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Originally published In Press as doi:10.1074/jbc.M305935200 on December 4, 2003

J. Biol. Chem., Vol. 279, Issue 9, 8343-8350, February 27, 2004
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A Novel Human Nucleolar Protein, Nop132, Binds to the G Proteins, RRAG A/C/D*

Takeshi Sekiguchi{ddagger}, Yuko Todaka, Yonggang Wang, Eiji Hirose, Nobutaka Nakashima§, and Takeharu Nishimoto

From the Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Received for publication, June 5, 2003 , and in revised form, November 13, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RRAG A (Rag A)/Gtr1p is a member of the Ras-like small G protein family that genetically interacts with RCC1, a guanine nucleotide exchange factor for RanGTPase. RRAG A/Gtr1p forms a heterodimer with other G proteins, RRAG C and RRAG D/Gtr2p, in a nucleotide-independent manner. To further elucidate the function of RRAG A/Gtr1p, we isolated a protein that interacts with RRAG A. This protein is a novel nucleolar protein, Nop132. Nop132 is associated with the GTP form, but not the GDP form, of RRAG A, suggesting that RRAG A might regulate Nop132 function. Nop132 is also associated with RRAG C and RRAG D. The Nop132 amino acid sequence is similar to the Saccharomyces cerevisiae nucleolar Nop8p, which is associated with Gtr1p, Gtr2p, and Nip7p. Nop132 also interacts with human Nip7 and is colocalized with RRAG A, RRAG C, and Nip7. RNA interference knockdown of Nop132 inhibited cell growth of HeLa cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Guanine nucleotide-binding proteins are a superfamily of regulatory GTP hydrolases composed of a large number of proteins, which include Ras family proteins, heterotrimeric G protein {alpha} subunits, and the elongation factors, TU and G, among others (1). Ras-like small G proteins such as Ras, Rab, Rho, ARF, and Ran are monomeric and bind to the guanine nucleotides, GTP or GDP, to function as molecular switches. They also have crucial roles in cell growth, differentiation, and protein trafficking between different intracellular compartments (2, 3).

Among the Ras superfamily of small G proteins, Ran is a nuclear protein with several functions, including nucleocytoplasmic transport of many types of protein and nucleic acids across nuclear membranes (4, 5). The guanine nucleotide exchange factor for Ran (RCC1) is bound to chromatin with histone H2A and H2B and confined within the nucleus (6, 7), whereas the Ran GTPase-activating protein is located both inside the cytosol and tethered to the external face of the nuclear pore complex with RanBP2. Compartmentalization of these factors is believed to create a gradient of GTPase Ran across the nuclear pore complex, which controls the stability of Imp{beta} interactions with particular cargo molecules. Ran is also a key factor that controls mitotic processes, including spindle assembly during metaphase and reformation of the nuclear envelope during telophase (8, 9).

gtr1-11suppresses srm1, a yeast Saccharomyces cerevisiae RCC1 functional homologue (10). Although the precise function of Gtr1p remains uncertain, it is thought to have a role in phosphate transport (11). Although yeast cells can grow even if GTR1 and GTR2 are deleted, they cannot grow at low temperature (12). Because many G proteins have multiple functions, Gtr1p and Gtr2p localization within the nucleus and cytoplasm suggests a role in both, possibly that of nucleocytoplasmic transport, as in the case of RanGTPase.

RRAG A/Rag A is a functional homologue to yeast Gtr1p (13) and belongs to the Ras superfamily of small G proteins. RRAG A/Rag A/FIP-1 was originally isolated using PCR methods during the search for novel G proteins (14). It was also independently isolated by two-hybrid screening using adenovirus E3 14.7K as the bait and is involved in tumor necrosis factor {alpha}-mediated cell death (15). We previously reported that RRAG A/Rag A forms a complex with other G proteins, RRAG C/Rag C and RRAG D/Rag D, which are homologues to Gtr2p (16). Heterodimer formation between RRAG A and RRAG C is a specific characteristic of these proteins, which are distinct from other Ras family proteins in that they are usually monomeric. RRAG A/Rag A/FIP-1 is associated with the dynein light chain, which is part of the molecular motor system located at the negative ends of microtubules, close to the nuclear membrane. This suggests a role for RRAG A/Rag A/FIP-1 in dynein-microtubule effects on the transcytoplasmic transport of organelles, viruses, and signal transduction molecules (17). The Human Genome Organization Gene Nomenclature Committee (www.hugo-international.org/hugo/) recently approved the root symbol of RRAG # for Ras-related GTP binding #. Thus, in the present study, RRAG A, RRAG B, RRAG C, and RRAG D were used for Rag A, Rag B, Rag C, and Rag D, respectively.

