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REPORT |
1 Department of Genetics and Molecular Biology, University of Rome "La Sapienza," Rome, Italy
2 Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
Reprint requests to: Alessandro Fatica, Department of Genetics and Molecular Biology, University of Rome "La Sapienza," Rome, Italy; e-mail: alessandro.fatica{at}uniroma1.it; fax: 0039-06-49912500.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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Keywords: pre-rRNA; ribosome synthesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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) to generate the mature 18S, 5.8S, and 25S rRNAs with concomitant modification of the mature rRNAs (for review, see Venema and Tollervey 1999). During these processing reactions, the rRNA intermediates associate with the 5S rRNA, about 80 ribosomal proteins, and a large number of nonribosomal proteins, to form preribosomal particles. The preribosomal particles are dynamically remodeled and matured as they transit from the nucleolus to the nucleoplasm and then are exported to the cytoplasm, where synthesis of the 60S and 40S ribosomal subunits is completed (Nissan et al. 2002; Saveanu et al. 2003; Schafer et al. 2003).
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; Milkereit et al. 2003). These analyses reveal that more than 160 different proteins are dynamically exchanged during ribosome maturation. A role in pre-rRNA processing had previously been demonstrated for many of these proteins, but a substantial number were either previously uncharacterized or had been identified as factors required for functions apparently unrelated to ribosome synthesis.
One such is Cic1p/Nsa3p, an essential protein previously reported to function as an adaptor for the 26S proteasome (Glickman and Ciechanover 2001) that is specifically required for degradation of SCF protein components (Jager et al. 2001). Despite this involvement in regulating the ubiquitin–proteasome system, Cic1p was localized to the nucleolus (Jager et al. 2001; Nissan et al. 2002), and the identification of Cic1p as a putative component of early pre-60S ribosomes (Baßler et al. 2001; Harnpicharnchai et al. 2001; Nissan et al. 2002; Saveanu et al. 2003) strongly suggested an additional or alternative role for Cic1p in ribosome synthesis.
Here we report that Cic1p is indeed required for maturation of large subunit rRNAs and is apparently also required for the release of the pre-60S ribosomal particles from the nucleus.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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). Growth of the cic1–2 strain was very similar to the otherwise isogenic wild-type strain at the permissive temperature of 25°C (data not shown), but almost ceased 6 h after transfer at the restrictive temperature of 37°C (Fig. 2A).
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) showed that in the wild-type strain, the 35S pre-rRNA was rapidly converted into the 27SA and 20S pre-rRNAs. The 27SA pre-rRNA is then converted to 27SB and the 25S rRNA, whereas 20S pre-rRNA is converted to 18S rRNA (see Fig. 1). By contrast, in the cic1–2 strain, processing of the 35S pre-rRNA was substantially slowed and the aberrant 23S RNA was readily detected. The 23S RNA originates from direct cleavage of the 35S pre-rRNA at site A3 when the cleavages at sites A0, A1, and A2 are delayed (Venema and Tollervey 1999). The 27SA pre-rRNA was synthesized with some delay but its conversion to 25S rRNA was strongly reduced. The 20S pre-rRNA and mature 18S rRNA appeared with delayed kinetics and reduced yield, but this reduction was much less marked than that seen for 27S and 25S.
