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Originally published In Press as doi:10.1074/jbc.M304198200 on June 4, 2003

J. Biol. Chem., Vol. 278, Issue 34, 32204-32211, August 22, 2003
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The NOG1 GTP-binding Protein Is Required for Biogenesis of the 60 S Ribosomal Subunit*

Bryan C. Jensen {ddagger} § , Qin Wang {ddagger} §, Charles T. Kifer {ddagger} and Marilyn Parsons {ddagger} § ||

From the {ddagger}Seattle Biomedical Research Institute, Seattle, Washington 98109 and §Department of Pathobiology, School of Community Medicine and Public Health, University of Washington, Seattle, Washington 98195

Received for publication, April 22, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
NOG1 is a nucleolar GTP-binding protein present in eukaryotes ranging from trypanosomes to humans. In this report we demonstrate that NOG1 is functionally linked to ribosome biogenesis. In sucrose density gradients Trypanosoma brucei NOG1 co-sediments with 60 S ribosomal subunits but not with monosomes. 60 S precursor RNAs are co-precipitated with NOG1. Together with the nucleolar localization of NOG1, these data indicate that NOG1 is associated with a precursor particle to the 60 S subunit. Disruption of NOG1 function through RNA interference led to a dramatic decrease in the levels of free 60 S particles and the appearance of an atypical rRNA intermediate in which ITS2 was not cleaved. Overexpression of mutant nog1 with a defect in its GTP binding motif on a wild type background caused a modest defect in 60 S biogenesis and a relative decrease in processing of the large subunit rRNAs. In contrast to the mutant protein, neither the N-terminal half of NOG1, which contains the GTP binding motifs, nor the C-terminal half of NOG1 associated with pre-ribosomal particles, although both localized to the nucleolus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Generation of a functional ribosome requires the transient association and function of non-ribosomal proteins in pre-ribosomal complexes located primarily in the nucleolus. The pathway of ribosome biogenesis appears to be generally conserved throughout eukaryotes but has been most well studied in yeast where a number of such complexes have been identified (1–3). The first is a nucleolar 90 S particle containing the 35 S rRNA, the precursor of the 18 S, 5.8 S, and 23 S rRNAs (4), plus many of the proteins required for biogenesis of both ribosomal subunits (5). After removal of the 5' external transcribed spacer, the first internal transcribed spacer (ITS1)1 is cleaved to release the precursor of the 18 S rRNA (6, 7). Separate particles then go on to form the 60 S and 40 S subunits (4). The 66 S precursor to the 60 S subunit is actually a series of particles that differ in the status of their rRNA processing and associated proteins. However, within the 66 S particle, cleavage of ITS2 releases the 5.8 S rRNA, and a defined series of rRNA cleavages results in the mature rRNAs. The 66 S precursor is further processed, then moves to the nucleoplasm and is exported to the cytoplasm as an immature 60 S subunit (8). As in Saccharomyces cerevisiae, cleavage of ITS1 and ITS2 are early events in Trypanosoma brucei (9). However, processing of large subunit sequences is considerably more complex, with mature transcript sizes of 1840 (LSU1), 1570 (LSU2), 220, 180, 140, and 70 nucleotides (9).

In addition to ribosome biogenesis, the nucleolus is the site of other processes such as tRNA processing or gene silencing (10–12). Although most of the nucleolar proteins studied thus far participate in ribosome biogenesis, the functions of many nucleolar proteins remain undefined (13). One such protein, NOG1, was originally identified in the protozoan T. brucei via a two-hybrid screen using the nucleolar phosphoprotein NOPP44/46 (14). NOG1 is a GTP-binding protein that is conserved among eukaryotes. In those species studied thus far (T. brucei, S. cerevisiae, and Homo sapiens), NOG1 is nucleolar (14). Data base searches with the G1 and putative effector motifs of the NOG1 GTP binding domain identified a larger family of related GTP-binding proteins called the ODN family, which also includes the DRG (15) and Obg proteins (16–18). DRGs and Obgs as well as prokaryotic NOG-like proteins are homologous throughout their length to the amino half of NOG1, which contains the GTP binding motifs. Phylogenetic analysis of ODN proteins shows that they are not closely related to other GTP-binding protein families.