In an effort to determine which cellular proteins might interact with RRAG A in the RCC1-Ran pathway, yeast two-hybrid screening was performed using RRAG A as the bait. This search identified a novel protein, which we named Nop132, because the protein has classic characteristics of nucleolar proteins, such as nucleolar localization and RNA-binding activity. Exogenous expression of the carboxyl terminus of Nop132 guided the formation of the R-rings induced by Nopp140 (18). Nop132 also interacted with RRAG C, RRAG D, and Nip7 and might be an anchoring protein that localizes these proteins to the nucleolus in the signal transduction pathway. Amino acid sequence comparison revealed that Nop132 is similar to the yeast Nop8p, an essential nucleolar protein involved in ribosome biogenesis (19). Nop132 knockdown by RNA interference revealed that Nop132 is an essential protein, as is Nop8.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transient Transfection—BHK21 cells and HeLa cells were grown at 37.5 °C in Dulbecco's modified Eagle medium containing 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) in a humidified atmosphere of 10% CO2-90% air. Cells were washed with TD buffer (25 mM Tris-HCl, pH 7.4, 136.8 mM NaCl, 5 mM KCl, and 0.7 mM Na2HPO4). BHK21 cells (2 x 105 cells) were transfected with DNA-lipid complex comprising 1 µg each of various vectors plus 7 µl (for a 35-mm dish) or 15 µl (for a 60-mm dish) of LipofectAMINETM reagent (Invitrogen, Carlsbad, CA) for 4 h in the absence of serum and antibiotics as recommended by the supplier, and incubated at 37.5 °C for 48 h as described previously (20).

Two pairs of small interfering RNAs (siRNA)1 were chemically synthesized (Hokkaido System Science, Sapporo, Japan) and annealed before transfection. Transfection was performed on 2 x 104 HeLa cells with a final concentration of 200 mM siRNA duplex using Oligofectamine reagent (Invitrogen) according to the manufacturer's guidelines. Using fluorescein isothiocyanate (FITC)-conjugated control oligonucleotide, there was nearly 100% incorporation of the oligonucleotide under our experimental conditions observed with a fluorescence microscope. The oligonucleotide sequences used for antisense and RNA interference experiments were: A-a (5'-UGAAGAACAGGUAAGACUCTT-3'), A-s (5'-GAGUCUUACCUGUUCUUCATT-3'), B (5'-CACUUCACCUAAUGAGCCATT-3'/5'-UGGCUCAUUAGGUGAAGUGTT-3'), and C (5'-CAGUUUCCUUAGGUGAGCCTT-3'/5'-GGCUCACCUAAGGAAACUGTT-3').

Generation of Recombinant Baculoviruses—Recombinant baculoviruses encoding Nop132, RRAG A, RRAG C, and RRAG D were generated in Sf9 cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen) according to the manufacturer's instructions. After the initial transfection, working virus stocks were generated by three sequential virus amplifications. Sf9 cells were cultured in 250-ml disposable Erlenmeyer flasks at 27 °C under rotation at 125 rpm in Grace's insect medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). Cells were maintained at a density of 0.5-6.0 x 106 cells/ml. Cells were seeded at 3.0 x 106 cells and infected with a 1:100 dilution of baculovirus stocks and cultured for 48-72 h before obtaining the cell extract.

Yeast Growth Media, Two-hybrid Screening—S. cerevisiae cells were grown in the following media: YPD (2% glucose, 2% peptone, and 1% yeast extract), SD-Trp, -Leu, and -His (2% glucose and 0.67% yeast nitrogen base without amino acids, supplemented with all essential amino acids except for tryptophan, leucine, and histidine), and GE-Leu and -Trp (3% glycerol, 2% ethanol, and 0.67% yeast nitrogen base without amino acids, supplemented with all essential amino acids except for leucine and tryptophan). Amino acids were added to a final concentration of 20-50 µg/ml. The solid media contained 2% agar in addition to the components described above. Large scale yeast transformation and two-hybrid screening were performed using the Y190 S. cerevisiae strain essentially as described before (16). The quantitative {beta}-galactosidase assay was performed as described previously (16). The {beta}-galactosidase chromogenic filter assays were performed by transferring the yeast colonies onto nitrocellulose filters (Protran BA85, Schleicher and Schuell, Germany). The yeast cells were partially lysed by submerging the filters in liquid nitrogen for 1 min. Filters were allowed to dry at room temperature for at least 5 min and placed onto filter paper presoaked in Z-buffer (100 mM sodium phosphate (pH 7.0), 10 mM KCl, 1 mM MgSO4) supplemented with 0.07 mg/ml 5-bromo-4-chloro-3-indolyl-D-galactoside. Filters were placed at 30 °C.

Purification of GST Fusion Proteins—Escherichia coli BL21 harboring a GST plasmid was grown in 750 ml of LB medium, treated with isopropyl {beta}-D-thiogalactoside (final concentration, 0.2 mM) for 4 h at 30 °C as described previously (21). Cells were dispersed in lysis solution at a ratio of 1:5 (cell volume: lysis solution (1x phosphate-buffered saline, 2 mM EDTA, 0.1% {beta}-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin)) and sonicated three times for 5 min on ice (SonicatorTM, Heat System-Ultrasonics Inc., Farmingdale, NY, with a microtip, 40% cycle, and output control 4). After centrifugation at 10,000 x g for 30 min at 4 °C, 10 ml of the supernatant was mixed with 1 ml of a 50% (v/v) slurry of glutathione Sepharose-4B beads (Amersham Biosciences, Buckinghamshire, UK) and rotated for 30 min at 4 °C. The beads were washed four times with the lysis buffer.

