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Address correspondence to Eduard Hurt, Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. Tel.: 49-6221-54-4173. Fax: 49-6221-54-4369. E-mail: cg5{at}ix.urz.uni-heidelberg.de
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Abstract |
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Key Words: rRNA processing; ribosome biogenesis; ribosome export; nucleolus; preribosome
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Introduction |
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Most steps in ribosome synthesis occur in the nucleolus, a specialized nuclear compartment. The nucleolus is a highly dynamic structure, and three subregions, fibrillar centers (FCs),* a dense fibrillar component (DFC), and a granular component (GC), can be distinguished in chemically fixed samples of many cells (for review see Shaw and Jordan, 1995; Scheer and Hock, 1999). Using these conventional methods of preparing samples, the ultrastructure of yeast nucleolus cannot be preserved, and it was described as dense crescent into the nucleus (Sillevis-Smitt et al., 1973). However, cryofixation and cryosubstitution can optimally preserve the cellular ultrastructure, allowing the three subnucleolar compartments to be observed in S. cerevisiae (Léger-Silvestre et al., 1999).
The relationship between the observed subnucleolar structures and the different steps of ribosome biogenesis is not well established and remains somewhat controversial (for review see Shaw and Jordan, 1995; Scheer and Hock, 1999). A plausible model is that pre-rRNA transcription occurs at the boundary between the FC and DFC. In the DFC, the pre-rRNA may assemble with the pre-rRNA processing and modification machinery, followed by small nucleolar RNA (snoRNA)-mediated rRNA modification and early processing reactions, with late processing and assembly reactions occurring in the GC. Final maturation of the subunits occurs after their release from the nucleolus and export to the cytoplasm via the nucleoplasm and nuclear pores. Many components required for the correct assembly and trafficking of the preribosomes have been identified, but how these function together in the various preribosomal particles remains unclear.
Three types of ribosomal precursor particles of different sizes were identified from yeast by sucrose gradient centrifugation (Trapman et al., 1975). The 90S preribosomal particle was reported to contain the 35S pre-rRNA (itself identified by sucrose gradient velocity) and many early-assembling ribosomal proteins. However, this size is substantially smaller than that expected for a pre-rRNA associated with the many modification guide snoRNPs, and a more recent analysis (Milkereit et al., 2001) indicates that the 35S pre-rRNA is actually found distributed in much higher weight gradient fractions. This particle is then divided into two smaller particles, presumably by cleavage of the pre-rRNA at site A2 in ITS1 (below and see Fig. 4), generating the 66S and 43S preribosomes that are the precursors to the mature 60S and 40S subunits, respectively (Trapman et al., 1975). The 66S preribosomal particle contains the 27SA2, 27SB, and 7S pre-rRNA species, whereas the 43S particle contains the 20S pre-rRNA (Milkereit et al., 2001). Many nonribosomal proteins were shown to be associated with the preribosomal particles (Bassler et al., 2001; Harnpicharnchai et al., 2001; Saveanu et al., 2001). For example, the Noc1pNoc2p and the Noc2pNoc3p protein complexes cofractionate with the 35S and the 27S/7S pre-rRNAs, respectively, under the extraction conditions used in this work (Milkereit et al., 2001).
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Results |
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Rlp7p is strikingly homologous to the conserved ribosomal protein L7 (Rpl7p), which has close homologues in archaea and bacteria (Fig. 2 A). Three conserved domains were identified in Rpl7p homologues, with domain II best conserved. The mutation in rlp7-1 alters a conserved residue within this domain (Fig. 2 B). Two RNA-binding domains have been mapped in human L7, designated RBD1 and RBD2 in Fig. 2 B. RBD2 is present in all homologues and binds preferentially to 28S rRNA (von Mikecz et al., 1999). These two RNA-binding domains are present in Rlp7p, and are not affected in the rlp7-1 mutant, raising the possibility that Rlp7p binds to the same rRNA sequence as Rpl7p (see Discussion).
