University of Cincinnati College of Medicine, Department of Pathology & Laboratory Medicine, Cincinnati, OH 45267-0529, USA1
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA2
Author for correspondence: David S. Askew. Tel: +1 513 558 2395. Fax: +1 513 558 2141. e-mail: David.Askew{at}uc.edu
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ABSTRACT |
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Keywords: yeast, nucleolus, ribosome biogenesis
Abbreviations: ETS, external transcribed spacer; ITS, internal transcribed spacer
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INTRODUCTION |
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METHODS |
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Gel electrophoresis and blotting.
For analysis of gene expression, RNA was isolated from yeast cultures by disruption with 0·5 mm glass beads (Fisher Scientific) according to standard procedures. Twenty micrograms of total RNA was fractionated by formaldehyde gel electrophoresis as previously described (Li et al., 1997 ), transferred to positively charged nylon membranes (MSI) and hybridized to 32P-labelled DNA probes under stringent conditions in 50% (v/v) formamide/5xSSC (1xSSC is 0·15 M NaCl, 0·015 M sodium citrate, pH 7·6), 2x Denhardts solution, 10% (w/v) dextran sulfate, 1% (w/v) SDS. Hybridization intensity was quantified with a Phosphorimager (Molecular Dynamics) and normalized for loading by quantitating the relative levels of SYBR green (Molecular Probes) stained 18S rRNA on a Phosphorimager.
Genomic DNA was extracted by cell disruption in the presence of 0·5 mm diameter glass beads (Fisher Scientific) according to standard protocols. For genomic Southern blot analysis, 10 µg digested DNA was fractionated on a 1% (w/v) agarose gel, transferred to a nylon membrane (MSI) and hybridized to a [32P]dCTP-labelled probe under conditions of high stringency as previously described (Li et al., 1997 ). Hybridization was monitored using a Phosphorimager (Molecular Dynamics).
Construction of a cgr1 truncation mutant and a cgr1 depletion mutant.
A mutant expressing a C-terminal truncation of Cgr1p, YDA110, was constructed by one-step PCR-mediated gene disruption. The truncation cassette was obtained by PCR amplification of the kanamycin-resistance gene (kanr) using plasmid pFA6-3HA-kanMX6 (Wach et al., 1994 ) as the template and primers containing 50 bp CGR1-homologous sequences. The upstream primer was 5'-GATGAGAAGGAAGAAGCTCGTCAAGCTAAAAT-AACCATGTTAAAGTGAGGCGCGCCACTTCTAAA-3' and the downstream primer was 5'-TATGAGCCTTCTATAATGCTTTATACCATTGTGCTTATCCGAATTCG-AGCTCGTTTAAAC-3' (sequences in italic are specific to pFA6-3HA-kanMX6). This construction places a stop codon in place of amino acid 81. The wild-type haploid yeast strain cry1 (Table 1
) was transformed with the PCR product, selected on YPD plates containing 200 µg G418 ml-1 (Gibco-BRL) and the transformants were genotyped by PCR using a CGR1 primer outside the recombination site (5'-ATAACTGTCTAGGGATGCCC-3') and the kanr-specific primer (5'-ATCGGGCTTCCCATACAATC-3'). Confirmation of single-copy modification of CGR1 was obtained by genomic Southern blot analysis of EcoRI-digested genomic DNA using CGR1- and kanr-specific probes.