Recombinant DNA—To obtain a full-length cDNA of Nop132, we performed a DNA data base search (BLAST) of human expressed sequence tag clones using a two-hybrid clone cDNA sequence. Clones HEP08473(DDBJ/GenBankTM/EMBL accession no. AK000743 [GenBank] ; provided by Dr. Sumio Sugano, University of Tokyo) and HEMBA1004596 (DDBJ/GenBankTM/EMBL accession no. AK001049 [GenBank] ; provided by Dr. Takao Isogai, Helix Research Institute) were derived from the same gene. Because there were gaps between these two clones, we amplified a 1.0-kbp middle region using the reverse transcription-PCR method with HeLa poly(A)+ RNA (DDBJ/GenBankTM/EMBL accession number, AB109030 [GenBank] ). We also amplified human Nopp140, Clk, and RNPS1 cDNAs with the RT-PCR method using HeLa poly(A)+ RNA. The BglII/SalI fragment of Nopp140 was inserted into the BglII/SalI site of pDsRed1. The XhoI/BamHI fragment of Clk was inserted into the XhoI/BamHI site of pEGFPc1. The BamHI/HindIII fragment of RNPS1 was inserted into the BamHI/HindIII site of pDsRed1. GST-Nop132N was constructed by inserting the BglII/BamHI fragment of Nop132 (1-197 aa) into the BamHI site of pGEX-KG. Fusion proteins with a GAL4 DNA-binding domain (GAL4BD) were constructed in pAS1 or pAS404. Those with the GAL4 activation-binding domain (GAL4AD) were constructed in pACT2, as described previously (16). Each construct was verified by automated DNA sequencing using an ABI PRISMR 3100 sequencer (Applied Biosystems, Foster City, CA).

Immunoblotting and Antibodies—Protein samples were electrophoresed on a 10% or 12.5% SDS-polyacrylamide slab gel or 5-20% gradient gel (Pagel, Atto, Japan) and analyzed by immunoblotting. Immunoblotting was performed as described previously (22) using an ECL kit (Amersham Biosciences) as recommended by the supplier. The Nop132 antibody was prepared as follows. Based on the sequence of human Nop132 cDNA, a peptide with the sequence HYDPTKQDHATYERKRDDK was synthesized. This peptide was coupled to the carrier protein and used to immunize rabbits. The Nop132N, RRAG A, and RRAG C antibodies were prepared by immunizing rabbits with GST-Nop132N, GST-RRAG A, or GST-RRAG C proteins.

Rabbit anti-HA antibody (catalog no. 561) was purchased from the MBL Institute. Mouse anti-T7 antibody (Cat.no.69522) was purchased from Novagen Inc. (Madison, WI). Rabbit anti-myc antibody (A-14) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The nucleolar-specific antiserum (catalog no. ANA-N) was purchased from Sigma-Aldrich Co. (St. Louis, MO).

In Vitro Binding Assay—Nop8p and portions of Nop132 were synthesized for the in vitro binding assay. Nop8p in the pET28a vector and the fragments of Nop132 cDNA in the pCR2 vector (Invitrogen) were incubated with 20 µCi of [35S]methionine (PerkinElmer Life Sciences, Boston, MA) plus 40 µl of a quick master mix of TNT® T7 Quick Coupled Transcription/Translation Systems (Promega Corp., Madison, WI) for 90 min at 30 °C as recommended by the supplier. The resultant extract was diluted to 500 µl with the immunoprecipitation buffer (50 mM Tris-Cl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.1 µg/ml aprotinin, and 1 mM DTT). GST, GST-RRAG A, GST-RRAG C, or GST-Nip7 (20 µg), which were bound to the glutathione Sepharose-4B beads, were mixed with the 35S-labeled proteins. After incubation at 4 °C for 30 min, the beads were spun down, washed four times with the immunoprecipitation buffer, and suspended in 50 µl of the sample buffer. Bound proteins were run on an SDS-polyacrylamide gel and analyzed using the Fuji BAS2000 Image Analyzer (Fuji Photo Film Co. Ltd., Japan).

Immunofluorescence—Cells (2 x 105) that were seeded on coverslips in a 35-mm dish were transfected with 1 µg of DNA using 7 µl of LipofectAMINE. The transfected cells on coverslips were fixed with 1 ml of cold methanol/acetone (1:1) for 5 min at -20 °C or 1 ml 4% paraformaldehyde for 5 min. They were processed for immunostaining as described previously (20). Cells were stained with Hoechst33342 (1 µg/ml) and mounted on Vectashield (Vector Laboratories, Burlingame, CA)-treated slides. A Zeiss Axiophot microscope (Germany) was used for sample analysis using standard microscopic methods. Digital images of the stained cells were obtained using the Olympus laser-scanning microscope LSM-GB200 system, as described previously (13).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Novel Protein, Nop132, That Interacts with RRAG A—The identification of interacting proteins with known functions is helpful for determining the function of RRAG A/Gtr1p and RRAG C/Gtr2p proteins. To search for interacting proteins for RRAG A, we employed yeast two-hybrid screens with RRAG A. Such screenings revealed that 2 of 30 clones were derived from cDNA fragments from the same gene. The full-length gene was isolated as described under "Experimental Procedures" and subcloned into the pME18SFL3 vector. The gene had the predicted molecular mass of 132 kDa and was named Nop132, because the gene product was localized within the nucleolus (see Fig. 2, a and b). The deduced amino acid sequence analysis of Nop132 protein indicated that Nop132 has a eukaryotic RNA recognition motif at its amino-terminal region (8-89 aa residues; Fig. 1a), suggesting that the Nop132 protein is an RNA-binding protein. A BLAST search identified the putative mouse homologue of Nop132 (GenBankTM accession no. XM127302). Sequence similarity comparisons between human Nop132 and the mouse homologue (Fig. 1a) indicated a higher similarity at both the amino-terminal region (1-240 aa; 83.8%) and the carboxyl-terminal region (750-1168 aa; 70.7%). A BLAST search also revealed that yeast Nop8p (GenBankTM accession no. Q08287 [GenBank] ) has similarity with human Nop132 in both the amino-terminal region (1-90 aa; 29.8%) and carboxyl-terminal region (750-1168 aa; 16%), where a coiled-coil structure for protein interaction was predicted in both proteins. The basic amino acid stretch of Nop132 (958-976 aa) was predicted to be a nuclear localization signal (Fig. 1, a and b). Three coiled-coil structures, one at its amino-terminal region and the other two at its carboxyl-terminal region, were predicted regions for protein-protein interaction (Fig. 1a).