The function of Obg proteins has been studied to some degree. Intriguingly, Bacillus subtilis Obg, which is required for the stress activation of transcription factor {sigma}B (19), was found to associate with ribosomal particles, possibly via rpl13 (20). Given these data and the fact that NOG1 is nucleolar, we postulated that NOG1 is involved in ribosomal biogenesis. During the course of these studies, other workers show that NOG1 is co-precipitated with tagged proteins known to be involved in 60 S biogenesis in yeast (21–23). In the studies presented, we show that RNA interference (RNAi)-mediated disruption of NOG1 function causes defects in 60 S biogenesis and that the GTP binding motif is required for function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Media—Initial studies were done using the procyclic form T. brucei TREU667, grown in Cunningham's media (24) containing 10% fetal calf serum. For in vivo expression of NOG1 constructs, we employed procyclic form 29-13 strain (25), which expresses T7 RNA polymerase and the Tet repressor to allow regulated expression of the epitope-tagged genes or double-stranded RNA. The 29-13 cells were cultured in SDM-79 (26) with 15% fetal calf serum containing 15 µg/ml G418 and 50 µg/ml hygromycin to maintain the T7 RNA polymerase and Tet repressor constructs. After transfection, stable transfectants were selected using 2.5 µg/ml phleomycin as described (25). Expression was induced with 2–2.5 µg/ml Tet.

NOG1 Constructs—The plasmid pLEW79-NOG1-Myc was made by amplifying the cloned NOG1 gene with the sense primer 5'-CGTTTCTAAGCTTATGTCCACAATCTACAAT and antisense primer 5'-TACTTACTCGAGTTATCTAGAGCGTCTGTCGCGTC, which contain the restriction sites HindIII and XbaI in their 5' ends. The PCR product was cloned into pBS-KS+. As with other coding regions, the NOG1 gene in the plasmid was sequenced to confirm the integrity of the open reading frame. To generate GTP-binding-deficient nog1-g1, we performed site-directed mutagenesis using primers 5'-GGGTTCCCCAACGTCGCTGCGGCAAGTTTCATGAACAA and 5'-CTTGTTCATGAAACTTGCCGCAGCGACGTTGGGGAACCC, which specify a GKS to AAA mutation in the G1 motif (amino acids 181–183) using the QuikChange site-directed mutagenesis kit from Stratagene. The wild type and mutant NOG1 genes were excised using HindIII and XbaI and used to replace the CK2a1 gene in pLew-CK2{alpha}-Myc (27), a derivative of Tet-regulated expression plasmid pLew79 (25). To generate tandem affinity purification (TAP)-tagged proteins (28), we used a pLew79 derivative modified by the addition of a sequence encoding the TAP tag (pLewTAP, a gift of Kenneth Stuart and Achim Schnaufer). Full-length NOG1 (sense primer above and antisense primer 5'-CCGGATCCGCGTCTGTCGCGTCTGCCGTTAG) and codons 335–667 (sense primer 5'-CGTTTCTAAGCTTATGTCCACAATCTACAAT and antisense primer as for full-length) were amplified and cloned into pGEM-T Easy vector (Promega). The fragments were excised with HindIII and BamHI and subcloned into the same sites in pLewTAP.

A NOG1 gene fragment (nucleotides 4–488 relative to the start codon) was amplified by PCR with primers containing HindIII and XhoI restriction sites (sense primer, 5'-GCACTAAGCTTTCCACAATCTACAATTTCAAAACG and antisense primer 5'-GCTTACTCGAGGCAGTCGAGACATGTGTTGC). The PCR products were cloned into pGEM-T Easy and subcloned into the HindIII and XhoI sites of pZJM, an RNAi vector that contains convergent Tet-regulated promoters (a gift of Dr. Paul Englund) (29).

Polysome Analysis and Particle Purification—Polysome analysis was performed as described (30) with the following modifications. NaCl was omitted from the buffer (10 mM Tris, pH 7.4, 300 mM KCl, 10 mM MgCl2) since it disrupted the association of NOG1 with the particle (similar to the NaCl-sensitive association of Obg with the ribosome (20)). Sucrose gradients were adjusted to 10–40%. Protease inhibitors were included (1 mM phenylmethylsulfonyl fluoride, 8.5 µg/ml aprotinin, 50 µg/ml leupeptin, 1 µM pepstatin, 50 µg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone, and 10 µM E-64). No GTP or GDP was present. Approximately 4 x 108 cells were separated on each 12-ml gradient by centrifugation in a Beckman SW40 rotor at 230,000 x g for 2 h unless otherwise stated. Fractions were collected using an ISCO model 185 density gradient fractionator, with the polysome profile monitored at A254. Aliquots of each fraction (20 µl, except where noted) were loaded onto SDS-PAGE gels for immunoblot analysis.

