the interaction tests. B, autoradiogram of in vitro translated coilin C1 fragment after SDS-PAGE, showing that the fragment interacts with GST-CINAP (lanes 4 and 5). Importantly, the C1 fragment is absent from the pellet of the pull-down (lane 3) when it is incubated with bacterial lysate of an uninduced culture that only contains negligible background concentration of GST-CINAP. T, total in vitro transcribed product; S, supernatant; P, pellet. The combination of these data revealed that an essential CINAP interaction domain lies within the carboxyl-terminal 214 amino acid residues of coilin.

 
We next examined the cellular localization of hCINAP. An amino-terminal YFP fusion of hCINAP was constructed (see "Materials and Methods"), and the fusion protein transiently expressed following transfection into HeLa cells (Figs. 4 and 5). Protein blotting analysis confirmed that the YFP-hCINAP, as well as endogenous hCINAP, were both recognized by the rabbit anti-hCINAP serum (Fig. 4D, lanes 3 and 4, arrows show YFP-hCINAP, arrowhead shows endogenous hCINAP), whereas an anti-fluorescent protein antiserum specifically recognized the fusion protein but not endogenous hCINAP (Fig. 4D, lanes 5 and 6). Fluorescence microscopy analysis of cells transiently expressing YFP-hCINAP showed that the fusion protein is specifically nuclear, with a widespread diffuse nucleoplasmic distribution, excluding nucleoli, and some additional concentration in nuclear bodies (Fig. 5, panel A1). A similar widespread nucleoplasmic distribution for endogenous hCINAP was observed in untransfected HeLa cells using both the polyclonal antiserum to the full-length recombinant protein and the serum raised against the hCINAP peptide (Fig. 5, panels B1 and C1). The specific nuclear localization of hCINAP was further supported by the detection of the endogenous protein in HeLa nuclear extract by immunoblotting (data not shown). As we observed in some cells additional concentration of hCINAP in nuclear bodies, we performed double-labeling immunofluorescence experiments to identify these subnuclear structures (Fig. 5, panels A2-C2 and A3-C3). The nuclear bodies containing hCINAP co-localized with anti-coilin antibodies, indicating that the structures are Cajal bodies. This is consistent with the original observation that hCINAP interacts with coilin in the yeast two-hybrid assay.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 8.
Overexpression of hCINAP affects the number of Cajal bodies per nucleus. A, fluorescence micrographs of HeLa cells transfected with either YFP-hCINAP (A1, A2, and A3) or unfused YFP control (B1, B2, and B3). Tagged proteins were detected by fluorescence (A1 and B1), whereas coilin was detected using immunofluorescence (A2 and B2) with a rabbit anti-coilin antibody as described under "Materials and Methods." The merged YFP and coilin images are presented in A3 and B3. Untransfected nuclei show the characteristic coilin staining pattern in both A2 and B2 with both bright foci (arrows) and a diffuse nucleoplasmic staining. Transfected nuclei, as assessed by detection of the fluorescent proteins in A1 and B1 (dashed outline in A2 and B2), only show a reduction in the number of Cajal bodies when transfected with YFP-hCINAP (A2) and not YFP control (B2, see arrowheads for examples of Cajal bodies in a cell transfected with YFP control). Scale bars in all images are 20 µm. B, three-dimensional images of random fields of cells were collected and presented as maximum intensity projections to allow all Cajal bodies within nuclei to be displayed onto a two-dimensional space. The number of Cajal bodies (see A for example) were counted from over 200 nuclei resulting from more than 20 images from each of three conditions: untransfected control (top graph); YFP alone transfected control (bottom graph); YFP-hCINAP transfected (middle graph). The results are displayed as frequency distributions showing the number of Cajal bodies in each nucleus (x-axis) and the number of nuclei (y-axis). Untransfected control nuclei (8.0 ± 0.23 Cajal bodies) (average ± S.E., n = 267) and YFP alone control nuclei (8.5 ± 0.21, n = 288) do not have significantly different numbers of Cajal bodies, whereas those nuclei transfected with YFP-hCINAP (3.3 ± 0.16, n = 221) have a reduced number of Cajal bodies per nucleus. This difference was confirmed to be statistically significant by performing a one-tailed t test (p < 0.0001).

