Vol. 12, No. 9
MOLECULAR AND CELLULAR BIOLOGY, Sept. 1992, p. 3865-3871
0270-7306/92/093865-07$02.00/0
Copyright X) 1992, American Society for Microbiology
NSR1 Is Required for Pre-rRNA Processing and for the Proper Maintenance of Steady-State Levels of Ribosomal Subunits WEN-CHING LEE, DAN ZABETAKIS, AND TERI MELESE* Department of Biological Sciences, Columbia University, New York, New York 10027 Received 12 May 1992/Accepted 18 June 1992
NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsrl mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsrl mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.
(11, 15, 22, 30). When yeast strains were depleted of these nucleolar proteins or snoRNAs, they all displayed the same phenotype; the processing of 35S pre-rRNA was impaired and the amount of 18S rRNA was dramatically reduced. Although it is not clear what role these nucleolar components play in the processing of rRNA, it has been suggested that they may function together as small nucleolar ribosomal particles (snoRNPs) (11, 30). Other evidence supports the notion that the assembly of newly formed ribosomal proteins on the pre-rRNA is also essential for the maturation of yeast pre-rRNA (27, 36). Therefore, a defect in pre-rRNA processing can result from improper assembly of the preribosomal particle as well as from blockage at the specific cleavage
In eukaryotic cells, the nucleolus is a specialized subcompartment in which ribosome biogenesis occurs. Pre-rRNA first is transcribed from rDNA genes located in the nucleolus and then undergoes a series of modifications. Ribosomal proteins are imported from the cytoplasm and packed onto the RNA molecule. At the same time, the primary transcript goes through sequential cleavages to generate mature forms of rRNA. Finally, the newly formed ribosomal particles are exported to the cytoplasm. In the yeast Saccharomyces cerevisiae, the largest detectable transcript from the rDNA genes is a 35S pre-rRNA (17), which is rapidly processed into three molecules: the 18S rRNA assembled into 40S ribosomal subunits and the 5.8S and 25S rRNAs found in 60S subunits (Fig. 1). During or immediately after transcription of the rDNA genes, the pre-rRNA is modified, mainly by methylation (32, 34). Meanwhile, a large number of ribosomal proteins and nonribosomal components (including proteins and RNA) associate with the pre-rRNA to form a 90S preribosomal particle. This particle is then split into 66S and 43S preribosomal particles, which are the precursors of the 60S and 40S subunits, respectively (31). While the 66S particles mature completely within the nucleus, the final maturation steps of the 43S particles, including the processing of 20S to 18S rRNA, are completed in the cytoplasm (31, 33). Only a few mutations that block specific steps in the pre-rRNA processing pathway have been found. One example is a temperature-sensitive mutation in a gene designated RRPJ that was shown to prevent the 27S-to-25S rRNA cleavage step specifically (1). In another mutant, CLP-8, the efficiency of the processing of 20S to 18S rRNA is greatly reduced, apparently because of a defect in 20S pre-rRNA export (5). Recently, two nucleolar proteins, NOP1 and GAR1, and two small nucleolar RNAs (snoRNAs), U3 and U14, were shown to be required for pre-rRNA processing
*
steps.
Ribosomal proteins
are
translated in the cytoplasm and
must be imported into the nucleus for assembly. From these ribosomal proteins and the rRNA components, ribosomal particles are then assembled in the nucleolus and exported to the cytoplasm to carry out the process of protein synthesis.
The tremendous flux of ribosomal proteins and ribosomal particles across the nuclear envelope likely accounts for a large portion of the nucleocytoplasmic traffic in exponentially growing cells. However, little is known about this import-export process. In recent years, two mammalian nucleolar proteins, nucleolin and No38, were shown to shuttle between the nucleus and the cytoplasm (3). Nucleolin, a major nucleolar protein in mammalian cells, has been thought to play a role in ribosome biogenesis (4, 14, 19). The phenomenon of shuttling raised the possibility that nucleolin participates in the nucleocytoplasmic transport of ribosomal components. In searching for proteins involved in the initial stage of protein import into the nucleus, we have identified a 67-kDa yeast protein that specifically recognizes nuclear localization sequences (NLS) (20). The gene that encodes this 67-kDa protein was cloned and named NSRI, for nuclear signal recognition protein (21). Surprisingly, this NLS-binding protein was localized to the nucleolar region by indirect immu-
Corresponding author. 3865
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:
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Ay
a I 18S
5.8S
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FIG. 1. Major pre-rRNA processing pathway in S. cerevisiae. The 35S pre-rRNA is the largest detectable transcript from the rRNA operon. The 32S, 27S, and 20S pre-rRNAs are the major processing intermediates. Processing from 20S to 18S rRNA is carried out in the cytoplasm. The 18S rRNA is found in the 40S ribosomal subunits, while the 5.8S and 27S rRNAs are hydrogen bonded together in the 60S ribosomal subunits. nofluorescence (21) and contains a glycine- and arginine-rich (GAR) domain, a common structural motif among the nucleolar proteins sequenced so far (11). Moreover, the NSR1 protein bears a strong resemblance to nucleolin in domain structure, but without a high degree of amino acid sequence similarity. Both proteins contain a GAR domain at their C termini, and both contain consensus RNA recognition motifs; nucleolin has four such motifs, while NSR1 has two. In particular, nucleolin and NSR1 contain N termini with serine stretches flanked by acidic amino acid residues, which are not present in the previously identified yeast nucleolar proteins (SSB1, GAR1, and NOP1) (11, 13, 16, 26). the structural similarity between NSR1 and nuclesb1k,'i hammalian nucleolin gene expressed in yeast cells a*nplent the slow-growth phenotype of an nsrl -e muIWtP'. IXi the same study, it was also found that nucIffl NSR1, is an NLS-binding protein; the recognitift tbe NLS occurs in the N-terminal regions of both '° ioteii1:)n the basis of features shared with nucleolin and tsloition in the nucledit1t, it is reasonable to suspect that NSR1 rnay be involved in ribosome biogenesis. We present evidence here to show that NSR1 is required not only for normal pre-rRNA processing but also for the
normal distribution of free ribosomal subunits. To assess the
physiological significance of this ribosome imbalance, we tested the nsrl mutant for sensitivity to a variety of drugs known to interfere with ribosome function. In comparison with the wild type, the only difference observed was in sensitivity to paromomycin. Paromomycin causes misreading of the RNA codons in vitro for both prokaryotic and eukaryotic systems (7, 24, 28). We have found that the nsrl mutant cannot grow at concentrations of paromomycin at which the growth of the wild-type strain is unaffected. MATERIALS AND METHODS Yeast strains. W303-1A (MALTa ade2-1 canl-100 ura3-1 leu2-3,112 trpl-1 his3-11,15) and WLY353 (same as W303-