Analysis of low molecular weight RNA (Fig. 2C) showed the processing of the 7S pre-rRNA to mature 5.8S rRNA in the wild-type strain. In the cic1–2 strain, the 7S pre-rRNAs was not detected and synthesis of 5.8S rRNA was strongly depleted. Synthesis of 5S rRNA was mildly reduced, presumably as a consequence of the growth inhibition. These data show that Cic1p is required for synthesis of the large subunit rRNAs. As seen for almost all other mutations defective in synthesis of the 60S subunit rRNAs, a delay in the early pre-rRNA processing steps was also observed. To further characterize the function of Cic1p in pre-rRNA processing, steady-state levels of mature rRNAs and precursors were assessed by Northern hybridization (Fig. 3). After transfer of the cic1–2 strain to 37°C for 2 h, the 35S pre-rRNA and 23S RNA were accumulated (Fig. 3B), consistent with the pulse-chase analysis, whereas the 27SA2 and 20S pre-rRNAs were reduced. The 27SB and 27SA3 pre-rRNA were depleted as well. In good agreement with the reduced levels of 27SA2 and 27SB, primer extension analysis showed that in the cic1–2 strain, there is a strong reduction of the stops at site A2, B1L and B1S (Fig. 3D). This suggests that the processing of all 27S pre-rRNAs is affected by Cic1p inactivation. Steady-state levels of mature 25S and, to a lesser extent, of 18S rRNAs were decreased. The defect in 18S synthesis is a common feature of strains with defects in 60S synthesis and may be secondary consequence of the reduced growth rate (Venema and Tollervey 1999). In the cic1–2 strain at 37°C there was a strong reduction of the 7S and 6S pre-rRNAs (Fig. 3C), precursors of the 5.8S rRNA. In addition, a fragment that extends from A2 to C2 was accumulated (Fig. 3C), indicating that inactivation of Cic1p allows an aberrant cleavage of 27SA2 at site C2 that reduce the conversion of 27SA2 into 27SB (Fig. 3B,D) and 7S pre-rRNAs (Fig. 3C). The A2-C2 molecule has been previously described for other components of early pre-60S ribosomal particles (Fatica et al. 2002) and indicates that Cic1p is required to maintain the normal order of processing in the 27SA2 molecule.
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), strongly supporting a direct role in ribosome synthesis.
Many factors required for ribosome assembly are necessary for the export of the preribosomal particles (Baßler et al. 2001; Gadal et al. 2001; Milkereit et al. 2001; Schafer et al. 2003).
To assess the involvement of Cic1p in subunit transport, the wild-type and cic1–2 strains were transformed with a plasmid expressing an eGFP-tagged form of the 60S subunit ribosomal protein Rpl11b, and analyzed as previously described (Stage-Zimmermann et al. 2000). In the wild-type strain and in the cic1–2 strain at permissive temperature, Rpl11b-eGFP was predominately cytoplasmic (Fig. 4b,e,h). Following transfer to 37°C for 4 h, the fusion protein showed nuclear localization (Fig. 4k). The GFP-decorated region did not fully coincide with the nucleoplasm, identified by DAPI staining (Fig. 4j), and generally showed the classic crescent shape of the yeast nucleolus.
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; Harnpicharnchai et al. 2001; Nissan et al. 2002; Saveanu et al. 2003). |
CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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) and was therefore predicted to be present in early pre-60S particles in the yeast nucleolus (Baßler et al. 2001; Harnpicharnchai et al. 2001; Nissan et al. 2002). Consistent with this model, we report that the cic1–2 mutant allele (Jager et al. 2001) inhibits the synthesis of the large subunit rRNAs. Pulse-chase, Northern, and primer extension analyses showed that Cic1p is required for normal maturation of the 27SA2 pre-rRNA to the 25S and 5.8S rRNAs. In addition, cic1–2 strains accumulated a GFP-tagged 60S reporter protein within the nucleolus, indicating a defect in release of pre-60S particles from the nucleolus into the nucleoplasm. A similar defect was previously seen for strains carrying a mutation in Noc1p (Milkereit et al. 2001), and Noc1p was coprecipitated with Cic1p (Nsa3p-TAP; Nissan et al. 2002) indicating their presence in the same early pre-60S particle.