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Pre-rRNA processing is defective in rlp7-1 mutant strains
To test whether the depletion of 60S ribosomal subunits observed in the rlp7-1 strain is a consequence of defects in pre-rRNA processing (for the structure of the pre-rRNA and processing scheme see Fig. 4), we performed Northern hybridization (Figs. 5, A and B), primer extension (Fig. 5 C), and pulse-chase analyses (Fig. 6). After transfer of the rlp7-1 strain to 37°C for 2 h, the 35S pre-rRNA was mildly accumulated (Fig. 5 A). The 27SA2 and 20S pre-rRNAs were reduced, indicating that processing at sites A1 and A2 was partially inhibited, but there was little accumulation of the 23S RNA (the product of cleavage of 35S at site A3 in the absence of prior cleavage at sites A0 to A2), which is seen in many other processing mutants. The 27SB pre-rRNA was also reduced (Fig. 5 A), and primer-extension analysis suggested that 27SBS was reduced to a greater extent than 27SBL (Fig. 5 C, stops at B1S and B1L, respectively). In the rlp7-1 strain at 37°C, a rapid and strong reduction was seen in the levels of the 7S pre-rRNAs (Fig. 5 B) which are generated from the 27SB pre-rRNAs by cleavage at site C2. The other product of C2 cleavage, the 26S pre-rRNA, cannot be detected by Northern hybridization, but primer extension through site C2 (Fig. 5 C) shows that this pre-rRNA is rapidly lost, within 2 h of transfer to 37°C. In strains depleted of a recently reported processing factor, Ssf1p, cleavage at site C2 leads to the appearance of the A2C2 fragment (Fatica et al., 2002), but this does not occur in the rlp7-1 strain (Fig. 5 B). The 25S' pre-rRNA, a short 5' extended form of 25S shown by the primer extension stop at site C1', was strongly depleted in the rlp7-1 strain. This is consistent with the loss of cleavage at site C2, which acts as an entry site for the 5' exonucleases Rat1p and Xrn1p that generate the 5' end of 25S rRNA.
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Primer extension analysis through site A0 showed that the 33S pre-rRNA was little affected in the rlp7-1 strain, whereas the reduction in the stop at A2 was consistent with the reduced level of 27SA2 seen in Northern analyses. Cleavage at all sites was accurate at the nucleotide level.
Pulse-chase labeling of the rlp7-1 strain 2 h after transfer to 37°C, with either [H3]-uracil or [H3]-methyl methionine, showed a dramatic reduction in the synthesis of both the 25S and 5.8S rRNAs (Fig. 6). In contrast, 18S synthesis continued, but with some delay consistent with the results of Northern hybridization. The 7S pre-rRNA was not detected in the rlp7-1 strain. Northern hybridization of the filter shown in Fig. 6 C showed that the weak band visible in the rlp7-1 strain at the approximate position of 7S does not hybridize with a 7S pre-rRNA probe and migrates above 7S on the gel.
From these results, we conclude that the rlp7-1 mutation leads to a delay in exonuclease processing from site A3, together with strong inhibition of cleavage at site C2. The delay in cleavage at sites A0 to A2 and 18S synthesis is a common feature of strains with defects in 60S subunit synthesis (Venema and Tollervey, 1999) and is likely to be an indirect effect.
Pre-rRNA containing ITS2 accumulates in the rlp7-1 ts mutant
Ultrastructural detection of pre-60S ribosomal precursors by in situ hybridization was performed in wild-type and mutant rlp7-1 cells with an ITS2-specific probe (Gleizes et al., 2001). As shown in Fig. 7, the labeling in rlp7-1 cells at permissive temperature was found in the nucleolus, and its intensity was the same as in wild-type cells. Upon a shift to 37°C, rlp7-1 cells displayed a strong buildup of ITS2 containing pre-rRNAs. Quantitation of these immuno-EM data (in total 30 nuclei were analyzed) yielded in the nucleoplasm 15 gold particles/µm2 for wild-type and 19 gold particles/µm2 for rlp7-1 at 37°C. Moreover, the density of the gold in the nucleolus is 99 particles/µm2 in wild-type cells and 207 particles/µm2 in the rlp7-1 ts mutant at 37°C. Thus, accumulating pre-rRNAs did not appear to move to the nucleoplasm, but remained confined in the nucleolus. Similar observations were made by FISH with oligonucleotidic probes complementary to the ITS2 (unpublished data). These results are consistent with the localization of Rpl25p-eGFP in the nucleolus at 37°C, and with a defect in 27S pre-rRNA processing.