1A, except nsrl::HIS3) with plasmid pBM272 were used in the [3H]uracil labelling experiment. pBM272 is a yeast plasmid derived from YCp5O, which carries the URA3 gene. Pulse-chase labelling of pre-rRNA. Yeast cells were cultured in glucose minimal medium lacking either methionine or uracil, as determined by the labelling reagent to be used in the experiment. Cells were first grown at 30°C to an optical density at 600 nm (OD6.) of 0.5 to 1.0 and then switched to room temperature for 1 h before being pelleted and resuspended in 1/10 the original volume. Each milliliter of cells was labelled with 30 ,Ci of [methyl-3H]methionine (NEN) or [3H]uracil (NEN) at room temperature for 1 min and then chased with cold methionine at a final concentration of 5 mM or with uracil at a final concentration of 240 ,ug/ml, respectively. At various times after the chase, 1-ml aliquots were withdrawn and placed in tubes containing crushed ice to stop the reaction. The cells were then collected by centrifugation and resuspended in 1 ml of RNA isolation buffer (100 mM NaCl, 50 mM sodium acetate [pH 5.3], 1 mM EDTA), and total RNA was extracted. Extraction and analysis of RNA. Total RNA was extracted by the method of Kohrer and Domdey (18) with modifications. Cells in 1 ml of RNA isolation buffer were mixed with 50 ,ul of 20% sodiutn dodecyl sulfate and 1 ml of 65°C phenol and immediately shaken vigorously in a 65°C water bath for 10 min. The tube was then quickly chilled in a dry ice bath and centrifuged to separate the aqueous phase from the phenol. The phenol was removed from the tube, and the aqueous phase with the cells was extracted with phenol again as described above. After centrifugation, the aqueous phase was transferred to a new tube and extracted twice with 25:24:1 phenol-chloroform-isoamyl alcohol and once with 24:1 chloroform-isoamyl alcohol. RNA was precipitated with ethanol and redissolved in diethylpyrocarbonatetreated water. The concentration of RNA was estimated by measuring the A260, and it was assumed that 1 A260 unit equals 40 p,g of RNA. RNA was separated in 1.2% agarose gels containing 6% formaldehyde, 50 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES) (pH 7.8), and 1 mM EDTA. The gels were stained with 5 ,ug of ethidium bromide per ml after electrophoresis. To detect 3H labelling, we soaked the gels in Amplify (Amersham) for 1 h, rinsed them with water, and dried them. The dried gels were exposed to XAR5 film. The intensities of the bands on the fluorograms (see Fig. 2) or the positive images of the ethidium bromide-stained gels (see Fig. 3A) were quantitated with a Bio Image Visage 110 image quantification system. Ribosomal profiles. Ribosomes were prepared essentially as described by Baim et al. (2). Yeast cells were grown to an OD6. of 0.2 to 0.8 at 30°C in 200 ml of YPD (1% yeast extract, 2% peptone, 2% glucose) (rich medium). Crushed ice and 10 mg of cycloheximide were placed in a 500-ml centrifuge tube. The yeast culture was poured into the tube, shaken, and immediately pelleted in a GS-3 rotor (DuPont) at 4,000 x g. The cells were resuspended in 10 ml of buffer A (10 mM Tris [pH 7.4], 100 mM NaCl, 30 mM MgCl2, 50 p,g of cycloheximide per ml, and 200 ,ug of heparin per ml in diethylpyrocarbonate-treated water) and repelleted. This step was repeated, and the cells were resuspended in 0.5 ml of buffer A and transferred into a 15-ml plastic centrifuge tube. Approximately 0.25 ml of glass beads was added, and the cells were lysed by 10 alterations of 15 s of vortexing and 30 s of cooling in an ice bucket. Buffer A was then added to bring the total volume to about 4 ml. Cell debris and glass beads were removed by centrifugation for 5 min at 3,600 x g
NSR1 IS REQUIRED FOR PRE-rRNA PROCESSING
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35Sb 32S"
27S vE _" *m* a 25S, 0
20S) 18S'
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FIG. 2. Pulse-chase labelling of pre-rRNA from the wild-type strain and the nsrl mutant. Wild-type (NSR1+) and nsrl mutant (nsrl-) cells were pulse-labelled with [methyl-3H]methionine (A) or [3H]uracil (B) for 1 min at room temperature and chased with a large excess of unlabelled methionine or uracil, respectively. Aliquots were withdrawn at various times after the chase, as indicated at the top of each lane. Total RNA was isolated from the aliquots and analyzed by agarose-formaldehyde gel electrophoresis and then by fluorography. The positions of the major pre-rRNA species (35S, 32S, 27S, and 20S) as well as 25S and 18S rRNAs are marked.
in an SA-600 rotor (DuPont). The supernatant was further clarified by another round of centrifugation at 10,000 x g for 10 min. This supernatant was divided into aliquots and frozen at -70°C. No differences were detected between samples frozen in this manner and fresh samples analyzed immediately. Ribosomes were analyzed by centrifugation through a sucrose step gradient. The sucrose solution contained 50 mM Tris-acetate (pH 7.0), 50 mM NH4C1, 12 mM MgCl2, 1 mM dithiothreitol, and 7, 17, 27, 37, or 47% sucrose. The gradient was prepared by pipetting 7 ml of each layer onto the bottom of a 40-ml open-top centrifuge tube. The ribosomal sample was loaded on top, and the gradient was centrifuged at 100,000 x g and 4°C for 4 h in an SW27 rotor (Beckman). The sample concentration was measured as the OD260. Approximately 40 OD260 units per ml was loaded on each gradient. Finally, the gradient was extracted and analyzed with an ISCO model 640 gradient fractionator, with monitoring of the effluent at OD254. Drug sensitivity assay. Yeast cells were grown to the stationary phase in YPD and diluted to an ODwo of 0.3. The cells (200 ,ul) were spread on YPD plates. Fifty microliters of various concentrations of paromomycin, cycloheximide, streptomycin, or erythromycin (each from Sigma) was pipetted onto sterile filter disks (diameter, 3/8 in. [ca. 1 cm]; Schleicher & Schuell). After the solutions were absorbed, the filter disks were placed on top of the spread cells. The plates were incubated at 30°C for 5 to 6 days and then photographed. RESULTS Pre-rRNA processing in the nsrl mutant. In previous studies in our laboratory, a disruption of the chromosomal copy of the NSR1 gene was carried out in diploids by the one-step gene disruption method (25). Two segregants from each tetrad containing the disrupted NSRI gene (nsrl ::HIS3)
grew slower than two segregants containing the wild-type copy of the NSRI gene (21). Thus, NSR1 is required for normal cell growth but is not essential for cell viability. The slow growth of the nsrl mutant enabled us to examine how growing cells functioned in the absence of NSR1. The nucleolar localization of NSR1 and the resemblance to nucleolin prompted us to test whether the protein is involved in any aspect of ribosome biogenesis. First, prerRNA processing in the nsrl mutant was investigated by pulse-chase labelling experiments. Both the wild-type (NSR1 +) strain and the nsrl mutant were grown to log phase at 30°C before being switched to room temperature for 1 h to slow down cellular metabolism. Cells were labelled with [methyl-3H]methionine for 1 min and then chased with large amounts of cold methionine. Aliquots were withdrawn at various times after the chase, total RNA was extracted, and the samples were analyzed by agarose gel electrophoresis and then by fluorography (Fig. 2A). Although the nsrl mutant grows much more slowly than the wild-type strain under optimal growth conditions, the doubling time of the nsrl mutant under the conditions used for pulse-chase labelling (synthetic minimal medium, room temperature) is nearly identical to that of the wild-type strain, enabling us to compare the pre-rRNA processing rates directly. In the NSR+ strain, pre-rRNA methylation and processing occurred rapidly, and two major processing intermediates, 27S and 20S pre-rRNAs, were observed by the end of the 1-min labelling period. The final products, the 18S and 25S rRNA species, appeared in an approximately 1:1 ratio, as predicted from the processing pathway (see Fig. 1 for the pathway). In comparison, processing was less efficient in the nsrl mutant. The amounts of 32S and 20S pre-rRNAs over the same times as the NSR1+ strain were dramatically reduced (Fig. 2A); consequently, the final amount of 18S rRNA was greatly affected, since the 20S pre-rRNA is the direct precursor of the 18S rRNA. The ratio of the labelled 18S and 25S rRNAs
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A
A
A NSR1+ nsrlRNA (Rg) 7.6 3.8 7.6 3.8
25S)-
18S
B
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0
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0
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25S
E
E 18S
B 2.0Or
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7.6
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3.8
NSR1+ nsr1 FIG. 3. Analysis of the steady-state levels rRNAs in the wildtype strain and the nsrl mutant. Yeast cells (10 ml) were grown in YPD to an OD600 of 1.0. Total RNA was extracted and quantitated by measuring the A260 (as described in Materials and Methods). RNA (7.6 or 3.8 jig) from the wild-type strain (NSR1+) or the nsrl mutant (nsrl-) was separated by agarose-formaldehyde gel electrophoresis and is marked at the top of the lanes as 7.6 or 3.8. (A) Ethidium bromide staining of the 25S and 18S rRNA species. The intensities of the RNA bands on the positive image of the stained gel were quantitated with a Bio Image Visage 110 image quantification system. (B) The relative amounts of the 25S and 18S rRNA species were plotted.