 
We next tested whether hCINAP and coilin interact directly using purified, tagged recombinant proteins (Fig. 6). A GST-tagged form of hCINAP was constructed, expressed in E. coli, and incubated together with E. coli-expressed hexahistidine-tagged coilin for 30 min at room temperature in the presence of 0-3 mM ATP (Fig. 6, A and B). Following incubation, the proteins were mixed with glutathione-Sepharose beads, the beads were collected by centrifugation, washed, and bound and unbound fractions were separated by SDS-PAGE (Fig. 6A) and then transferred to nitrocellulose and probed with an anti-coilin antibody (Fig. 6B). This shows that His6-coilin was recovered in the pellet fraction only when incubated in the presence of GST-hCINAP, consistent with a direct interaction between the two proteins (Fig. 6, A and B, compare lanes 1-4 with the negative control in lanes 5 and 6, arrows indicate His6-coilin). In the negative control lacking GST-hCINAP, all of the His6-coilin was recovered in the supernatant fraction (lanes 7 and 8). Although hCINAP contains a predicted ATP-binding domain and exhibits ATPase activity, no major change in the level of coilin bound to hCINAP was observed using the concentrations of ATP tested. We conclude that ATP does not inhibit binding between hCINAP and coilin and that it is either not strictly required for binding, or it stimulates binding at very low concentrations. To confirm that interaction between hCINAP and coilin occurs also in mammalian cells, we tested for binding of endogenous coilin to YFP-tagged hCINAP in transiently transfected HeLa cells (Fig. 6C). Using a co-immunoprecipitation assay with anti-fluorescent protein antibodies, we observe that coilin is recovered in the antibody-bound fraction only in the presence of YFP-hCINAP (compare lanes 3 and 5, arrow shows endogenous coilin). Taken together, the data indicate strongly that hCINAP is a bona fide interaction partner for coilin.

To test whether the hCINAP-coilin complexes in vivo contain any other known coilin-interacting proteins or components of Cajal bodies or other nuclear antigens, we repeated these immunoprecipitation assays and screened the antibody-bound fractions with antibodies against the U1A snRNP protein, fibrillarin, SMN, Sm, and Sp100 proteins. In contrast with coilin, none of these factors were co-immunoprecipitated with CINAP (data not shown). From these results we infer that the CINAP-coilin interaction is specific and does not involve the other Cajal body proteins tested. To localize the domain of coilin sufficient for interaction with hCINAP we constructed a series of progressively truncated fragments of coilin and assayed for their interaction with full-length hCINAP by the yeast two hybrid assay (Fig. 7). In addition to the combination of full-length coilin and hCINAP only the combination of full-length hCINAP and a construct of coilin containing its 214 carboxyl-terminal amino acids (residues 362-576, construct C1) was able to grow on quadruple selection medium and also displayed {beta}-galactosidase activity (Fig. 7A). This result indicated interaction between hCINAP and the carboxyl-terminal coilin fragment that resulted in the activation of the three independent reporter genes (auxotrophy to adenine and histidine and {beta}-galactosidase activity). The interaction was independently confirmed by in vitro translating the C1 coilin fragment in the presence of [35S]methionine, and testing whether it could be recovered with glutathione-agarose beads after incubation with a lysate made from bacteria expressing GST-tagged CINAP (Fig. 7B, lanes 4 and 5). As a negative control, bacterial lysate carrying the GST-CINAP plasmid, but not induced to express GST-CINAP (Fig. 7B, lanes 2 and 3) was also incubated with the in vitro translated coilin fragment. As shown, the C1 fragment is recovered in the pellet of the glutathione agarose pull-down only in the presence of GST-CINAP and is absent from the pellet when GST-CINAP is not expressed. These results are consistent with and strengthen the results from yeast two-hybrid assay presented above.