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  		FIG. 2.
Nucleolar localization of Nop132. a, BHK21 cells stably expressing DsRed-tagged Nop132 were immunostained with the anti-ANA-N antibody followed by a FITC-conjugated anti-mouse antibody. DNA was stained by Hoechst 33157 dye. The image was taken with a fluorescence microscope. Scale bar, 10 µm. b, paraformaldehyde-fixed HeLa cells were stained with affinity-purified rabbit anti-Nop132N (red) and human anti-ANA-N antibodies (green) and then with Alexa Fluor 568-conjugated anti-rabbit (left panel) and FITC-conjugated anti-human antibodies (middle panel). The images were taken with a confocal microscope. The merged image is shown in the right panel. c, BHK21 cells were transiently cotransfected with Nop132 in pME18S-FL and myc-RRAG A in pcDEB{Delta}. The cells were fixed and immunostained first with rabbit anti-Nop132 and the mouse anti-myc-tag antibodies, and then with Texas red-conjugated anti-mouse (middle panel) and FITC-conjugated anti-rabbit antibodies (left panel), before being processed for confocal microscopy imaging. The merged image is shown in the right panel. d, BHK21 cells were transiently cotransfected with the HA- carboxyl terminus of Nop132 (883-1168 aa) in pcDEB{Delta} and T7-RRAG A in pcDEB{Delta}. The cells were fixed and immunostained with rabbit anti-HA and mouse anti-T7-tag antibodies and then with Texas red-conjugated anti-rabbit (left panel) and FITC-conjugated anti-rabbit antibodies (middle panel), before being processed for confocal microscopy imaging. The merged image is shown in the right panel. e, BHK21 cells were transiently cotransfected with Nop132 in pME18S-FL and HA-RRAG C in pCDNA3. The cells were fixed and immunostained with rabbit anti-Nop132 and mouse anti-HA-tag antibodies and then with Texas red-conjugated anti-mouse (middle panel) and FITC-conjugated anti-rabbit antibodies (left panel), before being processed for confocal microscopy imaging. f, BHK21 cells were transiently transfected with Nop132 in pME18S-FL and pDsRed-Nopp140 expression plasmids. Forty-eight hours later, the cells were fixed and processed as described above. Merging of the Nop132 (green) and DsRed-Nopp140 (red) staining patterns is shown in the right panel, where colocalization of the two proteins yields a yellow signal. g, BHK21 cells were transiently transfected with pDsRed-Nop132 and pEGFP-clk-1 expression plasmids and processed as above. h, BHK21 cells were transiently transfected with pEGFP-Nop132 and pDsRed-RNPS1 expression plasmids and processed as above.

 



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  		FIG. 1.
a, schematic representation of Nop132 motifs. Nop132 motifs were predicted using Pfam (32) and the Coil program (33). RRM, RNA recognition motif; Coil, coiled-coil domain; NLS, nuclear localization signal. Sequence similarity (% similarity to human Nop132) was calculated using DNASIS software (Hitachi Software, Japan). b, the NLS is present in a portion of Nop132 (958-RKRDDKPRESKAKRKKKR-976). BHK21 cells were transiently transfected with the vectors as described below. The cells were fixed with 4% paraformaldehyde for 5 min and processed for fluorescence microscopy. The carboxyl terminus of Nop132 with NLS was localized within the nucleus (i). The carboxyl terminus of Nop132 without NLS and the EGFP vector were localized within both the cytoplasm and the nucleus (ii and iii). i, pEGFPc1-carrying carboxyl terminus of Nop132 (958-1168 aa); ii, pEGFPc1-carrying carboxyl terminus of Nop132 (977-1168 aa); iii, pEGFPc1 vector. Left panels are phase images. Right panels are fluorescence images. Arrows in the phase image panels indicate cells expressing EGFP. Scale bar, 10 µm. c, Western blot analysis of viral coinfection. Left panel: samples were run on SDS-polyacrylamide gels. Sf9 insect cells coinfected with the Nop132 recombinant baculovirus and one of the following recombinant baculovirus vectors: RRAG A, RRAG C, and/or RRAG D. The cell lysates were immunoprecipitated with the anti-Nop132 antibody. The precipitates were run on SDS-polyacrylamide gels and transferred onto a polyvinylidene difluoride membrane and blotted with the anti-His tag antibody to detect His-tagged proteins, as described under "Experimental Procedures". The positions of Nop132, RRAG A, RRAG C, and RRAG D are indicated by arrows. IgG bands are indicated by an asterisk. Right panel: total cell extract and the supernatant fraction used above were run on Pagel and immunoblotted with the anti-His antibody. d, the association of wild-type, T21L, and Q66L forms of RRAG A with Nop132. Extracts were obtained from cultures of S. cerevisiae colonies harboring two human genes, the RRAG A wild-type (lanes 1 and 4), T21L-(gtr1-11) (lane 2), or the Q66L form (lane 3) in the pAS404 vector, and the carboxyl terminus of Nop132 (883-1168 aa) in the pACT2 vector (lanes 1-3), as shown in the figure. Their {beta}-galactosidase activities were measured as described under "Experimental Procedures" and are shown as means of duplicate values with standard deviations. Lower panel: two independent transformants were assayed for {beta}-galactosidase activity in filter assay. e, colony growth was tested on a plate lacking tryptophan and leucine or on a plate lacking tryptophan, leucine, and histidine, but with the addition of 3-AT. Two independent colonies of each clone were picked up. The experiment was performed at least twice and representative results are shown.