approximately 1:2 after 6 min of chasing (the quantitation method is described in Materials and Methods). To exclude the possibility that the observed phenomenon in the nsrl mutant was merely due to a change in the normal methylation pattern of the rRNA, we measured pre-rRNA processing in the presence of [3H]uracil as the labelling reagent. In this experiment, longer chase times were used. Aside from the fact that the background was higher because nascent pre-rRNA transcripts and mRNA are also labelled, the results of these experiments (Fig. 2B) were basically identical to those shown in Fig. 2A. In the nsrl mutant, 35S pre-rRNA accumulated, 32S and 20S pre-rRNAs almost disappeared, and the amount of 18S rRNA was less than one-half that of 25S rRNA after 20 min of chasing with cold uracil. Steady-state levels of rRNAs. The kinetic studies discussed above demonstrate that the nsrl mutant is defective in making 18S rRNA, resulting in an altered ratio of newly formed 25S and 18S rRNAs. To test whether this kinetic alteration was reflected at steady-state levels, we compared the major rRNAs in the wild-type strain and the nsrl mutant. Total RNA was isolated from both strains, separated by agarose-formaldehyde gel electrophoresis, and then stained with ethidium bromide. The stained gel is shown in Fig. 3A, and quantitation and comparison of the two rRNA bands are shown in Fig. 3B. The results are consistent with the findings
was
In a
1,.0 _
0
top
bottom
FIG. 4. Ribosomal profiles of the wild-type strain and the nsrl mutant. Yeast cells were grown in YPD to mid-log phase. Ribosomes and ribosomal subunits were isolated and separated on a 7 to 47% sucrose gradient as described in Materials and Methods. The gradient was analyzed with an ISCO model 640 gradient fractionator by monitoring OD254. The OD scale is approximate. (A) Ribosomal profile of the wild-type strain. (B) Ribosomal profile of the nsrl mutant. The top and bottom of the gradient and the ribosomes and subunits are marked. The numbered peaks are polysomes containing the indicated numbers of ribosomes.
described for the pulse-chase labelling experiments. The ratio of 18S to 25S rRNAs in the nsrl mutant was different from that in the wild-type strain, and the amount of 18S rRNA was reduced. Ribosomal profiles in the nsrl mutant. The observation that the nsrl mutant was defective in its ability to produce 18S rRNA made us consider whether this defect was reflected in the composition of ribosomal subunits in the cells. If a cell cannot efficiently produce 18S rRNA, it may display this phenotype as a lack of 40S ribosomal subunits. It has been found that a deficiency in the production of a ribosomal protein results in the failure to assemble the subunit of which that protein is a component. Subsequently, this failure is observed as a deficiency of that subunit relative to the other subunit (23). Ribosomes were prepared as described by Baim et al. (2). Yeast cells were grown to the mid-log phase and harvested in the presence of cycloheximide. The cells were lysed with glass beads, and the supernatant was separated on a 7 to 47% sucrose gradient. The gradient was fractionated and moni-
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FIG. 5. Paromomycin sensitivity of the wild-type strain (top) and the nsrl mutant (bottom). The wild-type strain and the nsrl mutant were spread on YPD plates. Filter disks containing 10, 2, and 0.4 mg of paromomycin sulfate (labelled 1, 2, and 3, respectively) were placed on each plate. The cells were allowed to grow at 30°C for 5 days.
tored by measuring theA254. The results for both the NSR1I strain and the nsrl mutant are shown in Fig. 4. Compared with the NSR1+ strain, the nsrl mutant had little or no free 40S ribosomal subunits and a large surplus of free 60S subunits. There was essentially no difference in the amount and distribution of polysomes between the two strains. This result is consistent with the hypothesis that NSR1 is involved in the production of 40S ribosomal subunits. The nsrl mutant is sensitive to paromomycin. In wild-type strains, the two ribosomal subunits are present in equimolar quantities, but this balance is altered in the absence of NSR1. Since ribosomes are the machinery that carries out protein synthesis, we were curious to know whether this ribosomal imbalance affects protein synthesis in the nsrl mutant. To address this question, we tested the sensitivity of the nsrl mutant to a variety of drugs known to interfere with translation: cycloheximide, paromomycin, streptomycin, and erythromycin. Cultures of wild-type or nsrl mutant cells were spread on YPD plates, and sterile filter disks with various amounts of the drugs were placed on top of the spread cells. If the yeast cells are sensitive to the drug in the filter, their growth will be inhibited and a clear halo will form around the filter. The degree of sensitivity to the drug can be estimated by the size of the halo. Both strains were sensitive to cycloheximide to the same degree, and neither of the strains was sensitive to up to 10 mg (in the filter) of erythromycin or streptomycin (data not shown). The wild-type strain was quite resistant to paromomycin and did not form any sizeable halo around the filter, even when exposed to the highest amount of paromomycin (10 mg) used in this test. The nsrl mutant was extremely sensitive to this antibiotic, forming a significant halo around a filter containing only 2 mg of paromomycin (Fig- 5).
We have found the nsrl mutant to be defective in the processing of pre-rRNA, such that the production of 18S rRNA is reduced. Furthermore, the amount of free 40S ribosomal subunits, of which 18S rRNA is the only RNA component, is greatly reduced compared with that in the wild-type strain. In fact, in the nsrl mutant, there are no detectable free 40S ribosomal subunits in the cytosol, whereas a large excess of free 60S subunits is accumulated. Other investigators studying different yeast nucleolar proteins (GAR1 and NOP1) have also observed a defect in pre-rRNA processing in strains depleted of these proteins (11, 30). In the case of GAR1 and NOP1, pre-rRNA processing is slowed and 18S rRNA is deficient, and the authors suggest that an alternative processing pathway that results in the appearance of a new species, 23S RNA, occurs. The same phenotype has also been observed in strains depleted of snoRNAs U3 and U14 (15, 22). The phenotype observed for a strain lacking NSRJ, a nonessential gene, is essentially identical to that observed for strains depleted of the products of the essential genes NOPI, GAR1, U3, and U14. The processing of the 35S pre-rRNA is delayed, as evidenced by its accumulation. When the 35S pre-rRNA is finally processed, it does not form equimolar amounts of 20S and 27S pre-rRNAs, as observed in wild-type cells. Instead, the amount of the 20S pre-rRNA is greatly reduced and, consequently, the amount of the 18S rRNA is decreased. The decrease in the amount of the 20S rRNA may result from either degradation of the newly formed 20S pre-rRNA or degradation of the 5' region of the 35S pre-rRNA (where the 20S pre-rRNA resides). It was previously suggested that the 5' end of the 35S prerRNA is unstable in the absence of some nucleolar proteins or snoRNAs (22, 30). We also prefer the explanation of the instability of the 5' end of the 35S pre-rRNA because the accumulation of the 35S pre-rRNA and the decrease in the amounts of the 32S and 20S pre-rRNAs (Fig. 2) indicate that the cleavage steps between the 35S pre-rRNA to the 20S and 27S rRNAs are blocked or delayed. Specifically, we suggest that before a reasonable amount of 20S rRNA can be produced from the 35S pre-rRNA, a period of rapid degradation removes the 5' end of the 35S pre-rRNA, completely or partially, up to the cleavage site that normally separates the 20S from the 27S rRNA. Since we observed no smearing between the 35S pre-rRNA and 27S rRNA bands, the truncated pre-rRNA is rapidly cleaved into a normal 27S rRNA, and processing can proceed normally to produce the 5.8S and 25S rRNAs. Our observation that pre-rRNA processing does occur, albeit abnormally, in the nsrl mutant indicates that the blockage of 35S pre-rRNA processing discussed above is not absolute. Thus, cells are able to bypass the need for NSR1 function. An explanation for this could be that NSR1 is functionally redundant in cells. However, we did not detect a second copy of NSR1. When probes derived from various regions of NSR1 were used in a low-stringency Southern blot analysis, no specific hybridization was observed (data not shown). The fact that the depletion of several different nucleolar components results in the same phenotype has prompted investigators to propose the existence of a snoRNP or snoRNPs containing nucleolar proteins and snoRNA components required for proper pre-rRNA processing (11, 30). This suggestion is supported by observations that antibodies against several different nucleolar proteins (GAR1, NOP1,