Finally, having established the connection between hCINAP and coilin and mapped the CINAP interaction domain on coilin, we examined whether altering the expression level of hCINAP in vivo might have a corresponding effect on either coilin or Cajal bodies (Fig. 8). HeLa cells were transiently transfected with plasmids encoding either full-length YFP-hCINAP or YFP alone and the number of Cajal bodies, as detected using an anti-coilin antibody, scored in more than 200 transfected and 200 non-transfected control cells. The transfected cells expressing YFP-hCINAP were analyzed by direct fluorescence microscopy detection of the YFP (Fig. 8A). These data show that there is an approximate 3-fold reduction in the average number of Cajal bodies per nucleus in the cells that are transiently expressing exogenous hCINAP (Fig. 8B). To confirm this finding, a similar experiment was performed using a myc-tagged hCINAP construct, which also showed an approximate 3-fold reduction in the number of Cajal bodies per nucleus (supplementary Fig. S3). These data indicate that hCINAP expression levels can influence Cajal body number and suggest that hCINAP may have a functional role in modulating either the formation or stability of Cajal bodies.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Here we have identified a novel interaction partner for the Cajal body marker protein, coilin, using a yeast two-hybrid assay with coilin as the bait. The novel protein, termed hCINAP for human coilin interacting nuclear ATPase protein, is highly conserved across its full-length in all species examined, including yeast, plants, and invertebrates. In budding yeast, the orthologue of hCINAP, termed Fap7p (23), is essential for viability. hCINAP (whose nucleotide sequence had previously been reported in cDNA sequencing studies (25-27)) is a predicted ATP-binding protein that is not highly homologous to any other known proteins, but the amino-terminal P-loop motif shows similarity to the adenylate kinase family consensus motif. A very recent study (28) reported the crystal structure of the same protein and proposed to designate it an adenylate kinase (AK6). It was assigned to a distinct subfamily due to its unusually broad substrate specificity and distinct structural features as compared with known human adenylate kinase classes AK1-5. Although in our view the assignment of hCINAP as a bona fide adenylate kinase remains to be validated, our present data confirm its status as an ATPase. We show here that recombinant hCINAP exhibits high apparent affinity ATPase activity (Km = 75.3 ± 5 µM and Vmax = 1.27 ± 0.2 µmol of ADP formed per min·mg).

Interestingly, hCINAP is expressed as an alternatively spliced transcript from the TAF9 locus on human chromosome 5 and shares the two 5'-terminal exons with the mRNA encoding TAFIID32, despite there being no common amino acid sequences in the hCINAP and TAFIID32 proteins. Tagged hCINAP interacts with coilin in mammalian cells and recombinant, E. coli-expressed hCINAP, and coilin bind each other in vitro. hCINAP-coilin complexes in vivo do not contain other known coilin-interacting proteins or Cajal body components, including U1A, fibrillarin, SMN, Sm proteins, and Sp100. Hence the interaction seems to be distinct and independent of the other proteins tested. hCINAP appears to interact with the carboxyl terminus of coilin. It is interesting to note that earlier studies have shown that the carboxyl-terminal 96 amino acids of human coilin seem to be capable of down-regulating the number of Cajal bodies per nucleus (29). Furthermore, post-translational modifications such as phosphorylation and methylation at the carboxyl terminus of coilin influence Cajal body assembly (8, 11). Here we report that the carboxyl terminus of coilin, which appears essential for hCINAP-coilin interaction, also appears to influence Cajal body formation and/or stability, because overexpression of hCINAP causes a reduction in the average number of Cajal bodies in the nucleus of HeLa cells. The combination of these findings suggests that carboxyl terminus of coilin is important for the regulation of Cajal body number in the nucleus of human cells both through its intrinsic properties (structure and/or post-translational modifications) and also through protein-protein interactions with other nuclear proteins (for example hCINAP).

The hCINAP protein shows the hallmarks of a bona fide coilin-interacting protein. This is evident in its biochemical properties, because it interacts with coilin in all of the binding assays we have tested and is also present in complexes containing coilin in vivo. Like coilin, it is also a specifically nuclear factor. hCINAP is present in a widespread, diffuse nucleoplasmic pool, excluding nucleoli, and shows some enrichment in Cajal bodies, although not in all cells. Coilin is a marker protein for Cajal bodies. However, although it is more obviously concentrated in Cajal bodies than is evident for hCINAP, previous studies have shown that it is still a minor fraction of the total pool of nuclear coilin that is located within Cajal bodies at any one time (30). The majority of coilin is present instead in a diffuse nucleoplasmic pool that dynamically exchanges with the Cajal body fraction. It is possible that the hCINAP and coilin proteins interact therefore mainly in the diffuse nucleoplasmic compartment outside of Cajal bodies. The hCINAP protein was identified as a putative coilin interaction partner using a yeast two-hybrid screen with coilin as the bait. This same screen identified a number of other clones, of which the best characterized to date corresponded to cDNAs encoding nuclear import receptors of the importin {alpha}/karyopherin family (10, 22). The interaction of these factors with coilin in vivo is physiologically significant, because they transport coilin into the nucleus. Nonetheless, the interaction of these import receptors with coilin is transient, and they do not colocalize with coilin in vivo. It remains to be established whether hCINAP is a stable or transient interaction partner for coilin, despite its ability to bind to coilin in vitro. One possibility that merits future investigation is that coilin could be a substrate for hCINAP. For example, the predicted ATP binding of hCINAP and its homology with the adenylate kinase motif suggests that it may have kinase activity, which is supported by our finding that hCINAP hydrolyzes ATP in vitro. Consistent with this idea, coilin is known to be a phosphoprotein in vivo. Alternatively, the binding of ATP to hCINAP might affect its conformation and regulate its participation in specific complexes and/or activity.