 
To observe Nop132 and RRAG A interaction in vivo, His tag fusion proteins were produced in Sf9 cells by infection with recombinant baculovirus, which expressed Nop132 and RRAG A, as shown in Fig. 1c (right panel, lanes 4). Nop132 was bound to RRAG A (Fig. 1c, left panel, lanes 3). As a control, normal rabbit serum failed to pull down RRAG A (Fig. 1c, left panel, lanes 4). Previously, we suggested that RRAG C and RRAG D probably form a heterodimer with RRAG A (16). Therefore, we examined whether RRAG C and RRAG D could also associate with Nop132. Nop132 was associated with RRAG C and RRAG D, as well as with RRAG A (Fig. 1c, left panel, lanes 5 and 7).

In general, Ras-like small G proteins function as binary switches by cycling between an inactive form bound to GDP and the active GTP-bound state, thereby modulating the structure of their target proteins (effectors) by binding to and activating or inhibiting their biologic activity. A candidate effector protein for RRAG A has not yet been identified. To determine whether Nop132 was a candidate effector protein of RRAG A, wild-type and T21L and Q66L forms of RRAG A were used as bait to examine the interaction with the carboxyl terminus of Nop132 (883-1168 aa; Fig. 1, d and e). The T21L mutant of RRAG A (mutation of the 21st threonine to leucine), corresponded to a dominant negative form of Ran, T24N (GDP form), whereas the Q66L mutant of RRAG A (mutation of the 66th glutamine to leucine) corresponded to a dominant positive form of Ran, Q69L (GTP form) (12, 13). Positive associations between constructs were monitored by transcriptional activation of the His3 gene by growth on defined media lacking histidine and activation of the lacZ reporter gene, which resulted in the expression of {beta}-galactosidase activity. The control Y190 strain harboring pAS404-RRAG A and pACT2 had fewer than 8.0 {beta}-galactosidase units. There were 472 {beta}-galactosidase units in the Y190 strain harboring pAS404-RRAG A and pACT2-Nop132 (Fig. 1d). There were 732 {beta}-galactosidase units in pAS404-RRAG A (GTP form)-pACT2-Nop132 (Fig. 1d), whereas there were fewer than 10 {beta}-galactosidase units in pAS404-RRAG A (GDP form) and pACT2-Nop132, suggesting that Nop132 does not interact with the RRAG A GDP form. Y190 strains, harboring pAS404-RRAG A (GTP form)-pACT2- Nop132 but not pAS404-RRAG A (GDP form)-pACT2- Nop132, grew in SD medium lacking tryptophan, leucine, and histidine but containing 3-AT (Fig. 1e).

Nop132 Is a Novel Nucleolar Protein—Subcellular localization of Nop132 was studied with immunofluorescence staining. BHK21 cells were transfected with DsRed-tagged Nop132 to establish a cell line that stably expressed DsRed-tagged Nop132. Double labeling of BHK21 cells with the nucleolar-specific antiserum ANA-N (23) and Nop132 indicated that they were colocalized (Fig. 2a). In this experiment, Nop132 appears red, whereas the nucleolus is labeled green. Consistently, regions of red spots in DsRed-Nop132 (left panel) were colocalized with faintly stained regions in a DNA panel and dark regions of the nucleus in a phase image panel (right panel), which are characteristic findings for the nucleolus. Moreover, double labeling of HeLa cells with ANA-N and an affinity-purified antibody against Nop132 revealed a colocalization pattern, suggesting that Nop132 is mainly localized in the nucleolus (Fig. 2b). Consistently, RRAG A colocalized with Nop132 in BHK21 cells following transient transfection (Fig. 2c, right panel). Nop132 had a nuclear localization signal (958-976 aa) in its carboxyl terminus (Fig. 1, a and b); consistently, the carboxyl terminus of Nop132 (827-1168 aa) localized within the nucleus (Fig. 2d, left panel). The carboxyl terminus of Nop132 also colocalized with RRAG A (Fig. 2d, right panel). As shown by the arrowheads in Fig. 2d, the carboxyl terminus of Nop132 and RRAG A were localized in a unique ring-like structure, which is similar to R-rings (18, 24). Nop132 probably guided the formation of the R-rings in the nucleus, because RRAG A could not guide the formation of the R-rings. Only Nopp140 can guide the formation of the R-rings. Consistently, Nopp140 localized at the periphery of Nop132, suggesting that Nop132 and Nopp140 have similar roles in the nucleus and nucleolus (Fig. 2f). RRAG C also colocalized with Nop132 in BHK21 cells, although RRAG C in the nucleus localized at the periphery of Nop132 (Fig. 2e, right panel). When overexpressed by transient transfection, Nop132 sometimes colocalized with other nuclear structures, as indicated by colocalization of Nop132 with the proteins localized in the nuclear speckles such as Clk-1 and RNPS1 (Fig. 2, g and h). These findings suggest that Nop132 has multiple roles in the nucleus.