Cic1p is one of a surprisingly large number of yeast ribosome synthesis factors that apparently function in additional cellular processes (Buscemi et al. 2000; Valasek et al. 2001; Zimmerman and Kellogg 2001; Du and Stillman 2002; Tone and Toh-E 2002; Zhang et al. 2002). Cic1p was initially identified as a protein required for degradation of specific substrates by the 26S proteasome (Jager et al. 2001). Interestingly, this is not the first preribosome component to show functional interactions with the ubiquitin–proteasome system. Nob1p was shown to be required for biogenesis of both the 40S ribosomal subunit and the 26S proteasome (Tone and Toh-E 2002; Fatica et al. 2003; Schafer et al. 2003). The nucleolar protein Krr1p, which is also required for 40S synthesis, was identified in a two-hybrid screen using Tom1p, a member of the E3 ubiquitin-ligase family (Sasaki et al. 2000). Moreover, three ribosomal proteins are synthesized as fusions with ubiquitin, whereas others undergo posttranslational ubiquitination that is regulated during the cell cycle (Finley et al. 1989; Spence et al. 2000). In these latter cases, ubiquitination does not function to target the acceptor protein for degradation but facilitates incorporation of the ribosomal proteins into functional subunits (Finley et al. 1989; Spence et al. 2000).
Many interactions between ribosome synthesis and the ubiquitin–proteasome system can be envisioned. Stoichiometry must be maintained between synthesis of the rRNAs and proteins and, at least under some conditions, this is largely ensured by the rapid degradation of excess ribosomal proteins (Ju and Warner 1994; Warner 1999), presumably via the proteasome. Moreover, preribosomes that are misassembled or in which the RNA is not correctly processed are generally very rapidly degraded, probably including the ribosomal protein components. Finally, ribosome biogenesis plays an important role in the regulation of cell growth and proliferation (Jorgensen et al. 2002; Ruggero and Pandolfi 2003). Communication between ribosome assembly factors and proteasome activity could be an important mechanism to synchronize ribosome synthesis with cell growth and division.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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). Northern hybridization and primer extension were as described (Kufel et al. 2000). Standard 1.2% agarose/formaldehyde and 6% acrylamide/urea gels were used to analyze the high and low molecular weight RNA species, respectively.
Oligonucleotides
For RNA hybridizations and prime extensions, the following oligonucleotides were used:
001: 5'-CCAGTTACGAAAATTCTTG; 002: 5'-GCTCTTTGCTCT TGCC
003: 5'-TGTTACCTCTGGGCCC; 006: 5'-GGCCAGCAATTTCA AGTTA;
007: 5'-CTCCGCTTATTGATATGC; 008: 5'-CATGGCTTAATCT TTGAGAC;
013: 5'-GCGTTGTTCATCGATGC; 020: 5'-TGAGAAGGAAATG ACGCT;
033: 5'-CGCTGCTCACCAATGG; 041: 5'-CTACTCGGTCAGG CTC.
Pulse-chase labeling
Metabolic labeling of RNA was performed as described previously (Fatica et al. 2002). The strains cic1–2 and W303 (Jager et al. 2001) were transformed with a plasmid containing the URA3 gene and grown in glucose minimal medium lacking uracil. Cells were pregrown at permissive temperature (23°C) and transferred to restrictive temperature (37°C) for 4 h. Cells at 0.3 O.D.600 were labeled with [5,6-3H] uracil for 1 min followed by a chase with excess unlabeled uracil for 0, 1, 2.5, 5, 10, and 20 min. Standard 1.2% agarose/formaldehyde and 6% poly-acrylamide/urea gels were used to analyze the high and low molecular weight RNA species, respectively.
Rpl11b-eGFP localization assay
The assay was carried out as previously described (Stage-Zimmermann et al. 2000) with the following modifications. Cells were fixed in 3.7% formaldehyde for 30 min at room temperature before being washed in 1x PBS and were mounted using Vectashield-containing DAPI to stain the nuclei. Rpl11b-GFP was vizualized by fluorescence microscopy in the fluorescein channel using a Nikon microscope. Pictures were obtained using a Coolsnap HQ camera.
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ACKNOWLEDGMENTS |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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Footnotes |
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Received July 16, 2003; accepted September 2, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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