The primary amino acid sequence of hCINAP is highly conserved, and its orthologue in budding yeast shows 41% identity and ~62% similarity across its full length. As expected for such a highly conserved, single copy gene, knock-out of the gene in budding yeast is lethal, indicating that it has an essential and non-redundant function in the cell. The lethal phenotype of the budding yeast CINAP orthologue has been reported previously in a study of the transcriptional control of genes regulated in response to oxidative stress (23). The yeast POS9/SKN7 gene encodes a transcription factor that is required for the induction of genes activated upon oxidative stress. Juhnke et al. (23) conducted a screen for mutants in budding yeast that failed to activate a POS9-dependent reporter gene and thereby identified a gene they termed FAP7 (Factor activating Pos9p), which corresponds to the S. cerevisiae orthologue of hCINAP. They showed that Fap7/scCINAP, encoding a nuclear protein, is an essential gene in yeast and further showed that strains containing the mutant allele fap7-1, which is viable, fail to activate specifically oxidative stress-dependent genes, but not genes responding to other forms of stress. The fap7-1 allele results from a point mutation converting the conserved Gly-19 to Ser within the adenylate kinase-like P-loop motif. At present it is not clear by what mechanism Fap7p/scCINAP influences POS9 function or whether the effect results from a direct or indirect interaction.

Considering the implication from the analysis of Fap7p in yeast that hCINAP may play a role in the transcriptional control of oxidative stress-regulated target genes, it is interesting that we find here that hCINAP is encoded as an alternative transcript from the TAF9 locus that also encodes a basal transcription factor subunit. The TFIID complex is a multisubunit general transcription factor comprising the TATA box-binding protein TBP and multiple TBP-associated factors, called TAFs (31). It binds to the TATA box element and helps to recruit the TFIIB and TFIIF components of the transcription machinery, forming a protein-DNA complex (preinitiation complex) to which RNA polymerase II subsequently binds. The TAF9 locus on human chromosome 5 encodes a 32-kDa TAFIID subunit (TAFIID32). We show here that the coilin-interacting protein hCINAP is encoded by an alternatively spliced transcript from this same TAF9 locus and shares the first 2 exons with TAFIID32 at the 5' terminus of both mRNAs. However, because translation of TAFIID32 initiates at an internal ATG codon downstream of the first 2 exons that are shared with hCINAP, with a concomitant shift in the reading frame of TAFIID32 as compared with the reading frame used by hCINAP, the respective translation products of hCINAP and TAFIID32 share no common amino acid sequences. Furthermore, alignment of hCINAP and TAFIID32 protein sequences shows that they are unrelated. This represents an unusual situation where two alternative transcripts from the same gene encode completely distinct protein products and is conserved in mammals but not other vertebrates. We observe that a similar ratio of hCINAP and TAFIID32 transcripts is expressed in all the human tissues and cell lines tested, consistent with their being coordinately regulated. However, although the lack of sequence relationship does not exclude hCINAP from playing a possible role in some aspect of transcriptional regulation or related mechanisms, at present there is no direct evidence that hCINAP is a transcription factor. We note that in the genome annotation provided by Ensembl the hCINAP transcript is called "transcription factor TAFIID31," reflecting its genetic relationship with the known transcription factor TAFIID32. Based on our present data, we suggest that it is premature to assign the function of "transcription factor" to hCINAP. Nonetheless, it is possible that hCINAP could be a transcription factor and still play other roles. For example, several nuclear factors have been shown to play dual roles in transcription and RNA binding or processing, including TFIIIA and WT1 (for review see Ref. 32). The role of hCINAP as a coilin interaction partner could be consistent with possible connections to either transcription or RNA-processing mechanisms, or both. Thus Cajal bodies play a role as sites of RNA-protein complex maturation and RNP assembly (1). However, they also contain several transcription factors and have been shown to associate with, and potentially regulate the activity of, specific gene loci, including histones and U snRNA genes (2-4). Future studies will address further the potential role of hCINAP in Cajal body assembly and function.