Procyclic form lysates expressing NOG1-TAP (2 x 108 cells) were prepared using the same buffers as for polysome analysis (except that RNasin was increased 3-fold) and added to a preincubated complex of rabbit anti-horseradish peroxidase (Sigma) and sheep anti-rabbit IgG conjugated to magnetic beads (Dynal). After 1 h at 4 °C, the complexes were washed 3 times, and the associated RNA was isolated with TRIzol reagent (Invitrogen).

Northern Analysis—Procyclic form RNA, prepared with TRIzol reagent, was separated on formaldehyde-agarose gels, blotted, and hybridized with various RNA probes for ITS2 (a riboprobe), ITS3 (5'-ACGACAATCACTCACACACACATGGC), ITS5 (5'-TCATAAATGTTGTGTGCTGAGATGGC), and ITS7 (5'-TATGTAGTACCACACAGTGTGACGCG) as well as {alpha}-tubulin (a riboprobe) derived from the insert of plasmid pZJM. The probe spanning the junction between 5.8 S and ITS2 is 5'-TTGTTTTTATATTCGACACTGAGAA, where the underlined portion corresponds to ITS2 sequences.

Immunoblot and Immunofluorescence Analysis—Proteins were transferred from SDS-PAGE 10% acrylamide gels onto nitrocellulose. The blots were incubated with antibodies or antisera followed by horseradish peroxidase coupled to goat anti-rabbit Ig, goat anti-mouse Ig, or protein A and developed with the ECL or ECL Plus systems (Amersham Biosciences). Anti-TbNOG1 629L (14) was used at 1:500 to 1:5000, anti-Nopp44/46 (31) was used at 100 ng/ml, anti-T. brucei phosphoglycerate kinase (32) was used at 1:2000, anti-Myc 9E10 (Covance) (33) was used at 1 µg/ml (ECL) or 0.2 µg/ml (ECL Plus), and anti-Trypanosoma cruzi P0 (34) was used at 1:2500. For detection of TAP-tagged protein, blots were incubated with a mixture of rabbit anti-horseradish peroxidase plus horseradish peroxidase coupled to goat anti-rabbit IgG. For quantitative immunoblots, 125I-labeled protein A was used as the detection agent, and radioactivity was quantitated by phosphorimaging. Immunofluorescence was performed as described (35), except that the blocking solution was 10% nonfat milk plus 3% bovine serum albumin in phosphate-buffered saline. Anti-NOG1, anti-Nopp44/46, and anti-Myc were used at 1:500, 1 µg/ml, and 5 µg/ml respectively. The TAP tag was detected using rabbit anti-horseradish peroxidase (1 µg/ml) followed by fluorescein isothiocyanate-goat anti-rabbit IgG.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
NOG1 Co-sediments with the 60 S Ribosomal Subunit—Because the related B. subtilis Obg protein is associated with ribosomes, we tested whether NOG1 co-sediments with ribosomal particles in T. brucei. Cell lysates were prepared and fractionated over sucrose gradients. A substantial fraction of NOG1 was degraded in the cell lysate (Fig. 1B, lane T) despite the presence of a broad spectrum of protease inhibitors. The smaller immunoreactive species were not seen when cells were immediately boiled in SDS sample buffer (14). Several other proteins did not show this high susceptibility to degradation (Fig. 1B).



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  		FIG. 1.
NOG1 cosediments with the 60 S ribosomal subunit. A, polysome profile of T. brucei. Lysates from procyclic form TREU667 cells were centrifuged on 10–40% sucrose gradients for 4 h. Shown is the A254 trace from the gradient with the top of the gradient on the left. The 40 S, 60 S, and 80 S peaks are labeled. B, immunoblot analysis. Membranes were probed with antisera directed against T. brucei NOG1, the non-ribosomal protein PGK, and the ribosomal protein P0 as indicated. The migration of molecular mass markers is indicated. For NOG1 blots, the closed arrowhead marks full-length TbNOG1, and the open arrowhead marks a 55-kDa NOG1 fragment associated with the ribosomal subunit. For PGK blots, the upper species is the glycosomal isoform, and the lower is the cytosolic isoform. T, total lysate. Fraction numbers are indicated.

 

Shown in Fig. 1A is a polysome profile from a gradient in which the centrifugation time was increased over previous studies (30) to better separate the 60 S and 80 S particles. Under these conditions the polysomes, which represent mRNA molecules being translated by increasing numbers of ribosomes, are not well resolved. Fractions from the gradient were analyzed by immunoblotting to determine where the NOG1 proteins localized within the gradient (Fig. 1B). Two peaks of NOG1 (Fig. 1B) were seen in the gradient fractions. The first peak, which contained the bulk of the degraded protein and little intact NOG1, was at the top of the gradient with the soluble proteins (Fig. 1B, fractions 1–7). The second peak, which contained the full-length protein (closed arrowhead) and a specific degradation product (open arrowhead), was further down the gradient in the region of the 60 S subunit (Fig. 1B, fractions 15–18). No significant amount of NOG1 was found in the fractions containing polysomes, indicating that NOG1 is not associated with translating ribosomes (Fig. 1B, fractions 23–24, fractions 25–31 are not shown).