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Discussion |
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Subnucleolar structures have long been identified by EM and are the subject of a vast body of research (for review see Shaw and Jordan, 1995; Scheer and Hock, 1999). However, the relationship between nucleolar structures and the steps in ribosome synthesis are not well established. Transcription of the rRNA is likely to occur in the FCs or at the interface between the FCs and the DF). The FCs contain RNA polymerase I and transcription factors, whereas the DFC contains processing factors including fibrillarin (Nop1p in yeast) (Benavente et al., 1988; Pierron et al., 1989; Ochs and Smetana, 1991; Puvion-Dutilleul et al., 1991; Thiry and Goessens, 1992; Léger-Silvestre et al., 1999), which is associated with the box C/D snoRNAs, including U3, that are involved in early pre-rRNA processing and modification steps. In contrast to the FCs and DFC, very little is known about molecular markers for the GC, where late assembly and processing reactions are believed to occur. As an example, ribocharin, which is a nuclear 40-kD protein, was reported to specifically associate with the GC of the nucleolus and with a nucleoplasmic 65S particles (Hügle et al., 1985). Here we identify Rlp7p as a specific GC marker in yeast. Previously, Rlp7p was shown to be present in three different pre-60S particles (Fig. 10; Bassler et al., 2001; Harnpicharnchai et al., 2001; Saveanu et al., 2001; Fatica et al., 2002), indicating that each of these are concentrated in the GC. The 90S preribosomes contain the 35S pre-rRNA and are strongly predicted to be associated with the box C/D snoRNAs that direct the 35S pre-rRNA 2'-O-methylation. Therefore, the localization of the 2'-O-methylase, Nop1p/fibrillarin, to the DFC is good evidence for the localization of the 90S preribosomes to this region. Nop1p is not associated with the characterized pre-60S particles, and we predict that release from the DFC coincides with the formation of an early pre-60S particle, e.g., the pre-60S E1 complex shown in Fig. 10. Maturation through pre-60S E2 and pre-60S M is proposed to occur in the GC, with release of a complex located on the pathway between pre-60S M and pre-60S L from the GC into the nucleoplasm.
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The inhibition of cleavage at site C2 is a relatively specific phenotype, as several other mutations that inhibit 5.8S and 25S synthesis did not prevent C2 cleavage. These include the mutations noc2-1, rpl10-1, and rix7-1, which inhibit export of the 60S preribosomes, suggesting that the inhibition of C2 cleavage in the rlp7-1 strain is not the direct cause of the transport defect.
While this work was in progress, Dunbar et al. (2000) reported an analysis of the function of Rlp7p in pre-rRNA processing using a depletion approach. Rlp7p depletion was shown to inhibit 5.8S and 25S rRNA production, consistent with our observations.
Rlp7p is highly homologous to ribosomal protein L7, suggesting that they may compete for the same binding site on the rRNA. Because the 3' end of the 5.8S and the 5' end of the 25S rRNA are distant from the L7 binding site on the mature ribosome, it is unlikely that Rlp7p binds the ribosome close to the C2 cleavage site (Spahn et al., 2001). Following its function in pre-rRNA cleavage, Rlp7p could be dissociated from the rRNA by the binding of Rpl7p, potentially ensuring the correct succession of processing and assembly events. This strategy may be common in ribosome biogenesis, as three other known pre-rRNAprocessing and assembly factors show high similarity to proteins involved in translation. The U3 snoRNP protein Imp3p, which functions in 40S subunit synthesis, is homologous to the 40S protein Rps9p (Dunbar et al., 2000), the 60S preribosome components Yhr052p and Rlp24p are homologous to Rpl1p and Rpl24p, respectively (Bassler et al., 2001; Saveanu et al., 2001), and Efl1p is homologous to the translation elongation factor EF-2 (Senger et al., 2001).