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

* 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. Back

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

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.

3 The abbreviations used are: snRNP, small nucleolar ribonucleoprotein; snoRNP, small nucleolar ribonucleoprotein; hCINAP, human coilin-interacting nuclear ATPase protein; mAb, monoclonal antibody; RT, reverse transcription; ORF, open reading frame; YFP, yellow fluorescent protein; IPTG, isopropylgalactopyranoside; PBS, phosphate-buffered saline; GST, glutathione S-transferase; Pipes, 1,4-piperazinediethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank the EMBL DNA sequencing and oligonucleotide services and Doros Panayi at the photolab for excellent photographic assistance. We also thank Ursula Ryder for expert technical assistance with the two-hybrid screen. We acknowledge R. Brent (Massachusetts General Hospital, Harvard Medical School, Boston, MA) for kindly providing the two-hybrid vectors, strains, and HeLa cDNA library and G. Christopoulos at the Cyprus Institute of Neurology and Genetics for the gift of a chorionic villi sample. A human motor cortex biopsy was obtained from the University of Miami Brain and Tissue Bank.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ogg, S. C., and Lamond, A. I. (2002) J. Cell Biol. 159, 17-21[Abstract/Free Full Text]
  2. Frey, M. R., Bailey, A. D., Weiner, A. M., and Matera, A. G. (1999) Curr. Biol. 9, 126-135[CrossRef][Medline] [Order article via Infotrieve]
  3. Frey, M. R., and Matera, A. G. (2001) J. Cell Biol. 154, 499-509[Abstract/Free Full Text]
  4. Shopland, L. S., Byron, M., Stein, J. L., Lian, J. B., Stein, G. S., and Lawrence, J. B. (2001) Mol. Biol. Cell 12, 565-576[Abstract/Free Full Text]
  5. Tucker, K. E., Berciano, M. T., Jacobs, E. Y., LePage, D. F., Shpargel, K. B., Rossire, J. J., Chan, E. K., Lafarga, M., Conlon, R. A., and Matera, A. G. (2001) J. Cell Biol. 154, 293-307[Abstract/Free Full Text]
  6. Bohmann, K., Ferreira, J. A., and Lamond, A. I. (1995) J. Cell Biol. 131, 817-831[Abstract/Free Full Text]
  7. Hebert, M. D., Szymczyk, P. W., Shpargel, K. B., and Matera, A. G. (2001) Genes Dev. 15, 2720-2729[Abstract/Free Full Text]
  8. Hebert, M. D., and Matera, A. G. (2000) Mol. Biol. Cell 11, 4159-4171[Abstract/Free Full Text]
  9. Isaac, C., Yang, Y., and Meier, U. T. (1998) J. Cell Biol. 142, 319-329[Abstract/Free Full Text]
  10. Weis, K., Mattaj, I. W., and Lamond, A. I. (1995) Science 268, 1049-1053[Abstract/Free Full Text]
  11. Hebert, M. D., Shpargel, K. B., Ospina, J. K., Tucker, K. E., and Matera, A. G. (2002) Dev. Cell 3, 329-337[CrossRef][Medline] [Order article via Infotrieve]
  12. Carvalho, T., Almeida, F., Calapez, A., Lafarga, M., Berciano, M. T., and Carmo-Fonseca, M. (1999) J. Cell Biol. 147, 715-728[Abstract/Free Full Text]
  13. Kambach, C., and Mattaj, I. W. (1992) J. Cell Biol. 118, 11-21[Abstract/Free Full Text]
  14. Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5, 3610-3616[Abstract/Free Full Text]
  15. Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993) Cell 75, 791-803[CrossRef][Medline] [Order article via Infotrieve]
  16. Kandels-Lewis, S., and Seraphin, B. (1993) Science 262, 2035-2039[Abstract/Free Full Text]
  17. Stade, K., Ford, C. S., Guthrie, C., and Weis, K. (1997) Cell 90, 1041-1050[CrossRef][Medline] [Order article via Infotrieve]
  18. Skamnaki, V. T., Owen, D. J., Noble, M. E., Lowe, E. D. Lowe, G., Oikonomakos, N. G., and Johnson, L. N. (1999) Biochemistry 38, 14718-14730[CrossRef][Medline] [Order article via Infotrieve]