To generate a conditional CGR1 depletion strain, the wild-type haploid strain cry1 was transformed with pDA361 and the chromosomal CGR1 allele was deleted by transformation with a CGR1 disruption cassette. The disruption cassette was constructed by PCR amplification of the kanamycin-resistance gene (kanr) using plasmid pFA6-kanMX2 (Wach et al., 1994 ) as the template with primers containing 50 bp CGR1-homologous sequences. The upstream primer was 5'-ACTGTCCATATTCATTGAAAGCAAAATAAAACA-TAACCAGGAAATCAGCTGAAGCTTCGTACGC-3' and the downstream primer was 5'-CTGTCTAGGGATGCCCTTATTTTCTCCTTGTCAGGACTTAATAATGCATAG-GCCACTAGTGGATCTG-3' (sequences in italic are specific to pFA6-3HA-kanMX2). Following selection on G418 plates, the colonies were genotyped by PCR and deletion of the CGR1 ORF was confirmed by genomic Southern blot analysis, using radiolabelled probes specific for the CGR1 and kanr genes. Down-regulation of the tetO-CYC1 promoter in pDA361 was accomplished by supplementing the medium with 2 µg ml-1 of the tetracycline analogue doxycycline, as described by Gari et al. (1997)
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Pulsechase analysis of rRNA processing.
For the [methyl-3H]methionine pulsechase experiments, wild-type and YDA110 cells were grown to a density of (12)x107 cells ml-1 in methionine-free media at 25 °C. Wild-type cells containing pCM183 (Table 1) and YDA128 were grown to a density of (24) x107 cells ml-1 in methionine-free media at 25 °C. The cells were diluted into media containing 2 µg doxycycline ml-1 and cultured for 29 h. A total of 108 cells was concentrated to a volume of 1 ml and pulse labelled with 250 µCi (9·25 MBq) [methyl-3H]methionine for 2 min (7085 Ci mmol-1, 5 mCiml-1, 185 MBq ml-1; Amersham-Pharmacia Biotech). For the zero time point, 250 µl cells was transferred to a new tube, washed in 1 ml ice-cold media and the cell pellet frozen on dry ice. Chase was initiated by adding unlabelled methionine to the culture at a concentration of 1 mg methionine ml-1. At time points of 2, 5 and 10 min of chase, 250 µl cells were removed and processed as described above. RNA was isolated by the hot acid phenol method (Ausubel et al., 1997
). A total of 20000 c.p.m. radioactivity was loaded per lane onto a 1·2% (w/v) agarose-formaldehyde gel. RNA was transferred to a nylon membrane (Hybond-N+, Amersham-Pharmacia), UV-cross-linked and sprayed with En3hance (NEN). The membrane was exposed to film for 1 day at -80 °C.
For [5,6-3H]uracil pulsechase experiments, wild-type and YDA110 cells containing pRS306 (URA3 CEN) were grown to a density of 1x107 cells ml-1 in media lacking uracil at 25 °C. A total of 5x108 cells was concentrated to a volume of 6 ml and labelled with 100 µCi (3·7 MBq) [5,6-3H]uracil (3050 Ci mmol-1, 1 mCi ml-1, 37 MBq ml-1; NEN). After labelling for 3 and 6 min, 1 ml culture was removed, centrifuged, supernatants removed, and cell pellets frozen. Chase was initiated by adding unlabelled uracil to a concentration of 20 mg uracil l-1. At time points of 5, 10, 30 and 60 min of chase (for the 6 min label), a 1 ml sample was processed as described above and 5 µg RNA from each time point was loaded onto a 8% polyacrylamide, 8 M urea gel. RNA was transferred to Hybond-N+ membranes by semi-dry electrophoresis (Owl Scientific) and visualized by fluorography as described above. The membrane was exposed to film for 15 days at -80 °C.
Polysome profile analysis.
Polysome profiles were performed as described by Kressler et al. (1997) with the following modifications. Cells were taken from the cultures used for the [methyl-3H]methionine pulsechase experiments described above, and the lysate (4 A260 units) was layered onto 10 ml linear 749% sucrose gradients. Samples were centrifuged in a Beckman SW41Ti rotor for 2 h at 39000 r.p.m. at 4 °C (Beckman Instruments). A Beckman fraction recovery system was used to pass the gradients through a Pharmacia UV-1 monitor to measure A254. Analysis of dissociated ribosomal subunits was performed as described by Kressler et al. (1997)
. Cell extracts were prepared in 50 mM Tris/HCl (pH 7·5), 50 mM NaCl, 1 mM DTT, and 2 absorbance units at 260 nm were layered onto 1030% sucrose gradients prepared in the same buffer and centrifuged as described above.