This finding has potential relevance for the evolution of ribosome biogenesis. Ribosome biogenesis in prokaryotic cells occurs in a single cell compartment, whereas the synthesis of eukaryotic ribosomes involves a succession of transport events from the nucleolus to the cytoplasm. We suggest that intracellular transport systems for preribosomes initially recognized ribosomal proteins. During evolution, duplication of these components led to the separation of the ribosomal proteins and homologous proteins that bind to the same sequence in preribosomes. Replacement of the preribosomal protein with the ribosomal protein could act as a signal for release of the preribosomal particle from a specific region. In the simplest model, Rlp7p might be physically associated with preribosomal particles during their assemblage in the GC. By analogy, Imp3p and perhaps the U3 snoRNP might be required for the retention of the 90S preribosomes in the DFC, whereas the replacement of Efl1p by EF-2 might signal arrival of the subunits in the cytoplasm (Senger et al., 2001; unpublished data).
In summary, using a genetic screen to identify novel components required for the export of the large ribosomal particle, we identified a mutation in Rlp7p. We have shown that Rlp7p is enriched in a subcompartment of the nucleolus, the GC. Rlp7p is required for the C2 cleavage that occurs within a ribosomal precursor particle. Rlp7p belongs to the increasing family of maturation proteins with high homology to a component of the translation machinery. Therefore, we suggest that this family of proteins could allow retention of the premature particles in specific areas of the nucleus during their maturation. Using this strategy eukaryotic cells could achieve precise coupling between ribosome maturation and its nuclear export.
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Materials and methods |
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Plasmid constructions
Plasmids pUN100-DsRed-Nop1p, pRS314-DsRed-Nop1p, pRS316-Rpl25p-eGFP, pFA6a-(2*ProtA-TEV)-TRP1, and pFA6a-GFP(S65T)-kanMX6 were described previously (Longtine et al., 1998; Gadal et al., 2001). pFA6-YFP-TRP1, pFA6-CFP-TRP1, pFA6-YFP-HIS3MX6, and pFA6-CFP-HIS3MX6 are derivative of pFA6a-GFP(S65T)-HIS3MX6 and pFA6-GFP-TRP1 (Longtine et al., 1998), where the Pac1-Asc1 fragment, bearing the GFP-coding sequence, was PCR exchanged with the corresponding eYFP and eCFP spectral variant of GFP, using vectors pECFP-C1 and pEYFP-C1 from CLONTECH Laboratories, Inc. Plasmids pUN100-YFP-Nop1p and pUN100-CFP-Nop1p are derivative of pUN100-DsRed-Nop1p where the SphI-SphI fragment, bearing the GFP-coding sequence, was PCR exchanged with the corresponding eYFP and eCFP spectral variant of GFP, using vectors pECFP-C1 and pEYFP-C1 from CLONTECH Laboratories, Inc.
Strain constructions
Genomic integration of GFP in frame with RLP7 was obtained as described previously (Longtine et al., 1998). Genomic integration of ProtA in frame with RLP7, YFP, and CFP in frame with RLP7, GAR1, and NUG2 were done in the same way, but using, respectively, the pFA6a-(2*ProtA-TEV)-TRP1, pFA6-YFP-TRP1, pFA6-CFP-TRP1, pFA6-YFP-HIS3MX6, and pFA6-CFP-HIS3MX6 vectors.
Cloning of RIX9/RLP7
A yeast genomic library in an LEU2-containing ARS/CEN plasmid (Gautier et al., 1997) was transformed into the rix9-1 strain. From colonies growing at the restrictive temperature (37°C), plasmid pRIX9 with a genomic insert was isolated. The complementing plasmid contained the RLP7 gene. pRLP7 harboring only the RLP7 gene was cloned and shown to complement the ts growth defect of the rix9-1 mutant.