  19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd. Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  20. Laemmli, U. K. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
  21. Stelzer, E. H., Wacker, I., and De Mey, J. R. (1991) Semin. Cell Biol. 2, 145-152[Medline] [Order article via Infotrieve]
  22. Nachury, M. V., Ryder, U. W., Lamond, A. I., and Weis, K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 582-587[Abstract/Free Full Text]
  23. Juhnke, H., Charizanis, C., Latifi, F., Krems, B., and Entian, K. D. (2000) Mol. Microbiol. 35, 936-948[CrossRef][Medline] [Order article via Infotrieve]
  24. Klemm, R. D., Goodrich, J. A., Zhou, S., and Tjian, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5788-5792[Abstract/Free Full Text]
  25. Lai, C. H., Chou, C. Y., Ch'ang, L. Y., Liu, C. S., and Lin, W. (2000) Genome Res. 10, 703-713[Abstract/Free Full Text]
  26. Hu, R. M., Han, Z. G., Song, H. D., Peng, Y. D., Huang, Q. H., Ren, S. X., Gu, Y. J., Huang, C. H., Li, Y. B., Jiang, C. L., Fu, G., Zhang, Q. H., Gu, B. W., Dai, M., Mao, Y. F., Gao, G. F., Rong, R., Ye, M., Zhou, J., Xu, S. H., Gu, J., Shi, J. X., Jin, W. R., Zhang, C. K., Wu, T. M., Huang, G. Y., Chen, Z., Chen, M. D., and Chen, J. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9543-9548[Abstract/Free Full Text]
  27. Strausberg, R. L., Feingold, E. A., Grouse, L. H., Derge, J. G., Klausner, R. D., Collins, F. S., Wagner, L., Shenmen, C. M., Schuler, G. D., Altschul, S. F., Zeeberg, B., Buetow, K. H., Schaefer, C. F., Bhat, N. K., Hopkins, R. F., Jordan, H., Moore, T., Max, S. I., Wang, J., Hsieh, F., Diatchenko, L., Marusina, K., Farmer, A. A., Rubin, G. M., Hong, L., Stapleton, M., Soares, M. B., Bonaldo, M. F., Casavant, T. L., Scheetz, T. E., Brownstein, M. J., Usdin, T. B., Toshiyuki, S., Carninci, P., Prange, C., Raha, S. S., Loquellano, N. A., Peters, G. J., Abramson, R. D., Mullahy, S. J., Bosak, S. A., McEwan, P. J., McKernan, K. J., Malek, J. A., Gunaratne, P. H., Richards, S., Worley, K. C., Hale, S., Garcia, A. M., Gay, L. J., Hulyk, S. W., Villalon, D. K., Muzny, D. M., Sodergren, E. J., Lu, X., Gibbs, R. A., Fahey, J., Helton, E., Ketteman, M., Madan, A., Rodrigues, S., Sanchez, A., Whiting, M., Madan, A., Young, A. C., Shevchenko, Y., Bouffard, G. G., Blakesley, R. W., Touchman, J. W., Green, E. D., Dickson, M. C., Rodriguez, A. C., Grimwood, J., Schmutz, J., Myers, R. M., Butterfield, Y. S., Krzywinski, M. I., Skalska, U., Smailus, D. E., Schnerch, A., Schein, J. E., Jones, S. J., Marra, M. A. (the Mammalian Gene Collection Program Team) (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 16899-16903[Abstract/Free Full Text]
  28. Ren, H., Wang, L., Bennett, M., Liang, Y., Zheng, X., Lu, F., Li, L., Nan, J., Luo, M., Eriksson, S., Zhang, C., and Su, X. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 303-308[Abstract/Free Full Text]
  29. Shpargel, K. H., Ospina, J. K., Tucker, K. E., Matera, A. G., and Hebert, M. D. (2003) J. Cell Science 116, 3103-3110
  30. Bellini, M., and Gall, J. G. (1998) Mol. Biol. Cell 9, 2987-3001[Abstract/Free Full Text]
  31. Muller, F., and Tora, L. (2004) EMBO J. 23, 2-8[CrossRef][Medline] [Order article via Infotrieve]
  32. Ladomery, M. (1997) BioEssays 19, 903-909[CrossRef][Medline] [Order article via Infotrieve]
  33. Santama, N., Krijnse-Locker, J., Griffiths, G., Noda, Y., Hirokawa, N., and Dotti, C. G. (1998). EMBO J. 17, 5855-5867[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
280/43/36429    most recent
M501982200v2
M501982200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Santama, N.
Right arrow Articles by Lamond, A. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santama, N.
Right arrow Articles by Lamond, A. I.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement