Monoclonal Antibodies 37C12 and B47 Are Directed Against Nucleolar Proteins
Monoclonal antibodies raised against nucleolar antigens were evaluated by immunofluorescence (IF) staining. Monoclonal antibody 37C12 produced a bright and specific intranuclear IF pattern that substantially overlapped the distribution of the nucleolar protein Nop2p (). The mAb B47 also gave an IF staining pattern that coincided with the distribution of Nop2p (). The IF pattern in both cases was offset from the distribution of chromatin in many cells, depending on the orientation of the nucleus, which resulted in the appearance of a crescent shape that is characteristic of the nucleolus in yeast (, arrows). Thus, 37C12 and B47 recognized nucleolar antigens in yeast.
To identify the antigen(s) recognized by 37C12, immunoprecipitations were done with 35
S-labeled nuclear extracts. 37C12 immunoprecipitated two proteins of approximately 67 and 38 kDa, which were not observed in the absence of primary antibody (). 37C12 immunoprecipitates washed with IP buffer containing 0.5 m
NaCl, 2 m
urea, or 0.2% SDS, 1% Nonidet P-40 also contained the 67-kDa protein, but with lower relative amounts of the 38-kDa band (data not shown). This suggested that 37C12 recognized a 67-kDa protein in the nuclear fraction. Considering that Nop1p is known to migrate on SDS gels at 38 kDa (30
), we tested the possibility that this protein was Nop1p. The immunoprecipitate obtained with 37C12 was solubilized with SDS, diluted with non-ionic detergent, and re-immunoprecipitated with mAb A66, which is specific for Nop1p (30
). A66 quantitatively immunoprecipitated the 38-kDa protein, proving that it is Nop1p ().
Immunoprecipitation with mAb 37C12
Monoclonal 37C12 did not immunoblot yeast nuclear protein extracts, despite the use of protocols to renature proteins prior to transfer or after transfer.4
On the other hand, B47 was not very effective in protein immunoprecipitation experiments, but produced a specific signal on immunoblots (see and ).
Deletion of the COOH-terminal KKX repeat motif from Nop5p
Nop5p depletion affects the localization of the nucleolar protein Nop1p
NOP5 Encodes A Novel Protein That Is Localized to the Nucleolus
To molecularly clone the gene encoding the 67-kDa protein, a yeast expression library was screened with 37C12. Eight positives fell into two classes of overlapping clones, the largest one of which contained an insert that encoded approximately 70% (amino acids 71–436) of the YOR310C open reading frame present on chromosome XV (35
). A lacZ
gene fusion from a positive clone was expressed in a λ
-lysogen and yielded a protein of the expected size that was recognized by 37C12 on an immunoblot (). Thus, although full-length protein from a yeast nuclear extract did not react on immunoblots probed with 37C12 (see above), a fusion protein produced in E. coli
was recognized by 37C12. We refer to this gene as NOP5
rotein 5). NOP5
encodes a 511-amino acid protein of predicted molecular weight 56,953, with a predicted pI of 9.4, whose most notable sequence characteristic is a highly hydrophilic and charged KKX
repetitive motif at the carboxyl terminus (). The predicted molecular mass of 57.0 kDa is smaller than the size observed on SDS gels. This difference is likely due to the highly charged COOH terminus (see below). Other nucleolar proteins with clusters of charged amino acids also migrate anomalously on SDS gels (e.g.
). During the course of our studies, Gautier et al.