We monitored the presence of several other proteins in the gradients (Fig. 1B). The 60 S-associated protein P0 was present in the 60 S to 80 S region as well as in the polysomes, as expected for a protein functioning in the GTPase center of the ribosome (36). In contrast, both cytosolic and organellar forms of the monomeric protein phosphoglycerate kinase were localized predominantly at the top of the gradient. NOPP44/46, which can physically associate with NOG1 (14), sedimented as a soluble protein under the conditions employed (not shown).

NOG1 Association with Ribosomal RNA Precursors—To verify that the NOG1-containing particle was indeed the 60 S ribosomal precursor, we tested whether it contained rRNA precursors. The ribosomal DNA locus encodes the small subunit rRNA, 5.8S rRNA, and 6 RNAs that together correspond to the large subunit rRNA. Because neither the intermediates nor the full sequence of processing events have been defined in the parasite, we probed Northern blots with sequences corresponding to ITS2, ITS3, ITS5, and ITS7 (Fig. 2B), identifying RNA processing intermediates shown in Fig. 2A. We detected the previously described precursors of 10 (the primary transcript), 5.9, and 0.61 kb (a 5.8 S precursor) (37). Also seen was the 5.1-kb intermediate containing ITS3, ITS5, and ITS7 (but not ITS2). In addition, we identified two precursors that hybridized only to ITS3 (3.9 and 0.56 kb) and two that hybridized only to ITS7 (0.52 and 0.36 kb). No precursors that hybridized solely to ITS5 were detected using probes to two different regions.



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  		FIG. 2.
T. brucei rRNA processing intermediates. A, schematic of early events in rRNA processing in T. brucei (47). LSU1, LSU2, SR1, SR2, SR4, SR6, and 5.8 S are all components of the 60 S ribosome. In addition to those species previously described, transcripts marked by an asterisk were identified from the current study. Boxes delimit the regions of the mature rRNAs. Transcripts detected by the ITS7 probe have not been characterized to determine whether they are precursors of SR 4 or SR6. The intermediates are lettered in correspondence with Fig. 2B, and sizes are indicated. The transcript shown in brackets is seen in cells depleted for NOG1 (see Fig. 4). B, Northern analysis of rRNA precursors in total RNA. RNA was isolated from TREU667 procyclic forms separated on a 1% formaldehyde agarose gel and transferred to nylon membranes. Blots were hybridized with probes against the intervening sequences of ITS2, ITS3, ITS5, and ITS7. The migration of markers is indicated (kb). nt, nucleotides.

 



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  		FIG. 4.
NOG1 RNAi causes a defect in 60 S biogenesis. Procyclic forms (29-13) were stably transfected with a construct that allows the Tet-induced expression of double-stranded RNA corresponding to NOG1, causing partial depletion of NOG1. A, growth curve. The cell numbers were measured by particle counting, and cumulative cell density was calculated adjusting for dilutions required to maintain cultures. B, immunoblot analysis. Day 8 lysates were analyzed by immunoblotting using anti-NOG1 followed by 125I-labeled protein A. C, polysome analysis. Day 8 lysates (same cells as shown in panel B) were fractionated on analytical sucrose gradients. The inset is a magnification of the region of the gradient containing mRNAs translated by either two or three ribosomes. Arrowheads point to half-mer peaks seen in NOG1-depleted cells. D, Northern analysis. RNA was prepared on day 8 after the addition of Tet to the NOG1 RNAi line or its parent 29-13. The blots were probed with ITS2, ITS3, and a probe spanning the junction of 5.8 S rRNA and ITS2. A novel cleavage product appears at 2.6 kb.