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and SSB1) coimmunoprecipitate an overlapping set of snoRNAs (6, 11, 30). One well-characterized species is snRlO (29). The lack of detection of snoRNAs in immunoprecipitation experiments with anti-NSR1 antibody (data not shown) suggests, however, that NSR1 is not a snoRNP protein and may bind another species of RNA. Although NSRI, GAR1, NOPI, and SSBJ do appear to belong to the same family of genes containing a GAR domain (11), the overall structure of the NSR1 gene is different from those of the other genes. For example, like mammalian nucleolin (which also contains a GAR domain), NSR1 contains multiple RNA recognition motifs; nucleolin has four and NSR1 has two (21). Thus, NSR1 may recognize longer RNA species, possibly rRNAs. In addition, both NSR1 and nucleolin possess N-terminal domains that are highly acidic and specifically recognize NLS (37). The potential ability of NSR1 to recognize the NLS of nuclear proteins through the NLS-binding site and its multiple RNA recognition motifs may suggest a function that does not overlap those of the other characterized yeast nucleolar proteins. By way of its recognition of NLS and its multiple RNA recognition motifs, NSR1 may be involved in the formation of pre-rRNA particles in a direct fashion by facilitating the interaction between ribosomal proteins (containing NLS) and pre-rRNA in the nucleolus. An alternative is that NSR1 may play an indirect role in the formation of pre-RNA particles by regulating the nuclear import of ribosomal proteins or other components that are essential for the formation of preribosomal particles. In addition to the pre-rRNA processing defects seen in the nsrl mutant, we were also able to detect differences in the steady-state distribution of ribosomal subunits. We observed virtually no free pool of 40S subunits, and at the same time we also observed a large increase in the size of the free pool of 60S subunits. The concentration of ribosomes within yeast cells seems to be correlated with the growth rate (35), although in a less pronounced manner than is the case in Escherichia coli. Therefore, the lack of free 40S subunits makes it likely that the production of 40S subunits is the rate-limiting step in the growth of the nsrl mutant. Another mutant (ubi3) with a mutation in a nonessential yeast gene has been described, and it also lacks free 40S subunits and has defects in pre-rRNA processing (10). However, UBI3 encodes a ribosomal protein that is a component of the missing 40S subunits, whereas there is no evidence that NSR1 is a component of the mature 40S subunits. To study further the nature of the ribosomal subunit imbalance, we tested antibiotics that affect different steps in protein translation. The nsrl mutant was more sensitive to paromomycin than the NSRJ + strain. Sensitivity to paromomycin and other aminogylcoside antibiotics has been observed for the rrpl mutant, which has a defect in the processing of 27S to 25S rRNA (9). Paromomycin causes misreading of the RNA codons in vitro (7) and suppression of the mutant phenotypes of many nonsense or missense mutations in E. coli (12). In a more recent report, paromomycin was shown to disturb protein synthesis by interacting with the 16S rRNA in the small ribosomal subunit of E. coli (8). This antibiotic can also increase misreading in eukaryotic translation systems or produce phenotypic suppression of nonsense mutations in yeast cells (24, 28). Thus, the sensitivity of the nsrl mutant to paromomycin can be a result of the low number of 40S subunits. If the number of 40S subunits in the nsrl mutant is already the rate-limiting step in its growth, any circumstance that would cause further malfunction of the same subunits, e.g., by binding to paromomycin, would be expected to result in a lethal phenotype.
Another possibility is that the 40S subunits that are made are not assembled properly. In this case, the defective subunits are capable of protein synthesis but are sensitive to lower concentrations of the drug. The paromomycin-sensitive phenotype will enable us to search for extragenic suppressors. Through studies of such suppressors, we expect to identify proteins that interact or are functionally redundant with NSR1.
ACKNOWLEDGMENTS We thank Alberto L. Mancinelli, of our department, for graciously loaning us his ISCO gradient fractionator to carry out our studies. Special thanks are due to John Woolford and his laboratory staff at Carnegie-Mellon University, Pittsburgh, Pa., for their helpful discussions on obtaining ribosomal profiles from S. cerevisiae. We also thank John Woolford, Cathy Squires, Jim Manley, and members of our laboratory group for critical reading of the manuscript. This research was supported by an NSF Presidential Young Investigator Award to T. M. (DCB-88-58613) and a National Institutes of Health grant, also awarded to T. M. (GM44901-01).
REFERENCES 1. Andrew, C., A. K. Hopper, and B. D. Hall. 1976. A yeast mutant defective in the processing of 27S rRNA precursor. Mol. Gen. Genet. 144:29-35. 2. Baim, S. B., D. F. Pietras, D. C. Eustice, and F. Sherman. 1985. A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c. Mol. Cell. Biol. 5:1839-1846. 3. Borer, R. A., C. F. Lehner, H. M. Eppenberger, and E. A. Nigg. 1989. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56:379-390. 4. Bouche, G., M. Caizergues-Ferrer, B. Bugler, and F. Amalric. 1984. Interrelations between the maturation of a 1OOkDa nucleolar protein and pre rRNA synthesis in CHO cells. Nucleic Acids Res. 12:3025-3035. 5. Carter, C. J., and M. Cannon. 1980. Maturation of ribosomal precursor RNA in Saccharomyces cerevisiae. A mutant with a defect in both the transport and terminal processing of the 20S species. J. Mol. Biol. 143:179-199. 6. Clark, M. W., M. L. R. Yip, J. Campbell, and J. Abelson. 1990. SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolarspecific, silver-binding protein that is associated with the snRlO and snRll small nuclear RNAs. J. Cell Biol. 111:1741-1751. 7. Davies, J., L. Gorini, and B. D. Davis. 1965. Misreading of RNA codewords induced by aminoglycoside antibiotics. Mol. Pharmacol. 1:93-106. 8. De Stasio, E. A., D. Moazed, H. F. Noller, and A. E. Dahlberg. 1989. Mutations in 16S ribosomal RNA disrupt antibiotic-RNA interactions. EMBO J. 8:1213-1216. 9. Fabian, G. R., and A. K. Hopper. 1987. RRP1, a Saccharomyces cerevisiae gene affecting rRNA processing and production of mature ribosomal subunits. J. Bacteriol. 169:1571-1578. 10. Finley, D., B. Bartel, and A. Varchavsky. 1989. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature (London) 338: 394-401. 11. Girard, J.-P., H. Lehtonen, M. Caizergues-Ferrer, F. Amalric, D. Tollervey, and B. Lapeyre. 1992. GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast. EMBO J. 11:673-682. 12. Gorini, L. 1974. Streptomycin and misreading of the genetic code, p. 791-803. In M. Nomura, T. Tissieres, and P. Lengyel (ed.), Ribosomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 13. Henriquez, R., G. Blobel, and J. P. Aris. 1990. Isolation and sequencing of NOPI. J. Biol. Chem. 265:2209-2215. 14. Herrera, A. H., and M. 0. J. Olson. 1986. Association of protein
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20. 21.
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23. 24.