Pulse-chase and Northern analysis of rRNA
Pulse-chase labelling of rRNA, primer extension, and analysis of rRNA processing by Northern hybridization was performed as described (Tollervey, 1987; Tollervey et al., 1993). Oligonucleotides used were: 003, TGT TAC CTC TGG GCC C; 004, CGG TTT TAA TTG TCC TA; 007, CTC CGC TTA TTG ATA TGC; 008, CAT GGC TTA ATC TTT GAG AC; 013, GGC CAG CAA TTT CAA GTT A; 017, GCG TTG TTC ATC GAT GC; 020, TGA GAA GGA AAT GAC GCT; 219, GAA GCG CCA TCT AGA TG, and 5' A0L GGT CTC TCT GCT GCC GG.
Fluorescence microscopy
pRS315-Rpl25p-eGFP or pRS316-Rpl25p-eGFP was introduced into yeast cells by transformation and selected on SDC-leu or SDC-ura medium, respectively. Individual transformants were grown in liquid SDC-leu medium at 23°C to OD(600 nm) of 1, before shift to 37°C in liquid YPD medium. After centrifugation, cells were resuspended in water, mounted on a slide, and observed in the fluorescence microscope. In vivo, the GFP signal was examined in the FITC fluorescent channel; the DsRed used in fusion with Nop1p was examined in the rhodamine channel of a Zeiss Axioskop fluorescence microscope, and pictures were obtained with a Xillix Microimager CCD camera. Digital pictures were processed by software program Open lab (Improvision) and Adobe Photoshop® (v. 4.0.1).
Fluorescence microscopy of green fluorescent protein spectral variants was done on exponentially growing cells, which were washed in water and stained with Hoechst 33352 (5 ng/µl) for 5 min. Samples were examined using a Leica DMRXA fluorescence microscope. Fluorescent signals were collected with single-band pass filters for excitation of YFP (XF104; Omega Optical), CFP (XF114-2; Omega optical), and Hoechst (Leica). Images were acquired with a Hamamatsu C4742-95 cooled CCD camera controlled by the Openlab® software (Improvision) and processed with the Adobe Photoshop® software.
EM
Protein Atagged Rlp7p strains were prepared for EM using high-pressure freezing. After preembedding in lowmelting point agarose, cells moistened with the freezing medium (1-hexadecene) were loaded into specimen holder and frozen with liquid nitrogen under high pressure using the EM Pact system (Leica SA). The frozen samples were then transferred in 0.4% uranyl acetate in absolute acetone. Substitution fixation was carried out at -90°C for 3 d. The specimen were gradually transferred to -20°C, washed successively with absolute acetone and absolute ethanol at -20°C, infiltrated, and embedded with LR White resin at this temperature. For immunolocalization of the protein Atagged Rlp7p, we used a polyclonal antiprotein A antibody from Sigma-Aldrich. This antibody was labeled with gold-conjugated goat antirabbit IgG (British Bio-Cell). No nuclear labelling was detected when immunolocalization was performed on cells devoid of Protein Atagged protein, or when the primary antibodies were omitted. Nop1p was detected using a monoclonal antibody from Dr. J. Aris (University of Florida, Gainesville, FL) and goat antimouse IgG goldconjugated. In situ hybridization of pre-rRNAs using an ITS2-specific riboprobe was performed as previously described (Gleizes et al., 2001). The probe was generated by in vitro transcription in the presence of UTP-biotin and detected on sections with anti-biotin antibodies conjugated to 10-nm gold particles.
Miscellaneous
SDS-PAGE and Western blot analysis were performed according to (Siniossoglou et al., 1996), and isolation of ribosomes under low-salt conditions by sucrose gradient centrifugation as described in (Tollervey et al., 1993). Whole-cell lysates and fractions from the sucrose gradients were separated by SDS-PAGE and analysed by Western blotting using the indicated antibodies.
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Footnotes |
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* Abbreviations used in this paper: DFC, dense fibrillar component; FC, fibrillar center; GC, granular component; snoRNA, small nucleolar RNA.
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Acknowledgments |
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Submitted: 9 November 2001
Revised: 17 April 2002
Accepted: 18 April 2002
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References |
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