) also identified this gene in a screen for genes that are synthetically lethal with the nop1–3
allele and termed it NOP58
To confirm via an independent means that NOP5
encoded a nucleolar protein, Nop5p containing a carboxyl-terminal hemagglutinin antigen (HA-1) epitope tag was expressed under control of the GAL promoter in plasmid pPW73 in YPW38 (). Plasmid pPW73 complemented a nop5
null allele (data not shown), indicating that the epitope tag does not interfere with Nop5p function. Growth of YPW38 on galactose-induced expression of epitope-tagged Nop5p (). The protein band induced in the presence of galactose also reacted with mAb B47 (data not shown). IF analysis revealed a range of signal intensities (). A range of signal intensities has been observed in other experiments with YPW38 (data not shown), even though pPW73 carries CEN6
, which should limit plasmid copy number to 2–5 (37
). This range may be due to variation in plasmid copy number coupled with the effects of a strong GAL10
promoter. Cells expressing low to moderate levels of epitope-tagged Nop5p show colocalization with the nucleolar protein Nop2p and a typical crescent-shaped nucleolar pattern (, arrows
). Cells overexpressing Nop5p show signal distributed throughout the nucleus, including the nucleolus. Because overexpression of a protein can lead to an anomalous intracellular distribution, cells expressing low to moderate levels are the most reliable indicator of localization. YPW38 grown on glucose, and a control strain lacking pPW73, do not produce an IF signal with mAb 12CA5 (data not shown). Thus, we conclude that epitope-tagged Nop5p is localized to the nucleolus.
Localization of HA epitope-tagged Nop5p
The COOH-terminal KKX Repeat Motif Is Not Required for Cell Growth
Nop5p contains a KKX
repeat motif at its carboxyl terminus. Similar motifs are present in Cbf5p (at the COOH terminus; Ref. 38
) and Dbp3p (near the NH2
terminus; Ref. 39
). To examine the functional significance of the KKX
motif in Nop5p, two COOH-terminal truncations were made: nop5
(see and ). Western blotting with B47 and immunoprecipitation with 37C12 detected truncated proteins of apparent molecular masses 54 and 51 kDa from strains bearing nop5
, respectively (). The truncated forms of Nop5p corresponded more closely to the predicted molecular masses 52.1 and 49.3 kDa, respectively. This indicates that the highly charged COOH terminus of Nop5p is responsible for the larger than predicted size observed in SDS gels.
It is important to note that 37C12 immunoprecipitated a protein from YPW51 and YPW52 that comigrated with the 67-kDa band from YPW53 (). Thus, 37C12 recognized, or co-immunoprecipitated, an additional protein identical in size to Nop5p on SDS gels. Considering that Sik1p/Nop56p (36
) is 504 amino acids in length with a predicted size of 56.9 kDa, and is 43% identical to Nop5p, it is likely that Sik1p/Nop56p is the additional protein. In contrast, mAb B47 did not recognize a 67-kDa band in extracts from strains YPW51 and YPW52, and specifically recognized Nop5p ().
Both of the truncated alleles nop5Δ1 and nop5Δ2 complemented the nop5 disruption (). Growth rates of YPW51 (nop5Δ1), YPW52 (nop5Δ2), and YPW53 (NOP5) on different media and at different temperatures were compared. Growth of the COOH-terminal truncations was normal at 14 and 25 °C (). Measurements of doubling times on minimal and rich liquid medium at 30 °C did not reveal significant differences between YPW51, YPW52, and YPW53 (data not shown). However, growth of YPW51 and YPW52 was impaired at 37 °C (), implying a function for the KKX sequence. IF localization of Nop5p in YPW51, YPW52, and YPW53 grown at 25 °C revealed that the truncated forms of Nop5p were localized to the nucleolus in a manner indistinguishable from wild type (data not shown).
Nop5p Is a Member of an Evolutionarily Conserved Protein Family
Data base searches revealed that Nop5p is related to the yeast protein encoded by SIK1/NOP56
), and proteins in Methanococcus jannaschii, Arabidopsis thaliana
, Caenorhabditis elegans
, and human (). Sik1p/Nop56p is 43% identical to Nop5p (pairwise Lipman-Pearson alignment). Two proteins of unknown function from A. thaliana
are 52 and 47% identical to Nop5p. A C. elegans
protein is 39% identical to Nop5p and a M. jannaschii
protein is 35% identical to Nop5p. Six tentative human consensus sequences shared sequence similarity to Nop5p and may be grouped into two classes. One group aligns with a human homologue of Sik1p/Nop56p (hNop56; Ref. 36
), which is more similar to Sik1p/Nop56p (51% identity) than to Nop5p (38% identity). A putative human protein is encoded by a second grouping of three tentative human consensus (see ). We refer to this putative human protein as hNop5p. The putative hNop5p is 48% identical to Nop5p and is 38% identical to Sik1p/Nop56p.