C23 with rapidly labeled nucleolar RNA. Biochemistry 25:62586264. Hughes, J. M. X., and M. Ares, Jr. 1991. Depletion of U3 small nucleolar RNA inhibits cleavage in the 5' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA. EMBO J. 10:4231-4239. Jong, A. Y.-S., M. W. Clark, M. Gilbert, A. Oehm, and J. L. Campbell. 1987. Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins. Mol. Cell. Biol. 7:2947-2955. Klootwijk, J., P. de Jonge, and R. J. Planta. 1979. The primary transcript of the ribosomal repeating unit in yeast. Nucleic Acids Res. 6:27-39. Kohrer, K., and H. Domdey. 1991. Preparation of high molecular weight RNA, p. 398-405. In C. Guthrie and G. R. Fink (ed.), Guide to yeast genetics and molecular biology. Academic Press, Inc., New York. Lapeyre, B., H. Bourbon, and F. Amalic. 1987. Nucleolin, the major nucleolar protein of growing eukaryotic cells: an unusual protein structure revealed by the nucleotide sequence. Proc. Natl. Acad. Sci. USA 84:1472-1476. Lee, W.-C., and T. Melese. 1989. Identification and characterization of a nuclear localization sequence-binding protein in yeast. Proc. Natl. Acad. Sci. USA 86:8808-8812. Lee, W.-C., Z. Xue, and T. Melese. 1991. The NSRI gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs. J. Cell Biol. 113:1-12. Li, H. V., J. Zagorski, and M. J. Fournier. 1990. Depletion of U14 small nuclear RNA (snR128) disrupts production of 18S rRNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 10:11451152. Moritz, M., A. G. Paulovich, Y.-F. Tsay, and J. L. Woolford, Jr. 1990. Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly. J. Cell Biol. 111:2261-2274. Palmer, E., J. M. Wilhelm, and F. Sherman. 1979. Phenotypic suppression of nonsense mutants in yeast by aminoglycoside
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antibiotics. Nature (London) 277:148-150. 25. Rothstein, R. J. 1983. One-step gene disruption in yeast. Methods Enzymol. 101:202-211. 26. Schimmang, T., D. Tollervey, K. Hildegard, F. Rainer, and E. C. Hurt. 1989. A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 8:4015-4024. 27. Shulman, R. W., and J. R. Warner. 1978. Ribosomal RNA transcription in a mutant of S. cerevisiae defective in ribosomal protein synthesis. Mol. Gen. Genet. 161:221-223. 28. Singh, A., D. Ursic, and J. Davies. 1979. Phenotypic suppression and misreading in Saccharomyces cerevisiae. Nature (London) 277:146-148. 29. Tollervey, D. 1987. A yeast small nuclear RNA is required for normal processing of pre-ribosomal RNA. EMBO J. 6:41694175. 30. Tollervey, D., H. Lehtonen, M. Carmo-Fonseca, and E. C. Hurt. 1991. The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast. EMBO J. 10:573583. 31. Trapman, J., J. Retel, and R. J. Planta. 1975. Ribosomal precursor particles from yeast. Exp. Cell Res. 90:95-104. 32. Udem, S. A., and J. R. Warner. 1972. Ribosomal RNA synthesis in Saccharomyces cerevisiae. J. Mol. Biol. 65:227-242. 33. Udem, S. A., and J. R. Warner. 1973. The cytoplasmic maturation of a ribosomal precursor ribonucleic acid in yeast. J. Biol. Chem. 248:1412-1416. 34. Veinot-Drebot, L. M., R. A. Singer, and G. C. Johnston. 1988. Rapid initial cleavage of nascent pre-rRNA transcripts in yeast. J. Mol. Biol. 199:107-113. 35. Waldron, C., and F. Lacroute. 1975. Effect of growth rate on the amounts of ribosomal and transfer ribonucleic acids in yeast. J. Bacteriol. 122:855-865. 36. Warner, J. R., and S. A. Udem. 1972. Temperature sensitive mutations affecting ribosome synthesis in Saccharomyces cerevisiae. J. Mol. Biol. 65:243-257. 37. Xue, Z., X. Shan, B. LaPeyre, and T. M6lese. Unpublished data.
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0270-7306/92/093865-07$02.00/0
Copyright X) 1992, American Society for Microbiology
NSR1 Is Required for Pre-rRNA Processing and for the Proper Maintenance of Steady-State Levels of Ribosomal Subunits WEN-CHING LEE, DAN ZABETAKIS, AND TERI MELESE* Department of Biological Sciences, Columbia University, New York, New York 10027 Received 12 May 1992/Accepted 18 June 1992
NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsrl mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsrl mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.
(11, 15, 22, 30). When yeast strains were depleted of these nucleolar proteins or snoRNAs, they all displayed the same phenotype; the processing of 35S pre-rRNA was impaired and the amount of 18S rRNA was dramatically reduced. Although it is not clear what role these nucleolar components play in the processing of rRNA, it has been suggested that they may function together as small nucleolar ribosomal particles (snoRNPs) (11, 30). Other evidence supports the notion that the assembly of newly formed ribosomal proteins on the pre-rRNA is also essential for the maturation of yeast pre-rRNA (27, 36). Therefore, a defect in pre-rRNA processing can result from improper assembly of the preribosomal particle as well as from blockage at the specific cleavage
In eukaryotic cells, the nucleolus is a specialized subcompartment in which ribosome biogenesis occurs. Pre-rRNA first is transcribed from rDNA genes located in the nucleolus and then undergoes a series of modifications. Ribosomal proteins are imported from the cytoplasm and packed onto the RNA molecule. At the same time, the primary transcript goes through sequential cleavages to generate mature forms of rRNA. Finally, the newly formed ribosomal particles are exported to the cytoplasm. In the yeast Saccharomyces cerevisiae, the largest detectable transcript from the rDNA genes is a 35S pre-rRNA (17), which is rapidly processed into three molecules: the 18S rRNA assembled into 40S ribosomal subunits and the 5.8S and 25S rRNAs found in 60S subunits (Fig. 1). During or immediately after transcription of the rDNA genes, the pre-rRNA is modified, mainly by methylation (32, 34). Meanwhile, a large number of ribosomal proteins and nonribosomal components (including proteins and RNA) associate with the pre-rRNA to form a 90S preribosomal particle. This particle is then split into 66S and 43S preribosomal particles, which are the precursors of the 60S and 40S subunits, respectively (31). While the 66S particles mature completely within the nucleus, the final maturation steps of the 43S particles, including the processing of 20S to 18S rRNA, are completed in the cytoplasm (31, 33). Only a few mutations that block specific steps in the pre-rRNA processing pathway have been found. One example is a temperature-sensitive mutation in a gene designated RRPJ that was shown to prevent the 27S-to-25S rRNA cleavage step specifically (1). In another mutant, CLP-8, the efficiency of the processing of 20S to 18S rRNA is greatly reduced, apparently because of a defect in 20S pre-rRNA export (5). Recently, two nucleolar proteins, NOP1 and GAR1, and two small nucleolar RNAs (snoRNAs), U3 and U14, were shown to be required for pre-rRNA processing
*
steps.
Ribosomal proteins
are
translated in the cytoplasm and
must be imported into the nucleus for assembly. From these ribosomal proteins and the rRNA components, ribosomal particles are then assembled in the nucleolus and exported to the cytoplasm to carry out the process of protein synthesis.