Subcellular localization of Nop1p and 5'-ITS1.
Nop1 was localized with anti-Nop1 monoclonal antibodies as previously described (Moy & Silver 1999 ). 5'-ITS1 rRNA was localized by fluorescence in situ hybridization as described by Amberg et al. (1992)
with the following modifications. An oligonucleotide complementary to the first 50 bases of ITS1 rRNA, 5'- ATGCTCTTGCCAAAACAAAAAAATCCATTTTC-AAAATTATTAAATTTCTT-3', was synthesized with the Cy3 fluorophore at its 5' end (IDT). Samples were hybridized with this oligonucleotide at a concentration of 50 nM.
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RESULTS |
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cgr1 mutants are hypersensitive to translation inhibitors
Yeast mutants deficient in ribosome assembly or function often display increased sensitivity to aminoglycoside antibiotics (Zanchin et al., 1997 ; Benard et al., 1998
; Ho & Johnson, 1999
; Dresios et al., 2000
). We have previously shown that a strain expressing CGR1 from a glucose-repressible promoter is hypersensitive to the aminoglycoside paromomycin under repressing conditions (Sun et al., 2001a
). To determine whether the cgr1 truncation mutant also confers increased susceptibility to translational inhibitors, the sensitivity of YDA110 cells to paromomycin and hygromycin was examined. Glass filter discs containing various concentrations of each antibiotic were placed onto the surface of a plate of confluent cells and cultured for 4 days at 30 °C. As shown by the zone of growth inhibition surrounding the filter paper discs, strain YDA110 was more sensitive to both of these antibiotics than wild-type cells (Fig. 4
), indicating that inhibitors of translational fidelity synergize with loss of Cgr1 function to inhibit growth.
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The defect in 27S pre-rRNA processing in YDA110 and YDA128 predicted that the formation of the 5·8S rRNA should also be adversely affected (Fig. 1). We therefore examined 5·8S rRNA synthesis in YDA110 cells by pulse labelling with [3H]uracil for 3 or 6 min followed by a chase with an excess of cold uracil for 5, 10, 30 and 60 min (for the 6 min pulse). In wild-type cells, both forms of the 5·8 S RNA were evident within 5 min of chasing (Fig. 7
), indicating a normal rate of processing. By comparison, the formation of the 5·8S rRNA was delayed in YDA110 cells (Fig. 7
), consistent with the defect in 27S pre-rRNA processing that was observed in this strain (Fig. 6
). The processing of the 7S pre-rRNA was also delayed in YDA110 (Fig. 7
), suggesting that Cgr1p may have a role in the processing of this intermediate into the mature 5·8S rRNA. The 5S rRNA is transcribed from a separate transcription unit by RNA polymerase III (Raué & Planta, 1991
) and although it forms a stable complex with rpL1, and is incorporated into the 60S subunit (Deshmukh et al., 1993
), this experiment showed no major defects in 5S rRNA synthesis in YDA110 cells (Fig. 7
).
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DISCUSSION |
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To investigate the possibility that the nucleolar localization of S. cerevisiae Cgr1p is due to its role in yeast ribosome synthesis, we have examined mutants deficient in Cgr1 function for abnormalities in ribosomal subunits and pre-rRNA processing. Each of the two cgr1 mutants generated for this study was deficient in the steady-state levels of 60S ribosome subunits, resulting in abnormally high levels of the 40S subunit and an increase in half-mer polysomes. Similar profiles have been described for mutants defective in components required for pre-rRNA processing or 60S subunit assembly (Ripmaster et al., 1992 ; Hong et al., 1997
; de la Cruz et al., 1998a
; Basu et al., 2001
), suggesting that Cgr1p is also involved in this pathway.