Alignment of Nop5p with related proteins
NOP5 Is an Essential Gene
To determine if NOP5 is essential, ~90% of one copy of NOP5 in a diploid strain was replaced by TRP1 (; ). Southern blotting confirmed the transplacement of NOP5 with TRP1, and produced the predicted results: ClaI digestion of genomic DNA gives a 6.4 kb band corresponding to wild type NOP5, and an additional 5.7-kb band in YPW42 and YPW43; XbaI gives a 3.8-kb band from wild type, and an additional 2.95-kb band from the disrupted locus (). YPW42 was transformed with pPW80, sporulated, dissected, and a 5-FOA sensitive strain (YPW45) was obtained. Plasmid shuffling was used to replace pPW80 in YPW45 with pPW83, which carried NOP5 under GAL promoter control, to yield YPW48. YPW48 was viable when grown on galactose containing medium, but not in the presence of glucose, whereas YPW45 was viable on both carbon sources, but was inviable on 5-FOA containing medium (). This demonstrated that NOP5 is an essential gene.
NOP5 is an essential gene
YPW48 was used to genetically deplete Nop5p in vivo by shifting from YPGal to YPD medium. During the first 10 h in YPD, YPW48 grew slightly faster than cells in YPGal (). However, after approximately 10 h in YPD, cell growth was inhibited and the doubling time increased about 5-fold from ~2.0 to ~10.5 h. Northern blotting showed that NOP5 mRNA levels became undetectable after 1 h of depletion, whereas actin (ACT1) mRNA levels remained unchanged over the time course (data not shown). Immunoblotting with mAb B47 confirmed that Nop5p was depleted (see below, ). Thus, growth of YPW48 on glucose-containing medium substantially depletes the cell of Nop5p.
Depletion of Nop5p Leads to Reduced Levels of 40 S Subunits
Since Nop5p is localized in the nucleolus and its depletion leads to a reduction in growth rate, we reasoned that Nop5p was likely to play a role in ribosome synthesis. To test this, polysomes, ribosomes, and ribosomal subunits from cells depleted of Nop5p were analyzed on sucrose density gradients. YPW48 was grown in YPGal, shifted to YPD, and grown for 4, 8, or 12 h. The wild type haploid strain W303–1a grown in both YPGal and YPD showed typical levels of 40 S and 60 S subunits, 80 S monosomes, and polysomes corresponding to 2 to 10 ribosomes (). YPW48 showed reductions in 40 S, 80 S, and polysome peaks after 8 and 12 h in YPD, but the effect was not dramatic after 4 h in YPD (). The increase in the 60 S peak reflected an increase in the cytoplasmic pool of free subunits. Because the reductions in peak heights observed at 4 and 8 h precede the reduction in growth rate at about 10 h, these results cannot be attributed to a secondary effect of reduced growth rate.
Nop5p is required for 40 S subunit synthesis
Depletion of Nop5p Impairs Synthesis of 18 S rRNA and Processing of Pre-rRNA
The 18 S rRNA is synthesized by the pathway diagrammed in . To explore 18 S rRNA synthesis, the levels of 18 S and 25 S rRNAs from YPW48 grown in YPGal or shifted to YPD for 24 h were compared (). After growth in YPD for 24 h, the abundance of the 18 S rRNA was reduced by approximately 50%, whereas the abundance of the 25 S rRNA was unaffected (). This indicated that Nop5p depletion leads to a specific reduction of 18 S rRNA, which could either be at the level of reduced synthesis or stability, or both.
Nop5p depletion leads to reduced synthesis of 18 S rRNA
To investigate a role for Nop5p in pre-rRNA processing, YPW48 was analyzed by pulse-chase labeling with [methyl-3H]methionine (). In SGal, after 2 min of chase, there was little or no accumulation of 35 S pre-rRNA and levels of 27 S and 20 S intermediates were normal. By 8 min of chase, most of the 27 S and 20 S intermediates were processed to mature 25 S and 18 S rRNAs. At 12 min of chase only mature rRNAs were detected. After 4 h of growth in SD, accumulation of 35 S pre-rRNA became visible. Levels of 20 S and 18 S rRNAs were reduced as compared with 27 S and 25 S rRNAs. After 8 and 12 h, the 35 S pre-rRNA accumulation became more prominent, 20 S rRNA levels were reduced substantially, and 18 S rRNA levels decreased to very low levels. On the contrary, processing from 27 S to 25 S rRNA remained similar to results obtained with cells grown in SGal.