The tremendous flux of ribosomal proteins and ribosomal particles across the nuclear envelope likely accounts for a large portion of the nucleocytoplasmic traffic in exponentially growing cells. However, little is known about this import-export process. In recent years, two mammalian nucleolar proteins, nucleolin and No38, were shown to shuttle between the nucleus and the cytoplasm (3). Nucleolin, a major nucleolar protein in mammalian cells, has been thought to play a role in ribosome biogenesis (4, 14, 19). The phenomenon of shuttling raised the possibility that nucleolin participates in the nucleocytoplasmic transport of ribosomal components. In searching for proteins involved in the initial stage of protein import into the nucleus, we have identified a 67-kDa yeast protein that specifically recognizes nuclear localization sequences (NLS) (20). The gene that encodes this 67-kDa protein was cloned and named NSRI, for nuclear signal recognition protein (21). Surprisingly, this NLS-binding protein was localized to the nucleolar region by indirect immu-
Corresponding author. 3865
3866
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3Y
so
:
20S I
Ay
a I 18S
5.8S
25S
FIG. 1. Major pre-rRNA processing pathway in S. cerevisiae. The 35S pre-rRNA is the largest detectable transcript from the rRNA operon. The 32S, 27S, and 20S pre-rRNAs are the major processing intermediates. Processing from 20S to 18S rRNA is carried out in the cytoplasm. The 18S rRNA is found in the 40S ribosomal subunits, while the 5.8S and 27S rRNAs are hydrogen bonded together in the 60S ribosomal subunits. nofluorescence (21) and contains a glycine- and arginine-rich (GAR) domain, a common structural motif among the nucleolar proteins sequenced so far (11). Moreover, the NSR1 protein bears a strong resemblance to nucleolin in domain structure, but without a high degree of amino acid sequence similarity. Both proteins contain a GAR domain at their C termini, and both contain consensus RNA recognition motifs; nucleolin has four such motifs, while NSR1 has two. In particular, nucleolin and NSR1 contain N termini with serine stretches flanked by acidic amino acid residues, which are not present in the previously identified yeast nucleolar proteins (SSB1, GAR1, and NOP1) (11, 13, 16, 26). the structural similarity between NSR1 and nuclesb1k,'i hammalian nucleolin gene expressed in yeast cells a*nplent the slow-growth phenotype of an nsrl -e muIWtP'. IXi the same study, it was also found that nucIffl NSR1, is an NLS-binding protein; the recognitift tbe NLS occurs in the N-terminal regions of both '° ioteii1:)n the basis of features shared with nucleolin and tsloition in the nucledit1t, it is reasonable to suspect that NSR1 rnay be involved in ribosome biogenesis. We present evidence here to show that NSR1 is required not only for normal pre-rRNA processing but also for the
normal distribution of free ribosomal subunits. To assess the
physiological significance of this ribosome imbalance, we tested the nsrl mutant for sensitivity to a variety of drugs known to interfere with ribosome function. In comparison with the wild type, the only difference observed was in sensitivity to paromomycin. Paromomycin causes misreading of the RNA codons in vitro for both prokaryotic and eukaryotic systems (7, 24, 28). We have found that the nsrl mutant cannot grow at concentrations of paromomycin at which the growth of the wild-type strain is unaffected. MATERIALS AND METHODS Yeast strains. W303-1A (MALTa ade2-1 canl-100 ura3-1 leu2-3,112 trpl-1 his3-11,15) and WLY353 (same as W303-
1A, except nsrl::HIS3) with plasmid pBM272 were used in the [3H]uracil labelling experiment. pBM272 is a yeast plasmid derived from YCp5O, which carries the URA3 gene. Pulse-chase labelling of pre-rRNA. Yeast cells were cultured in glucose minimal medium lacking either methionine or uracil, as determined by the labelling reagent to be used in the experiment. Cells were first grown at 30°C to an optical density at 600 nm (OD6.) of 0.5 to 1.0 and then switched to room temperature for 1 h before being pelleted and resuspended in 1/10 the original volume. Each milliliter of cells was labelled with 30 ,Ci of [methyl-3H]methionine (NEN) or [3H]uracil (NEN) at room temperature for 1 min and then chased with cold methionine at a final concentration of 5 mM or with uracil at a final concentration of 240 ,ug/ml, respectively. At various times after the chase, 1-ml aliquots were withdrawn and placed in tubes containing crushed ice to stop the reaction. The cells were then collected by centrifugation and resuspended in 1 ml of RNA isolation buffer (100 mM NaCl, 50 mM sodium acetate [pH 5.3], 1 mM EDTA), and total RNA was extracted. Extraction and analysis of RNA. Total RNA was extracted by the method of Kohrer and Domdey (18) with modifications. Cells in 1 ml of RNA isolation buffer were mixed with 50 ,ul of 20% sodiutn dodecyl sulfate and 1 ml of 65°C phenol and immediately shaken vigorously in a 65°C water bath for 10 min. The tube was then quickly chilled in a dry ice bath and centrifuged to separate the aqueous phase from the phenol. The phenol was removed from the tube, and the aqueous phase with the cells was extracted with phenol again as described above. After centrifugation, the aqueous phase was transferred to a new tube and extracted twice with 25:24:1 phenol-chloroform-isoamyl alcohol and once with 24:1 chloroform-isoamyl alcohol. RNA was precipitated with ethanol and redissolved in diethylpyrocarbonatetreated water. The concentration of RNA was estimated by measuring the A260, and it was assumed that 1 A260 unit equals 40 p,g of RNA. RNA was separated in 1.2% agarose gels containing 6% formaldehyde, 50 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES) (pH 7.8), and 1 mM EDTA. The gels were stained with 5 ,ug of ethidium bromide per ml after electrophoresis. To detect 3H labelling, we soaked the gels in Amplify (Amersham) for 1 h, rinsed them with water, and dried them. The dried gels were exposed to XAR5 film. The intensities of the bands on the fluorograms (see Fig. 2) or the positive images of the ethidium bromide-stained gels (see Fig. 3A) were quantitated with a Bio Image Visage 110 image quantification system. Ribosomal profiles. Ribosomes were prepared essentially as described by Baim et al. (2). Yeast cells were grown to an OD6. of 0.2 to 0.8 at 30°C in 200 ml of YPD (1% yeast extract, 2% peptone, 2% glucose) (rich medium). Crushed ice and 10 mg of cycloheximide were placed in a 500-ml centrifuge tube. The yeast culture was poured into the tube, shaken, and immediately pelleted in a GS-3 rotor (DuPont) at 4,000 x g. The cells were resuspended in 10 ml of buffer A (10 mM Tris [pH 7.4], 100 mM NaCl, 30 mM MgCl2, 50 p,g of cycloheximide per ml, and 200 ,ug of heparin per ml in diethylpyrocarbonate-treated water) and repelleted. This step was repeated, and the cells were resuspended in 0.5 ml of buffer A and transferred into a 15-ml plastic centrifuge tube. Approximately 0.25 ml of glass beads was added, and the cells were lysed by 10 alterations of 15 s of vortexing and 30 s of cooling in an ice bucket. Buffer A was then added to bring the total volume to about 4 ml. Cell debris and glass beads were removed by centrifugation for 5 min at 3,600 x g
NSR1 IS REQUIRED FOR PRE-rRNA PROCESSING
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A
Chase (min)
B
[3H]-methionine label
nsrl -
0 0.5 1.5 6
0 0.5 1.5 6
35Sk_
[3H]-uracil label
NSR1+
NSR1
Chase(min)
__ _
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1
5 10 20 0 1
nsrl1 5 10 20
35Sb 32S"
27S vE _" *m* a 25S, 0
20S) 18S'
emw
lw
27S25S.
*104 _4
20S 18St
FIG. 2. Pulse-chase labelling of pre-rRNA from the wild-type strain and the nsrl mutant. Wild-type (NSR1+) and nsrl mutant (nsrl-) cells were pulse-labelled with [methyl-3H]methionine (A) or [3H]uracil (B) for 1 min at room temperature and chased with a large excess of unlabelled methionine or uracil, respectively. Aliquots were withdrawn at various times after the chase, as indicated at the top of each lane. Total RNA was isolated from the aliquots and analyzed by agarose-formaldehyde gel electrophoresis and then by fluorography. The positions of the major pre-rRNA species (35S, 32S, 27S, and 20S) as well as 25S and 18S rRNAs are marked.