Nop132 Interacts with Nip7—Nop132 has sequence similarity to yeast S. cerevisiae Nop8p (Fig. 1a). S. cerevisiae Gtr1p was used to determine whether it could bind to Nop8p. GST-Gtr1p and GST-Gtr2p were able to pull down in vitro synthesized 35S-labeled Nop8p (Fig. 3a, lanes 2 and 3). As a control, GST failed to pull down Nop8p (Fig. 3a, lane 1). GST-Gtr1p and GST-Gtr2p, which were prepared from the yeast cell lysate, pulled down HA-tagged Nop8p (data not shown). Yeast two-hybrid analysis revealed that the region that interacts with Gtr1p resides within amino acid residues 201-484 of Nop8p (data not shown).



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  		FIG. 3.
The association of Nop132 with Nip7. a, S. cerevisiae NOP8 was subcloned into pET28a. 35S-Labeled recombinant protein was produced using the TNT-Quick Coupled Reaction Kit as described under "Experimental Procedures" and pulled down with GST (lane 1), GST-Gtr1p (lane 2), and GST-Gtr2p (lane 3), which were bound to the glutathione Sepharose-4B beads. Lane 4 corresponds to 2% of labeled Nop8p input. Bound proteins were run on SDS-polyacrylamide gels and analyzed using the Fuji Image Analyzer. b, baculovirus-produced Nop132 was mixed with GST (lane 3) or GST-Nip7 (lane 4) beads. Bound Nop132 was detected by Western blotting analysis using the anti-Nop132 antibody. Lane 1 corresponds to 20% of Nop132 input. Lane 2 corresponds to 10% of Nop132 input. c, two-hybrid analysis for Nop132 and Nip7 interaction. Colony growth was tested on a plate lacking tryptophan and leucine or on a plate lacking tryptophan, leucine, and histidine, but with the addition of 3-AT. Three independent colonies of each clone were picked up. For the filter assay of {beta}-galactosidase activity, the color intensities of a representative transformant for each strain are shown (lower panel). Nop132C: 883-1168 aa; Nop132: 1-1168 aa. d, colocalization of Nop132 with Nip7. BHK21 cells were transiently cotransfected with Nop132-EGFP and Nip7-DsRed1. The cells were fixed and processed for confocal microscopy imaging. Scale bar, 10 µm.

 
Nop8p interacts with Nip7p in budding yeast (19). The human homologue of Nip7p (hypothetical protein, HSPC031; GenBankTM accession no. AAD40195 [GenBank] has high similarity to yeast Nip7p (overall amino acid similarity, 57%). If Nop132 has a homologous function with yeast Nop8p, Nop132 might interact with human Nip7. As expected, in vitro-produced GST-Nip7 fusion protein pulled down baculovirus-produced Nop132 (Fig. 3b). As a control, GST protein failed to pull down Nop132. Yeast two-hybrid analysis revealed that Nip7 interacted with the Nop132 carboxyl-terminal portion (883-1168 aa; Fig. 3c). Moreover, Nip7 was colocalized with Nop132 within the nucleus in BHK21 cells (Fig. 3d).

The Carboxyl Terminus Region of Nop132 Is the Binding Region for RRAG A, RRAG C, and Nip7—To determine the region of Nop132 that binds to RRAG A, RRAG C, and Nip7, GST-RRAG A, GST-RRAG C, and GST-Nip7 proteins were purified and used to pull down portions of 35S-labeled Nop132. RRAG A, RRAG C, and Nip7 bound within a relatively small region (958-1168 aa) of Nop132 (Fig. 4a). Serial deletion clones of Nop132 were used as targets to determine the minimum region of interaction with RRAG A using a two-hybrid assay (966-1116 aa; Fig. 4b). The G proteins, RRAG A and RRAG C, were signaling molecules and localized within the cytoplasm and the nucleus, but Nop132 was localized within the nucleolus. Thus, Nop132 protein could be an anchor protein, which is defined as a protein that localizes its binding partners to specific subcellular compartments or to specific substrates, thereby determining the specificity of signal transduction.