To ensure that these results were not due to a change in the methylation pattern of pre-rRNAs, pulse-chase labeling was repeated with [3H]uracil. YPW48 cells grown in SGal were shifted to SD for 12 h. The results were essentially the same as observed with [methyl-3H]methionine pulse-chase labeling: the 35 S pre-rRNA accumulated, and levels of 32 S, 20 S, and 18 S rRNAs were reduced dramatically (). This indicated that Nop5p depletion leads to a specific processing defect in the pathway that forms the 20 S rRNA intermediate.
Although processing from 27 S to 25 S rRNA does not seem to be affected by Nop5p depletion, 5.8 S rRNA processing could be affected nevertheless (e.g.
). Thus, we analyzed the synthesis of 5.8 S rRNA by [3
H]uracil pulse-chase labeling. At the different chase times examined (2, 8, 16, and 32 min) there was no observable decrease in 5.8 S rRNA levels relative to the control (data not shown).
Depletion of Nop5p Affects Processing of the 5′-Externally Transcribed Spacer
The defect in production of 18 S rRNA suggested an early defect in pre-rRNA processing. To determine the steady-state levels of pre-rRNAs and rRNAs in Nop5p-depleted cells, Northern blotting analysis was done (see for oligonucleotide positions). YPW48 cells depleted for 2, 4, 8, and 12 h showed an accumulation of 35 S pre-rRNA and a decrease in levels of 32 S, 20 S, and 18 S rRNAs (). The 23 S intermediate is usually present at very low levels in wild type cells and corresponds to an intermediate in which cleavage at A0
, and A2
has failed to take place (5
). Levels of the 27 S intermediate decreased by approximately 2.5-fold after 2 h of growth in glucose, but did not decrease dramatically at longer times in SD medium (). Levels of 18 S and 25 S rRNAs decreased only a small amount during the depletion time course (), and after 12 h of depletion were 60 and 68%, respectively, of the levels at the 0-h time point. Taken together, the Northern blotting results suggested a defect in early processing steps in the 5′-ETS and ITS1.
Northern blot analysis of rRNA processing during Nop5p depletion
To examine processing at sites in the 5′-ETS and ITS1, primer extension analysis was done (see for oligonucleotide positions). At 12 and 24 h of Nop5p depletion, processing at site A0 was progressively impaired (). At 24 h, the band corresponding to processing at A0 was decreased in intensity by 76% compared with the band at 0 h. In addition, a number of longer primer extension products were observed (, lanes 12 and 24), which was consistent with the accumulation of unprocessed 35 S pre-rRNA. Similarly, processing in ITS1 at site A2 was impaired (). At 24 h, the band corresponding to processing at A2 was decreased in intensity by 86% compared with the band at 0 h. To control for variables such as RNA yield, the relative abundance of the 18 S rRNA was determined. Bands corresponding to the 5′-end of 18 S rRNA (processing site A1) showed only small variations in intensities (). At 24 h, the band corresponding to processing at A1 was decreased in intensity by only 10% compared with the band at 0 h. We note that the primer extension method we use does not allow us to address processing at A1 during Nop5p depletion (see “Experimental Procedures”). To rule out the possibility that reductions in A0 and A2 band intensities could be attributed to reduced transcription of the 35 S precursor, the relative amounts of the 5′-end were determined (). At 24 h, the band corresponding to the 5′-end was decreased in intensity by only 4% compared with the band at 0 h. Thus, reductions in processing at sites A0 and A2 during Nop5p depletion cannot be accounted for by alterations in 35 S transcription.