Analysis of pre-rRNA processing showed that the deficit in 60S subunits in the cgr1 mutants was attributable to a primary defect in 27S pre-rRNA processing, thereby affecting the synthesis of the mature 25S and 5·8S rRNAs. The role of Cgr1p in 25S/5·8S rRNA synthesis appears to be specific because synthesis of the 18S rRNA and 5S rRNAs was unaffected in either of the cgr1 mutants. This places Cgr1p in a category of proteins that influence the production of the 25S rRNA (Moritz et al., 1991 ; Russell & Tollervey, 1992
; Deshmukh et al., 1993
; Berges et al., 1994
; Fabian & Hopper 1997
; Hong et al., 1997
; Basu et al., 2001
). However, in addition to the major effects on the synthesis of the 25S/5·8S rRNAs, we also observed delayed processing of the 35S pre-rRNA and accumulation of the 7S intermediate. This suggests that Cgr1p may influence several steps in the pathway to ribosome synthesis, for which there is precedence among factors involved in pre-rRNA processing and ribosome assembly (Kressler et al., 1999
), although it is also conceivable that the effect is indirect. It has been suggested that a delay in processing of 35S pre-rRNA may be a consequence of a negative feedback mechanism to slow production of the 18S rRNA whenever the formation of the 25S/5·8S rRNA is inhibited (Zanchin et al., 1997
; de la Cruz et al., 1998b
). However, the delay in 35S pre-rRNA processing observed in these cgr1 mutants had little effect on the overall synthesis of the 18S rRNA.
The phenotype of the cgr1 mutants resembles the phenotype described for a mutant of Dob1p, a putative ATP-dependent RNA helicase. Similar to cgr1 mutants, the dob1 mutant has a defect in the synthesis of the 5·8S and 25S rRNAs, a reduction in 60S subunits and an increase in 7S rRNA (de la Cruz, 1998b ). The processing of the 7S rRNA involves the activity of a complex of 3'
5' exonucleases called the exosome, and Dob1p acts as a cofactor for this complex (Allmang et al., 1999
). The similarity between the dob1 and cgr1 mutants raises the possibility that Cgr1p has a direct or indirect effect on the activity of the exosome.
The yeast nucleolus occupies a crescent-shaped region of the nucleus that is approximately one-third of the total nuclear volume (Warner, 1982 ). The assembly of the nucleolus is thought to involve the recruitment of various processing factors through direct or indirect interactions with rDNA, nascent rRNAs, or nucleolar proteins that already reside in the nucleolus (Melèse & Xue, 1995
; Carmo-Fonseca, 2000
). For example, Net1p preferentially binds to rDNA and is required for the recruitment of the silencing protein Sir2p into the nucleolus (Straight et al., 1999
). In net1
cells, Nop1p redistributes over the nucleus, suggesting that Net1p is required to maintain the integrity of the nucleolar compartment. Our results indicate that cells deficient in Cgr1p are unable to retain all Nop1p and 5'-ITS1 in the nucleolar region and this would also be consistent with a role for Cgr1p in nucleolar integrity.
In contrast to highly conserved nucleolar proteins such as Nop1p (Tollervey et al., 1991 , 1993
), there are no clear homologues of Cgr1p in higher eukaryotes, including Drosophila melanogaster and Caenorhabditis elegans, raising the possibility that Cgr1p is either fungal specific or highly divergent in metazoan species. If the A. fumigatus orthologue of Cgr1p proves to be essential for the growth of A. fumigatus, its apparent divergence in higher eukaryotes would make it an attractive antifungal target. Further understanding of the role for Cgr1p in fungal ribosome synthesis awaits mechanistic detail on how this protein contributes to pre-rRNA processing.
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ACKNOWLEDGEMENTS |
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Received 6 September 2001;
revised 22 October 2001;
accepted 3 December 2001.