in an SA-600 rotor (DuPont). The supernatant was further clarified by another round of centrifugation at 10,000 x g for 10 min. This supernatant was divided into aliquots and frozen at -70°C. No differences were detected between samples frozen in this manner and fresh samples analyzed immediately. Ribosomes were analyzed by centrifugation through a sucrose step gradient. The sucrose solution contained 50 mM Tris-acetate (pH 7.0), 50 mM NH4C1, 12 mM MgCl2, 1 mM dithiothreitol, and 7, 17, 27, 37, or 47% sucrose. The gradient was prepared by pipetting 7 ml of each layer onto the bottom of a 40-ml open-top centrifuge tube. The ribosomal sample was loaded on top, and the gradient was centrifuged at 100,000 x g and 4°C for 4 h in an SW27 rotor (Beckman). The sample concentration was measured as the OD260. Approximately 40 OD260 units per ml was loaded on each gradient. Finally, the gradient was extracted and analyzed with an ISCO model 640 gradient fractionator, with monitoring of the effluent at OD254. Drug sensitivity assay. Yeast cells were grown to the stationary phase in YPD and diluted to an ODwo of 0.3. The cells (200 ,ul) were spread on YPD plates. Fifty microliters of various concentrations of paromomycin, cycloheximide, streptomycin, or erythromycin (each from Sigma) was pipetted onto sterile filter disks (diameter, 3/8 in. [ca. 1 cm]; Schleicher & Schuell). After the solutions were absorbed, the filter disks were placed on top of the spread cells. The plates were incubated at 30°C for 5 to 6 days and then photographed. RESULTS Pre-rRNA processing in the nsrl mutant. In previous studies in our laboratory, a disruption of the chromosomal copy of the NSR1 gene was carried out in diploids by the one-step gene disruption method (25). Two segregants from each tetrad containing the disrupted NSRI gene (nsrl ::HIS3)
grew slower than two segregants containing the wild-type copy of the NSRI gene (21). Thus, NSR1 is required for normal cell growth but is not essential for cell viability. The slow growth of the nsrl mutant enabled us to examine how growing cells functioned in the absence of NSR1. The nucleolar localization of NSR1 and the resemblance to nucleolin prompted us to test whether the protein is involved in any aspect of ribosome biogenesis. First, prerRNA processing in the nsrl mutant was investigated by pulse-chase labelling experiments. Both the wild-type (NSR1 +) strain and the nsrl mutant were grown to log phase at 30°C before being switched to room temperature for 1 h to slow down cellular metabolism. Cells were labelled with [methyl-3H]methionine for 1 min and then chased with large amounts of cold methionine. Aliquots were withdrawn at various times after the chase, total RNA was extracted, and the samples were analyzed by agarose gel electrophoresis and then by fluorography (Fig. 2A). Although the nsrl mutant grows much more slowly than the wild-type strain under optimal growth conditions, the doubling time of the nsrl mutant under the conditions used for pulse-chase labelling (synthetic minimal medium, room temperature) is nearly identical to that of the wild-type strain, enabling us to compare the pre-rRNA processing rates directly. In the NSR+ strain, pre-rRNA methylation and processing occurred rapidly, and two major processing intermediates, 27S and 20S pre-rRNAs, were observed by the end of the 1-min labelling period. The final products, the 18S and 25S rRNA species, appeared in an approximately 1:1 ratio, as predicted from the processing pathway (see Fig. 1 for the pathway). In comparison, processing was less efficient in the nsrl mutant. The amounts of 32S and 20S pre-rRNAs over the same times as the NSR1+ strain were dramatically reduced (Fig. 2A); consequently, the final amount of 18S rRNA was greatly affected, since the 20S pre-rRNA is the direct precursor of the 18S rRNA. The ratio of the labelled 18S and 25S rRNAs
3868
LEE ET AL.
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A
A
A NSR1+ nsrlRNA (Rg) 7.6 3.8 7.6 3.8
25S)-
18S
B
U
0
I*
a
0
60S
25S
E
E 18S
B 2.0Or
'I)
7.6
3.8
7.6
3.8
NSR1+ nsr1 FIG. 3. Analysis of the steady-state levels rRNAs in the wildtype strain and the nsrl mutant. Yeast cells (10 ml) were grown in YPD to an OD600 of 1.0. Total RNA was extracted and quantitated by measuring the A260 (as described in Materials and Methods). RNA (7.6 or 3.8 jig) from the wild-type strain (NSR1+) or the nsrl mutant (nsrl-) was separated by agarose-formaldehyde gel electrophoresis and is marked at the top of the lanes as 7.6 or 3.8. (A) Ethidium bromide staining of the 25S and 18S rRNA species. The intensities of the RNA bands on the positive image of the stained gel were quantitated with a Bio Image Visage 110 image quantification system. (B) The relative amounts of the 25S and 18S rRNA species were plotted.
approximately 1:2 after 6 min of chasing (the quantitation method is described in Materials and Methods). To exclude the possibility that the observed phenomenon in the nsrl mutant was merely due to a change in the normal methylation pattern of the rRNA, we measured pre-rRNA processing in the presence of [3H]uracil as the labelling reagent. In this experiment, longer chase times were used. Aside from the fact that the background was higher because nascent pre-rRNA transcripts and mRNA are also labelled, the results of these experiments (Fig. 2B) were basically identical to those shown in Fig. 2A. In the nsrl mutant, 35S pre-rRNA accumulated, 32S and 20S pre-rRNAs almost disappeared, and the amount of 18S rRNA was less than one-half that of 25S rRNA after 20 min of chasing with cold uracil. Steady-state levels of rRNAs. The kinetic studies discussed above demonstrate that the nsrl mutant is defective in making 18S rRNA, resulting in an altered ratio of newly formed 25S and 18S rRNAs. To test whether this kinetic alteration was reflected at steady-state levels, we compared the major rRNAs in the wild-type strain and the nsrl mutant. Total RNA was isolated from both strains, separated by agarose-formaldehyde gel electrophoresis, and then stained with ethidium bromide. The stained gel is shown in Fig. 3A, and quantitation and comparison of the two rRNA bands are shown in Fig. 3B. The results are consistent with the findings
was
In a
1,.0 _
0
top
bottom
FIG. 4. Ribosomal profiles of the wild-type strain and the nsrl mutant. Yeast cells were grown in YPD to mid-log phase. Ribosomes and ribosomal subunits were isolated and separated on a 7 to 47% sucrose gradient as described in Materials and Methods. The gradient was analyzed with an ISCO model 640 gradient fractionator by monitoring OD254. The OD scale is approximate. (A) Ribosomal profile of the wild-type strain. (B) Ribosomal profile of the nsrl mutant. The top and bottom of the gradient and the ribosomes and subunits are marked. The numbered peaks are polysomes containing the indicated numbers of ribosomes.
described for the pulse-chase labelling experiments. The ratio of 18S to 25S rRNAs in the nsrl mutant was different from that in the wild-type strain, and the amount of 18S rRNA was reduced. Ribosomal profiles in the nsrl mutant. The observation that the nsrl mutant was defective in its ability to produce 18S rRNA made us consider whether this defect was reflected in the composition of ribosomal subunits in the cells. If a cell cannot efficiently produce 18S rRNA, it may display this phenotype as a lack of 40S ribosomal subunits. It has been found that a deficiency in the production of a ribosomal protein results in the failure to assemble the subunit of which that protein is a component. Subsequently, this failure is observed as a deficiency of that subunit relative to the other subunit (23). Ribosomes were prepared as described by Baim et al. (2). Yeast cells were grown to the mid-log phase and harvested in the presence of cycloheximide. The cells were lysed with glass beads, and the supernatant was separated on a 7 to 47% sucrose gradient. The gradient was fractionated and moni-
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DISCUSSION
FIG. 5. Paromomycin sensitivity of the wild-type strain (top) and the nsrl mutant (bottom). The wild-type strain and the nsrl mutant were spread on YPD plates. Filter disks containing 10, 2, and 0.4 mg of paromomycin sulfate (labelled 1, 2, and 3, respectively) were placed on each plate. The cells were allowed to grow at 30°C for 5 days.
tored by measuring theA254. The results for both the NSR1I strain and the nsrl mutant are shown in Fig. 4. Compared with the NSR1+ strain, the nsrl mutant had little or no free 40S ribosomal subunits and a large surplus of free 60S subunits. There was essentially no difference in the amount and distribution of polysomes between the two strains. This result is consistent with the hypothesis that NSR1 is involved in the production of 40S ribosomal subunits. The nsrl mutant is sensitive to paromomycin. In wild-type strains, the two ribosomal subunits are present in equimolar quantities, but this balance is altered in the absence of NSR1. Since ribosomes are the machinery that carries out protein synthesis, we were curious to know whether this ribosomal imbalance affects protein synthesis in the nsrl mutant. To address this question, we tested the sensitivity of the nsrl mutant to a variety of drugs known to interfere with translation: cycloheximide, paromomycin, streptomycin, and erythromycin. Cultures of wild-type or nsrl mutant cells were spread on YPD plates, and sterile filter disks with various amounts of the drugs were placed on top of the spread cells. If the yeast cells are sensitive to the drug in the filter, their growth will be inhibited and a clear halo will form around the filter. The degree of sensitivity to the drug can be estimated by the size of the halo. Both strains were sensitive to cycloheximide to the same degree, and neither of the strains was sensitive to up to 10 mg (in the filter) of erythromycin or streptomycin (data not shown). The wild-type strain was quite resistant to paromomycin and did not form any sizeable halo around the filter, even when exposed to the highest amount of paromomycin (10 mg) used in this test. The nsrl mutant was extremely sensitive to this antibiotic, forming a significant halo around a filter containing only 2 mg of paromomycin (Fig- 5).