Primer extension analysis of rRNA processing during Nop5p depletion
Nop5p Is Associated with Small Nucleolar RNAs
Given the effects of Nop5p depletion on pre-rRNA processing and the likely interaction between Nop5p and Nop1p, we tested whether Nop5p was associated with snoRNAs. RNAs immunoprecipitated by B47 and 37C12 were 3′-end-labeled and analyzed by denaturing PAGE. Identification of snoRNAs was based on RNA lengths determined by comparison with a DNA sequencing ladder (data not shown). B47 immunoprecipitated snoRNAs that migrated at positions corresponding to U3, U14, snR13, and U18 (). 37C12 immunoprecipitated snR13 and U18 strongly, but immunoprecipitated U3 only weakly, and immunoprecipitated only one of the U14 isoforms. Minor bands were also immunoprecipitated by the mAbs, especially by 37C12, and may be snoRNAs more loosely associated with Nop5p, or snoRNAs that were nonspecifically associated with Nop5p. Small amounts of 5.8 S and 5 S rRNAs were immunoprecipitated nonspecifically, and were also observed in a control immunoprecipitate (). These findings indicated that Nop5p is associated, either directly or indirectly, with the snoRNAs U3, U14, snR13, and U18.
Immunoprecipitation of small nucleolar RNAs
As mentioned above, B47 did not immunoprecipitate Nop5p from yeast nuclear extracts. To investigate this discrepancy, we compared immunoprecipitates obtained with B47 and 37C12 using the two different methods for protein and RNA immunoprecipitation (see “Experimental Procedures”). We found that B47 immunoprecipitated a 67-kDa band of moderate intensity with the RNA method, but not with the protein method (data not shown). Conversely, the band observed in 37C12 immunoprecipitates is considerably weaker with the RNA method compared with the protein method (data not shown). Thus, the difference in RNA and protein immunoprecipitation buffers was an important factor in the binding of antigens by B47 and 37C12, perhaps as a consequence of epitope conformation. In addition, the 67-kDa band immunoprecipitated by B47 comigrated with the band immunoprecipitated by 37C12, indicating that the 67-kDa band recognized in immunoprecipitations was the same as the 65-kDa band recognized by B47 on immunoblots. Immunoprecipitates contain a large amount of IgG heavy chain, which could influence the mobility of Nop5p during SDS-PAGE and result in a small difference in a apparent size in immunoblotting and immunoprecipitation experiments.
Nop5p Is Required for Localization of Nop1p to the Nucleolus
Of interest to us are the mechanisms by which nucleolar proteins are localized to, and interact within, the nucleolus. Immunofluorescence and cell fractionation approaches were used to determine the extent to which the nucleolar localization of Nop5p and Nop1p was interdependent.
Strikingly, Nop5p depletion affected the localization of Nop1p, and caused Nop1p to become distributed in the nucleus and cytoplasm (). After growth of YPW48 for 4 h in glucose, Nop5p was only faintly detected by mAb B47. Staining with mAb 37C12 also showed a decrease in intensity after 4 and 8 h, but faint staining remained even after 12 h in glucose medium. We attribute this residual staining to the recognition of Sik1p/Nop56p, whose levels may have decreased during Nop5p depletion. The effect on distribution was specific to Nop1p because the localization of Nsr1p was not affected by Nop5p depletion (). The distribution of the nucleolar protein Nop2p, the nuclear protein homocitrate synthase, and the nuclear pore complex protein Nsp1p were not affected by Nop5p depletion (data not shown). In addition, the localization of Nop1p was strictly nucleolar in strains carrying the nop5Δ1 and nop5Δ2 COOH-terminal truncation alleles grown at either 30 or 37 °C (data not shown).
To confirm the immunofluorescence results, immunoblotting was done using crude nuclear and cytoplasmic fractions (see “Experimental Procedures”). Depletion was rapid and Nop5p was barely detectable after only 4 h of growth on glucose (). During depletion, nuclear levels of Nop1p decreased, while cytoplasmic levels increased (). The level of nuclear Nop1p increased by 15% after 4 h on glucose, but Nop1p levels decreased by 25 and 31% at 8 and 12 h, respectively. The level of cytoplasmic Nop1p increased steadily between the 4 and 12 h time points, and at 12 h reached a level equal to 219% of the level at zero time. Thus, Western blotting results confirmed that efficient localization of Nop1p to the nucleolus requires normal levels of Nop5p.