 
Initial immunoprecipitates were performed using anti-NOG antibodies. These antibodies react with the C terminus of NOG1 and were able to efficiently precipitate TbNOG1 when expressed in S. cerevisiae.2 However, only a small percentage of the protein could be precipitated from T. brucei lysates even at higher antibody concentrations. This suggests that the NOG1 epitopes are obscured, potentially by being present in a particle, when expressed in its native organism. Therefore, we turned to a cell line expressing NOG1 with a TAP tag at the C terminus in a Tet-inducible system. The TAP tag contains a calmodulin binding domain followed two Ig binding domains of protein A (28). In cell lysates, the protein A domain was exposed to affinity reagents, whereas the calmodulin domain was not.2 We collected NOG1-TAP using a combination of rabbit antibodies (to bind protein A) and sheep anti-rabbit Ig coupled to magnetic beads. Similar purifications were done on the parental line 29-13 (Fig. 3) and uninduced cells (not shown). RNA was isolated and assayed for the presence of rRNA precursors and tubulin. Ethidium bromide staining showed that mature rRNAs were not present, but some higher molecular weight species were detected in the NOG1-TAP samples (not shown). The first cleavage event occurs in ITS2, and probing with this sequence showed that the 5.9-kb precursor as well as the 0.61-kb product was present in the NOG1-TAP complexes (Fig. 3). Probing with ITS3 (Fig. 3) also showed the 5.9- and 5.1-kb precursors, but the 3.9-kb transcript was not highly enriched. We could not assess whether the 0.56-kb ITS3-containing precursor was significantly enriched due to the weak signal in total RNA plus trailing of degraded RNA into the small RNA region. Probing with ITS7 confirmed the presence the two larger transcripts, but the presence of the smaller transcripts was equivocal.



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  		FIG. 3.
Northern analysis of RNA co-precipitated with NOG1. RNA was isolated from affinity purification with NOG1-TAP or untransfected 29-13 cells. As a control, 1 µg of total RNA was loaded (Tot); also shown is a 10-fold darker exposure of that lanes (10X Tot). After separation on a 1.5% agarose gel, the samples were hybridized with probes to ITS2, ITS3, and ITS7 to detect 60 S precursors as well as a probe to {alpha}-tubulin to test specificity. The band labeled {alpha}T in the ITS7 probing is a residual signal left from probing for {alpha}-tubulin. The sizes of the bands seen are indicated.

 

Quantitation by phosphorimaging showed that ~10-fold more of the 5.9 and 5.1 precursors were present in the NOG1-TAP precipitates than in 1 µg of total RNA. In contrast, in TAP purifications from the non-expressing parental line 29-13, the signal associated with these transcripts was 9-fold less than that seen in 1 µg of total RNA. As a specificity control, we assessed the abundance of a non-ribosomal RNA, {alpha}-tubulin, in the precipitates by Northern analysis and phosphorimaging. Whether isolated from control or NOG1-TAP purifications, the amount of {alpha} tubulin RNA was only a few percent that seen in 1 µg of RNA (NOG1-TAP averaged 2.5%, 29-13 averaged 0.2%). Thus, there is a cumulative 360-fold enrichment of ITS3-containing precursors in the NOG1-TAP purification compared with tubulin. The specific enrichment of 60 S ITS sequences in NOG1-TAP purification demonstrates that the NOG1-containing particles are precursors to the 60 S subunit.

Reduction in NOG1 Results in a Defect in Ribosome Biogenesis—To assess the functional association of NOG1 and ribosome biogenesis, we used double-stranded RNAi to reduce the level of NOG1. A fragment of NOG1 corresponding to the 5' end of the coding region was inserted into the RNAi vector pZJM, which allows expression of the double-stranded RNA to be regulated by Tet. When RNAi was induced by the addition of Tet, the growth of the cells slowed and eventually stopped (Fig. 4A). As with most other RNAi experiments in T. brucei where the construct confers a deleterious effect, a resistant population grew out somewhat later. At day 8 after induction, the levels of NOG1, estimated by quantitative immunoblot analysis and phosphorimaging, were reduced to 7% of the uninduced level (Fig. 4B). Data were normalized to the levels of the control protein, cytosolic phosphoglycerate kinase.

Sucrose density gradient fractionation of day-8 cells revealed a dramatic decrease in the abundance of 60 S subunits as well as the presence of half-mers (shoulders on the polysome peaks) (Fig. 4C). Half-mers are seen for S. cerevisiae mutants defective in 60 S biogenesis and result from a bottleneck in assembly of the 60 S subunit onto the 40 S-mRNA complex (see for example Refs. 38 and 39). Tet induction had no effect on the polysome profile of the parental line 29-13. These data demonstrate that NOG1 is required for 60 S biogenesis.

To assess whether knockdown of NOG1 was associated with specific defects in rRNA processing, RNA was isolated from cells on day 8 after induction of RNAi. Blots were hybridized with a probe to ITS2, which showed the accumulation of an atypical RNA at 2.6 kb. This species did not hybridize to ITS3 or to ITS1 probes, but it did hybridize to a probe spanning the junction of the 5.8 S rRNA and ITS2. This mapping indicates that the atypical intermediate likely spans 5.8 S through LSU1, with a cleavage in ITS3 just after LSU1 (see Fig. 2A). Although this molecule does not accumulate to a significant degree in wild type cells, a low level of hybridization in that region suggests that it may be an alternative processing intermediate.