We have found the nsrl mutant to be defective in the processing of pre-rRNA, such that the production of 18S rRNA is reduced. Furthermore, the amount of free 40S ribosomal subunits, of which 18S rRNA is the only RNA component, is greatly reduced compared with that in the wild-type strain. In fact, in the nsrl mutant, there are no detectable free 40S ribosomal subunits in the cytosol, whereas a large excess of free 60S subunits is accumulated. Other investigators studying different yeast nucleolar proteins (GAR1 and NOP1) have also observed a defect in pre-rRNA processing in strains depleted of these proteins (11, 30). In the case of GAR1 and NOP1, pre-rRNA processing is slowed and 18S rRNA is deficient, and the authors suggest that an alternative processing pathway that results in the appearance of a new species, 23S RNA, occurs. The same phenotype has also been observed in strains depleted of snoRNAs U3 and U14 (15, 22). The phenotype observed for a strain lacking NSRJ, a nonessential gene, is essentially identical to that observed for strains depleted of the products of the essential genes NOPI, GAR1, U3, and U14. The processing of the 35S pre-rRNA is delayed, as evidenced by its accumulation. When the 35S pre-rRNA is finally processed, it does not form equimolar amounts of 20S and 27S pre-rRNAs, as observed in wild-type cells. Instead, the amount of the 20S pre-rRNA is greatly reduced and, consequently, the amount of the 18S rRNA is decreased. The decrease in the amount of the 20S rRNA may result from either degradation of the newly formed 20S pre-rRNA or degradation of the 5' region of the 35S pre-rRNA (where the 20S pre-rRNA resides). It was previously suggested that the 5' end of the 35S prerRNA is unstable in the absence of some nucleolar proteins or snoRNAs (22, 30). We also prefer the explanation of the instability of the 5' end of the 35S pre-rRNA because the accumulation of the 35S pre-rRNA and the decrease in the amounts of the 32S and 20S pre-rRNAs (Fig. 2) indicate that the cleavage steps between the 35S pre-rRNA to the 20S and 27S rRNAs are blocked or delayed. Specifically, we suggest that before a reasonable amount of 20S rRNA can be produced from the 35S pre-rRNA, a period of rapid degradation removes the 5' end of the 35S pre-rRNA, completely or partially, up to the cleavage site that normally separates the 20S from the 27S rRNA. Since we observed no smearing between the 35S pre-rRNA and 27S rRNA bands, the truncated pre-rRNA is rapidly cleaved into a normal 27S rRNA, and processing can proceed normally to produce the 5.8S and 25S rRNAs. Our observation that pre-rRNA processing does occur, albeit abnormally, in the nsrl mutant indicates that the blockage of 35S pre-rRNA processing discussed above is not absolute. Thus, cells are able to bypass the need for NSR1 function. An explanation for this could be that NSR1 is functionally redundant in cells. However, we did not detect a second copy of NSR1. When probes derived from various regions of NSR1 were used in a low-stringency Southern blot analysis, no specific hybridization was observed (data not shown). The fact that the depletion of several different nucleolar components results in the same phenotype has prompted investigators to propose the existence of a snoRNP or snoRNPs containing nucleolar proteins and snoRNA components required for proper pre-rRNA processing (11, 30). This suggestion is supported by observations that antibodies against several different nucleolar proteins (GAR1, NOP1,
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and SSB1) coimmunoprecipitate an overlapping set of snoRNAs (6, 11, 30). One well-characterized species is snRlO (29). The lack of detection of snoRNAs in immunoprecipitation experiments with anti-NSR1 antibody (data not shown) suggests, however, that NSR1 is not a snoRNP protein and may bind another species of RNA. Although NSRI, GAR1, NOPI, and SSBJ do appear to belong to the same family of genes containing a GAR domain (11), the overall structure of the NSR1 gene is different from those of the other genes. For example, like mammalian nucleolin (which also contains a GAR domain), NSR1 contains multiple RNA recognition motifs; nucleolin has four and NSR1 has two (21). Thus, NSR1 may recognize longer RNA species, possibly rRNAs. In addition, both NSR1 and nucleolin possess N-terminal domains that are highly acidic and specifically recognize NLS (37). The potential ability of NSR1 to recognize the NLS of nuclear proteins through the NLS-binding site and its multiple RNA recognition motifs may suggest a function that does not overlap those of the other characterized yeast nucleolar proteins. By way of its recognition of NLS and its multiple RNA recognition motifs, NSR1 may be involved in the formation of pre-rRNA particles in a direct fashion by facilitating the interaction between ribosomal proteins (containing NLS) and pre-rRNA in the nucleolus. An alternative is that NSR1 may play an indirect role in the formation of pre-RNA particles by regulating the nuclear import of ribosomal proteins or other components that are essential for the formation of preribosomal particles. In addition to the pre-rRNA processing defects seen in the nsrl mutant, we were also able to detect differences in the steady-state distribution of ribosomal subunits. We observed virtually no free pool of 40S subunits, and at the same time we also observed a large increase in the size of the free pool of 60S subunits. The concentration of ribosomes within yeast cells seems to be correlated with the growth rate (35), although in a less pronounced manner than is the case in Escherichia coli. Therefore, the lack of free 40S subunits makes it likely that the production of 40S subunits is the rate-limiting step in the growth of the nsrl mutant. Another mutant (ubi3) with a mutation in a nonessential yeast gene has been described, and it also lacks free 40S subunits and has defects in pre-rRNA processing (10). However, UBI3 encodes a ribosomal protein that is a component of the missing 40S subunits, whereas there is no evidence that NSR1 is a component of the mature 40S subunits. To study further the nature of the ribosomal subunit imbalance, we tested antibiotics that affect different steps in protein translation. The nsrl mutant was more sensitive to paromomycin than the NSRJ + strain. Sensitivity to paromomycin and other aminogylcoside antibiotics has been observed for the rrpl mutant, which has a defect in the processing of 27S to 25S rRNA (9). Paromomycin causes misreading of the RNA codons in vitro (7) and suppression of the mutant phenotypes of many nonsense or missense mutations in E. coli (12). In a more recent report, paromomycin was shown to disturb protein synthesis by interacting with the 16S rRNA in the small ribosomal subunit of E. coli (8). This antibiotic can also increase misreading in eukaryotic translation systems or produce phenotypic suppression of nonsense mutations in yeast cells (24, 28). Thus, the sensitivity of the nsrl mutant to paromomycin can be a result of the low number of 40S subunits. If the number of 40S subunits in the nsrl mutant is already the rate-limiting step in its growth, any circumstance that would cause further malfunction of the same subunits, e.g., by binding to paromomycin, would be expected to result in a lethal phenotype.
Another possibility is that the 40S subunits that are made are not assembled properly. In this case, the defective subunits are capable of protein synthesis but are sensitive to lower concentrations of the drug. The paromomycin-sensitive phenotype will enable us to search for extragenic suppressors. Through studies of such suppressors, we expect to identify proteins that interact or are functionally redundant with NSR1.
ACKNOWLEDGMENTS We thank Alberto L. Mancinelli, of our department, for graciously loaning us his ISCO gradient fractionator to carry out our studies. Special thanks are due to John Woolford and his laboratory staff at Carnegie-Mellon University, Pittsburgh, Pa., for their helpful discussions on obtaining ribosomal profiles from S. cerevisiae. We also thank John Woolford, Cathy Squires, Jim Manley, and members of our laboratory group for critical reading of the manuscript. This research was supported by an NSF Presidential Young Investigator Award to T. M. (DCB-88-58613) and a National Institutes of Health grant, also awarded to T. M. (GM44901-01).
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