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  		FIG. 4.
Determination of the minimum essential region of Nop132 that interacts with RRAG A. a, the carboxyl terminus of Nop132 was subcloned in the pCR2 vector. 35S-Labeled recombinant protein were produced with the TNT-Quick Coupled Reaction Kit, as described in Fig. 3a, and pulled down with GST-RRAG A (lane 1), GST-RRAG C (lane 2), GST-Nip7 (lane 3), and GST (lane 4) proteins, which were bound to the glutathione Sepharose-4B beads. Bound proteins were run on SDS-polyacrylamide gels and analyzed using the Fuji Image Analyzer. Lane 5 corresponds to 2% of labeled Nop132 input. The experiment was performed at least twice, and representative results are shown. b, Y190 strains harboring RRAG A in pAS404-RRAG A and serial deletion fragments of Nop132 in pACT2 were tested for growth on plates containing SD medium lacking tryptophan, leucine, and histidine, but with 3-AT (12.5 mM). The minimum region is shown under the scale. c, colony growth was tested on a plate lacking tryptophan and leucine or on a plate lacking tryptophan, leucine, and histidine but containing 3-AT. Two independent colonies of each clone were picked. Y190 strains harboring RRAG ALG1 and pACT2 did not form colonies on the selection plates.

 
RRAG A has a leucine zipper structure (191-LRNFAQIIEADEVLLFERATFL-212). The leucine zipper structure is important for dimer formation of proteins. A point mutation was introduced to change one of the four residues underlined above into glycine (mutation at the 191st leucine to glycine). The L191G mutant of RRAG A (RRAG ALG1) did not interact with Nop132 (Fig. 4c). RRAG ALG1 did, however, interact with RRAG C and RRAG D. So, the leucine zipper structure of RRAG A is probably not required for heterodimer formation with RRAG C or RRAG D but is required for dimer formation of RRAG A. Thus, RRAG A forms a dimer and is considered to interact with Nop132.

Nop132 Is an Essential Protein for Cell Growth—Nop132 might have a role similar to that of yeast Nop8p, as described above. Because yeast Nop8p is essential for growth, we deduced that Nop132 is also an essential protein for growth. To address this issue, antisense oligonucleotides (A-a) were applied to human HeLa cells to knock down Nop132. Growth suppression was observed when antisense oligonucleotides were applied to HeLa cells (Fig. 5a), whereas sense oligonucleotides (A-s) did not have such an effect. Growth suppression by antisense oligonucleotides was observed in a dose-dependent manner. Furthermore, the siRNA duplex was applied to human HeLa cells. Growth of HeLa cells was also suppressed by oligonucleotides B and C. The Nop132 protein level was knocked down when oligonucleotides B and C were applied to HeLa cells (Fig. 5b, inset). When the siRNA duplex was applied to cells to knock down Nop132 for 1 day, cell number and RNA synthesis were not reduced, but the level of DNA synthesis was greatly reduced to 20-30% (Fig. 5c), implying that Nop132 is important for DNA synthesis. The above results suggest that Nop132 is not involved in ribosome RNA transcription per se but is involved in post-transcriptional events, such as ribosome RNA processing and/or ribosome assembly.



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  		FIG. 5.
Nop132 is essential for cell growth. a, triplicate dishes of HeLa cells (2 x 104 cells/dish) were transfected with antisense oligonucleotides (A-a) and sense oligonucleotides (A-s) as described under "Experimental Procedures." The number of cells was counted 3 days later with a Coulter counter and plotted. The means of triplicate values with standard deviations are shown. b, triplicate dishes of HeLa cells were transfected with double strand interference RNA oligonucleotides B and C. The number of cells was counted 3 days later with a Coulter counter and plotted and is shown as the mean of triplicate values with standard deviations. As a control, the number of cells without oligonucleotides was counted (arbitrary unit: 100) and is shown as minus oligos. Inset, amount of Nop132 and Ran as a control in each cell lysate as detected by Western blotting analyses. c, triplicate dishes of HeLa cells were transfected with double strand interference RNA oligonucleotides B and C as above. One day later, the number of cells was counted. RNA synthesis and DNA synthesis were measured by labeling cells with [3H]uridine (10 µCi/ml) and [3H]thymidine (10 µCi/ml) for 1 h as described previously (34).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Originally, yeast GTR1 was identified as a suppressor of the srm1 mutation of RCC1, which is a RanGTPase guanine nucleotide-exchange factor localized within the nucleus. Human RRAG A is a homologue to yeast GTR1. There are reports of several RCC1-interacting proteins, Ran (25), importin alpha 3 (26, 27), and histones H2A and H2B (6). If there are proteins that link RRAG A to RCC1 biochemically, previous genetic data will be confirmed. Accordingly, we attempted to identify RRAG A-interacting proteins. In the present study, a novel RRAG A-interacting protein, Nop132, was determined to be a nucleolar protein. Previously, we reported that RRAG A is a Ras-like small G protein that is localized within both the cytoplasm and the nucleus and might be involved in transport across the nuclear membrane. Two RRAG A-interacting proteins, adenovirus E3 14.7K and dynein light chain, are localized within the cytoplasm (15, 17). These proteins are probably cytoplasmic targets of RRAG A inside the cytoplasm. We were previously unable to determine why RRAG A is localized to the nucleus. In the present study, however, Nop132, a nucleolar protein, provided us with an explanation as to why this happens. It appears that RRAG A is a multifunctional G protein that functions in both the cytoplasm and nucleus.