Both Domains of NOG1, but Not GTP Binding, Are Required for Stable Association with the 60 S Precursor—The N-terminal half of NOG1 contains the GTP binding motifs and is conserved from Archaea to humans. The C-terminal half is highly charged but divergent in sequence among eukaryotes and absent in Archaea. To assess which portion of the molecule was required for interaction with the 60 S precursor and the role of GTP binding in the interaction, we expressed several tagged constructs in T. brucei using a Tet-inducible vector (Fig. 5A). The carboxyl half of NOG1 as well as wild-type NOG1 was expressed with a C-terminal TAP tag. Wild type NOG1, residues 1–334 of NOG1 (14), and the full-length protein mutated in the G1 motif of the GTP binding domain (GKS -> AAA, nog1-g1) were expressed with a C-terminal Myc tag. After induction with Tet, all proteins were expressed (Fig. 5B). Data for NOG1-Myc and NOG1-TAP are not shown because they behaved like wild type with respect to subcellular localization, polysome profile, and association with a 60 S particle. Previously, nog1 (1,334)-Myc was shown to localize to the nucleolus (14). Immunofluorescence analysis showed that both nog1 (335,667)-TAP and nog1-g1-Myc were nucleolar (Fig. 5C).



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  		FIG. 5.
Expression and localization of nog1 mutant proteins in T. brucei. A, schematic of constructs. The region homologous to GTP binding domains is shaded green, and red marks the site of the g1 mutation. B, immunoblot analysis. Stable transfectants containing Tet-regulated genes encoding nog1-g1-Myc, nog1 (1,334)-Myc, or nog1 (335,667)-TAP were generated, and expression of the tagged proteins was induced with Tet. The anti-NOG1 reacts almost exclusively with the C-terminal half of the molecule; therefore, anti-Myc was used to detect nog1 (1,334)-Myc. Its relative abundance is similar to that of the nog1-g1-Myc mutant protein, which in turn is expressed at somewhat higher than wild type levels as shown by immunodetection with anti-NOG1. The abundance of nog1 (335, 667)-TAP cannot be accurately assessed because the molecule binds both the Fab and Fc portions of anti-NOG1 (Fc binding is mediated by the TAP tag). C, subcellular localization. The indicated transfectants were stained with reagents to detect the tag plus fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. The nucleolus was stained with anti-NOPP44/46 plus Texas Red-conjugated goat anti-mouse IgG2a. DNA was visualized with 4,6-diamidino-2-phenylindole (DAPI). The merged image of the staining patterns is shown in the overlay with DAPI colored blue. The white bar represents 5 µM. Virtually all cells in the nog1-g1-Myc line stained with the antibody to the tag, whereas 76% of those in the nog1 (335,667)-TAP line were positive. D, association with 60 S particles. Lysates from cells expressing nog1 (1,334)-Myc, nog1 (335,667)-TAP, or nog1-g1-Myc were separated on analytical polysome gradients. The total cell lysate (T), plus fractions 1 through 15 (which contain the 40 S, 60 S, 80 S, and 2-mer peaks) were analyzed by immunoblotting using reagents to specifically detect the tagged proteins. The same samples were analyzed using anti-NOG1, verifying proper sedimentation of the wild type molecule. The anti-NOG1 panel shown is from the nog1-g1-Myc expressor, with a high molecular mass doublet corresponding to the wild type and tagged mutant. The location of the 60 S peak is marked.

 

Lysates from both induced and uninduced cells were separated on sucrose gradients, and fractions were analyzed by immunoblotting to determine whether the tagged proteins were associated with the precursor particle (Fig. 5D). In the gradients from cells expressing nog1-g1-Myc, probing with either anti-Myc or anti-NOG1 revealed the same two peaks, one with the soluble proteins and one in the 60 S region. Thus, NOG1 does not require GTP binding for its ability to interact with the ribosome. In contrast, nog1 (1,334)-Myc and nog1 (335,667)-TAP localized to the top of the gradient. However, the entire molecule is not required for the interaction. For example, in gradients such as those shown in Fig. 1, the 55-kDa degradation fragment of NOG1 (open arrowhead) cosedimented with the 60 S subunit.