Previously, we reported that RRAG A is associated with RRAG C and RRAG D, suggesting that RRAG A forms a heterodimer with RRAG C and/or RRAG D. G protein-heterodimer formation was reported for tubulin, which has two subunits, {alpha} and {beta}, both of which are G proteins. The subunits {alpha} and {beta} have separate functions: {alpha}-tubulin is important for heterodimer formation, whereas {beta}-tubulin is involved in microtubule assembly, although they always attach to each other (reviewed in (28)). By analogy with tubulin, RRAG A might have a different role in the cellular process than does RRAG C. Nop132 colocalizes with RRAG A more efficiently than with RRAG C (Fig. 2, c-e). We observed that GAL4 DNA-binding (DB)-RRAG C fusion protein contains significant GAL4 transactivation properties in a yeast two-hybrid system, whereas GAL4 DB-RRAG A, a fusion protein, does not contain GAL4 transactivation property.2

Sequence comparison between Nop132 and Nop8p revealed that they have ~30% homology in the amino-terminal region where they have RNA-binding motifs. Although the carboxyl-terminal regions of both proteins have only a 16% similarity, they have coiled-coil structure motifs, which are probably involved in RRAG A, RRAG C, and Nip7 interactions. Both human Nop132 and yeast Nop8p have similar characteristics for associating proteins Nip7/Nip7p, RRAG A/Gtr1p, and RRAG C, RRAG D/Gtr2p (Fig. 6). Moreover, both proteins are essential for cell growth and are localized within the nucleolus, implying that Nop132 is functionally similar to Nop8p. S. cerevisiae Nip7p also interacts with Rrp43p (exosome subunit) and is involved in rRNA and ribosome maturation (19). Because the carboxyl terminus of Nop132 (958-1168 aa) is evenly localized within the nucleus, other portions of Nop132 are required for its localization to the nucleolus. Expression of EGFP-Nop132 deletion proteins in BHK21 cells demonstrates which region is required for the nucleolus localization of Nop132. Anchor proteins localize their binding partners to specific subcellular compartments or to specific substrates (29). Nop132 might be an anchor protein that tethers the G proteins (RRAG A and RRAG C), Nip7, and the exosome to the nucleolus, where ribosome RNA is transcribed and processed (Fig. 6).



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  		FIG. 6.
Scheme of Nop132 interactions summarizing present and previous results and incorporating data from studies in yeast. Yeast gtr1-11 mutation and disruption of GTR2 suppress the prp20-1 mutation, suggesting that GTR1 and GTR2 negatively regulate prp20 function. Because we did not observe a direct interaction of Gtr1p and Gtr2p with Prp20p and Gsp1p, the suppression could have been indirect. Because Gsp1p interacts with Dis3p, a component of the exosome and ribosome, for export, Prp20p and Gsp1p are required for ribosome biogenesis. prp20-1 is a temperature-sensitive mutation that results in a defect of nuclear export of mRNA, proteins, and ribosomes. Thus, gtr1-11 or disruption of GTR2 releases the negative regulation for nuclear export. RRAG A, RRAG C, and Nip7 interact with the carboxyl terminus of Nop132. "?" indicates that these pathways are based on speculation.

 
Most G proteins influence a particular signal transduction pathway through the activation or inactivation of target proteins by binding and leading to conformational changes of those proteins. How RRAG A and/or RRAG C regulate the function of Nop132 is unknown. A yeast two-hybrid assay and mutants of RRAG A (GTP and GDP forms) were used to examine the nucleotide specificity of RRAG A binding to Nop132. Because Nop132 interacts with the GTP form of RRAG A, Nop132 could be a possible target effector protein of RRAG A. Nop132 might be involved in some signaling pathway for growth, most likely ribosome biogenesis, and might change its conformation by interacting with GTP-bound RRAG A and/or RRAG C. Because RRAG A is a functional homologue to yeast Gtr1p, the knowledge obtained using GTR1 mutant cells will provide some hints about the function of RRAG A. Pull-down experiments with GST-Gtr1p (Fig. 3a) and the yeast two-hybrid system revealed that Gtr1p interacts with Nop8p, suggesting that Gtr1p is involved in ribosome RNA processing. Because RNA synthesis was not reduced in siRNA-applied HeLa cells, Nop132 does not appear to be involved in ribosome RNA transcription per se (Fig. 5c) but in post-transcriptional events similar to those in which Nop8p is involved.

The nucleolus is critically involved in several cellular processes, including cell-cycle progression, gene silencing, and ribosome biogenesis (reviewed in Refs. 30 and 31). Nop132 might have a role in these nucleolar events.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB109030 [GenBank] .

* This work was supported by Grants-in-aid for Specially Promoted Research (to T. N.) and by a Grant-in-aid for Scientific Research on Priority Areas (to T. S.), from the Japan Ministry of Education, Science, Sport and Culture. 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

§ Present address: National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo 062-8517, Japan. Back

{ddagger} To whom correspondence should be addressed. Tel.: 81-92-642-6177; Fax: 81-92-642-6183; E-mail: sekigu{at}molbiol.med.kyushu-u.ac.jp.

1 The abbreviations used are: siRNA, small interference RNA; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; aa, amino acid(s); HA, hemagglutinin; DB, DNA binding; EGFP, enhance green fluorescent protein; 3-AT, 3-aminotriazole. Back

2 T. Sekiguchi, Y. Todaka, Y. Wang, E. Hirose, N. Nakashima, and T. Nishimoto, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. H. Kobayashi for a critical reading of the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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