No alteration in the polysome profile of cells expressing nog1 (1,334) was detected (not shown). The polysome profile of the nog1 (335,667) line did not show half-mers but did have a modest and reproducible decrease in the 60 S peak. Unfortunately, immunofluorescence revealed that 24% of the cells did not express significant levels of the mutant protein, making the profile difficult to interpret. In contrast, overexpression of nog1-g1-Myc (Tet+) led to a modest decrease in 60 S subunits (Fig. 6A). In addition the 60 S peak was broader with a flat top, suggesting the presence of two closely spaced peaks. Half-mers were visible on the 2-mer and 3-mer peaks. This dominant effect, observed in multiple gradients from two different inductions, suggests that the nog1-g1 mutant functions poorly if at all in ribosome biogenesis.



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  		FIG. 6.
Defect in 60 S biogenesis associated with a mutation in NOG1. A, polysome profile. Samples from nog1-g1-Myc transfectants were analyzed on sucrose gradients. Expression of the mutant protein was induced with Tet overnight (+Tet). A parallel analysis of the uninduced culture is shown (–Tet). The inset shows the 2-mer and 3-mer peaks, with half-mers present in the induced culture. B, rRNA processing. The wild type parental line 29-13 and nog1-g1-Myc transfectants were incubated overnight in the presence or absence of Tet. The isolated RNA was analyzed by Northern analysis using ITS2 and ITS7 as probes.

 

A Mutant NOG1 Affects Relative Abundance of rRNA Precursors—To assess the effect of dysfunctional NOG1 on early steps of 60 S rRNA maturation, we examined RNA isolated from trypanosomes expressing nog1-g1 (Fig. 6B). Unlike the NOG1 RNAi cells, these cells proliferate at the time of analysis. The left blot shows the ITS2 hybridization, which detects the 5.9-kb transcript containing all of the cotranscribed large subunit rRNAs and the 0.61-kb precursor of the 5.8 S RNA that results from cleavage in ITS2 (see Fig. 2A). In the parental line 29-13, after induction with Tet, there was little change in the ratios of the two transcripts detected with the ITS2 probe (0.8–1.1-fold) or other ITS probes. However, in two separate experiments, when Tet was used to induce nog1-g1, the relative abundance of the 0.61-kb cleavage product decreased. In the experiment shown in Fig. 6B, phosphorimaging indicated that the ratio of hybridization of the 0.61-kb transcript to the 5.9-kb transcript decreased more than 5-fold upon expression of the nog1-g1 protein. In a second experiment (not shown) it decreased ~4-fold. Thus, the precursor accumulated relative to the product. Probing with sequences corresponding to ITS7 (Fig. 6B, right) and IT3 (not shown) confirmed these findings. Upon expression of the mutant protein, the ratio of the ITS7 containing 0.52- and 0.36-kb transcripts to the 5.9-kb transcript both decreased 3-fold. This decrease was traced to a 1.6-fold decrease in the abundance of the 5.1-kb transcript relative to its 5.9-kb precursor and a 1.6-fold decrease in the small transcripts relative to the 5.1-kb precursor. The ratio of the two smaller transcripts to each other was the same under all conditions. The ITS3 probing showed a 5-fold decrease in the ratio of the smallest ITS3-containing transcript relative to the 5.9-kb precursor. Each step of the ITS3 pathway appeared to lag in the nog-g1-expressing cells relative to the uninduced cells and relative to the control cell line. Taken together, these data show that overexpression of nog-g1 led to a decline in the abundance of downstream rRNA-processing intermediates compared with the 5.9-kb precursor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There has been little data concerning the function of NOG1 other than its initial description as essential and nucleolar. However, it is a member of the ODN family of GTP-binding proteins that also includes the bacterial Obg and DRG proteins. Although little is known about DRGs, some functional data exist for the Obgs. Obg was originally identified in a B. subtilis operon containing genes involved in sporulation (16). Additional Obg homologs were identified in other prokaryotes, a number of which are also involved in morphological differentiation (17, 40, 41). In B. subtilis Obg is associated with the 50 S ribosomal subunit, the prokaryotic equivalent of the 60 S subunit (20), but studies on the related Escherichia coli molecule suggest that this property is not conserved (42). We therefore investigated whether NOG1 was associated with the 60 S ribosomal subunit.

T. brucei NOG1 co-sediments with the 60 S ribosomal particle on sucrose gradients. Co-immunoprecipitation of rRNA precursors demonstrated that the NOG1-containing particle is in fact a 60 S precursor. The NOG1 particles contain at least three rRNA precursors, the first identified precursor to the large subunit RNAs at 5.9 kb plus the two products of its cleavage in ITS2. The ITS2 cleavage is conserved among eukaryotes as is the NOG1 interaction with 60 S precursors. The T. brucei NOG1 antiserum identifies a unique, appropriately sized molecule in the distantly related parasite Leishmania, which sediments in the 60 S region.3 Recently, S. cerevisiae NOG1 was shown to be present in the same particle(s) as some other non-ribosomal proteins associated with the 60 S-pre-ribosome (1, 21–23).

Our data extend these observations by showing that strains with decreased NOG1 expression had a significant defect in 60 S biogenesis and accumulation of an atypical rRNA species that results from cleavage in ITS3 without cleavage in ITS2. Cells overexpressing a mutated nog1 protein defective in GTP binding showed an altered polysome profile and a relative decrease in the abundance of the precursors rRNAs. Products resulting from cleavage of ITS2 were decreased, as are smaller RNAs detected by probes for ITS3 or ITS7. Although NOG1 is clearly associated with particles that contain the first dedicated large subunit precursor and remains associated after cleavage in ITS2, we could not determine whether the subsequent cleavages occur in a NOG1-containing particle. If not, then the effects of mutant NOG1 on the later trypanosome-specific processing events could be indirect.

Nucleolar Localization—One surprising finding was that a T. brucei NOG1 fragment that corresponds to archaeal NOG1-like proteins was not capable of stable interaction with the ribosomal subunit. Although the archaeal proteins are presumably capable of interacting with the 60 S subunit, in eukaryotic NOG1s apparently elements in the C-terminal extension participate in the interaction. Both amino (14) and carboxyl halves of NOG1 localize to the nucleolus despite their lack of association with the ribosome. Accumulating data strongly suggest that localization to the nucleolus does not depend on a straightforward targeting motif but rather through interactions with other nucleolar components (13, 43). This leaves us with the question, What non-ribosomal components do these fragments interact with that allow localization to the nucleolus?

Role of GTP Binding—A major unanswered question is the role of GTP in the function of NOG1. Our previous studies showed that the GKS -> AAA mutation in S. cerevisiae NOG1, which should not bind GTP, abrogated function (14). Here we show that T. brucei nog1-g1, which carries the same mutation, is able to target to the nucleolus and interact with the nascent ribosome. These data argue against the possibility is that GTP binding is required for folding or transport. Additionally, removal of GTP does not release NOG1 from the particle. Thus, GTP binding by NOG1 is likely to more directly function in ribosomal biogenesis.

NOG1 and Obg contain a substitution that blocks GTPase activity when present in other G-proteins. Indeed, Obg has a low rate of GTP hydrolysis (44, 45) and an affinity low enough to promote rapid exchange. These features suggest that one function of Obg and NOG1 may be to sense the GTP level and regulate ribosome biogenesis accordingly. Because GTP does not regulate the binding of NOG1 to the complex, GTP may regulate function within the complex. There the GTP-bound form could be capable of recruiting or remodeling a protein(s), which in turn allows functional biogenesis. We speculate that the GDP-bound form cannot fulfill this function. It is interesting that overexpression of nog1-g1 and knockdown of NOG1 gave somewhat different phenotypes with respect to large subunit processing. The former showed a delay in multiple processing steps, including some specific for T. brucei, whereas the latter showed an accumulation of an atypical intermediate.

Although it is clear that defects in NOG1 can lead to defects in 60 S biogenesis, GTP/GDP binding to NOG1 could have additional roles by signaling to other pathways either directly or indirectly. For example, the stress response in B. subtilis requires both Obg and rpl11 (46). In a recent proteomic analysis of the mammalian nucleolus, 11% of identified proteins had functions or motifs that did not appear related to ribosome biogenesis (13). It remains feasible that NOG1 could, by analogy to Obg, allow for coordination of other cellular processes such as the cell cycle and ribosome biogenesis.


   FOOTNOTES
 
Note Added in Proof—During review of this report, NOGI was shown to be required for 60 S biogenesis in yeast (48).

* This work was supported in part by National Institutes of Heath Grant R01 AI31077. 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

Supported by National Institutes of Heath Grant 1T32 AI07509. Back

|| To whom correspondence should be addressed: Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98109. Tel.: 206-284-8846 (ext. 315); Fax: 206-284-0313; E-mail: mparsons{at}sbri.org.

1 The abbreviations used are: ITS1, first internal transcribed spacer; RNAi, RNA interference; TAP, tandem affinity purification; kb, kilobase(s). Back

2 B. C. Jensen, unpublished results. Back

3 B. C. Jensen, Q. Wang, C. T. Kifer, and M. Parsons, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Paul Englund and Mark Drew for the gift of pZJM, Achim Schnaufer and Kenneth Stuart for pLew-TAP, George Cross and Elizabeth Wirtz for 29-13 cells, and Steven Reed for anti-P0.



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 DISCUSSION
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