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Previous Article | Next Article 
Molecular and Cellular Biology, August 2007, p. 5414-5429, Vol. 27, No. 15
0270-7306/07/$08.00+0 doi:10.1128/MCB.00380-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer, CSIC-University of Salamanca, Campus Unamuno, E-37007 Salamanca, Spain
Received 2 March 2007/ Returned for modification 28 March 2007/ Accepted 10 May 2007
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INTRODUCTION |
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80 ribosomal proteins. Due to its amenability for genetic and proteomic analyses, Saccharomyces cerevisiae is the organism where the different steps of this pathway have been best characterized (4, 11, 13, 18, 32, 34). Thus, it is known that three of the four mature rRNAs that form the ribosome structure are generated from a common 35S pre-rRNA polycystronic precursor. After being transcribed in the nucleolus, this precursor is chemically modified and cleaved at three positions (known as the A0, A1, and A2 sites) of its 5'-terminal end to generate the intermediate 33S, 32S, 27SA2, and 20S pre-rRNA precursors (see Fig. S1 in the supplemental material). The 20S and 27SA2 pre-rRNAs then follow two independent maturation routes that lead to the generation of either the 18S rRNA (a component of the 40S ribosomal subunit) or the 5.8S and 25S rRNAs (two components of the 60S ribosomal subunit), respectively (see Fig. S1 in the supplemental material). These pre-rRNA maturation steps require the involvement of 170 nonribosomal proteins and 70 small nucleolar ribonucleoproteins (snoRNPs) (4, 11, 13, 32). Different subsets of these molecules form large ribonucleoprotein complexes with specific pre-rRNA precursors that, according to their specific Svedberg coefficients in gradient ultracentrifugation experiments, were initially referred to as 90S, 66S, and 43S preribosomal particles (31, 33). The 90S particle, also known as the "small-subunit processome," contains the 35S pre-rRNA and assembly/processing factors needed for the early cleavage of the 35S pre-rRNA precursor at A0, A1, and A2 sites, which is strictly required for the production of 40S ribosomal subunits. The 66S and 43S particles contain 35S pre-rRNA cleavage derivatives and proteins that mediate the downstream maturation steps of rRNA biosynthesis. Recent studies have shed light on the complex structure of preribosomal particles. In the case of the 90S preribosome, the combination of several biochemically, genetically, and proteomically based studies has shown that it contains the 35S pre-rRNA, the U3 small nucleolar RNA (snoRNA), and 50 nonribosomal proteins. These include enzymes that participate in RNA-processing functions and a large cohort of proteins containing protein-protein and RNA-binding motifs but lacking known catalytic domains (3, 7, 13, 17, 22, 29, 34, 36). The mechanism by which all these proteins are assembled together to form the final 90S particle is unknown. However, available evidence suggests that yeast cells might minimize the complexity of this process by preassembling 90S particle components in smaller building blocks prior to the binding to the 35S pre-rRNA. In favor of this model, electron microscopy studies have revealed that early preribosomal particles undergo time-dependent changes in size and shape upon binding to the primary pre-rRNA precursor, suggesting that their components are sequentially assembled (26). Other studies have revealed the presence of discrete 90S particle subcomplexes, such as the U3 snoRNA-containing U3 snoRNP, the Mpp10p complex, and the Pwp2p subunit. Some of these subcomplexes seem to represent true 90S preribosomal subunits, because they are detected as stable biochemical entities independently of the 90S particle (6, 17, 19, 36, 37). Recent high-throughput proteomic studies have also reported the presence in yeast cells of three small 90S particle subcomplexes that were designated U three protein (UTP)-A, UTP-B, and UTP-C (22). It is believed that these subcomplexes might represent stable preassembly or postassembly 90S modules because they are detected in cell lysates depleted of 90S particles by an ultracentrifugation step (22). This hypothesis is further supported by the observation that the UTP-B subcomplex is identical in protein composition to the Pwp2p subunit (and, as such, it will be referred to as the Pwp2p/UTP-B subunit hereafter). Finally, it has also been shown that t-UTP, an UTP-A-like subset of 90S preribosomal proteins, influences 35S rDNA transcription independently of other particle proteins (14).
In addition to the number of subunits, there are other questions related to 90S particle assembly that remain unanswered, such as those pertaining the order of incorporation of components onto the 35S pre-rRNA and the interconnections established by them, both during and after the formation of the particle. Given the large number of molecules and protein-protein interactions involved, it is likely that the only way to obtain a comprehensive view of the 90S particle architecture and assembly process is by studying the reciprocal connections among multiple components using different experimental techniques. In this work, we have used this strategy to delve in the 90S particle substructure and the mechanism of its assembly. This experimental avenue allowed us to characterize discrete 90S particle subunits, to dissect the hierarchy of assembly of those subunits onto the 35S pre-rRNA, to identify several assembly intermediaries of the 90S particle, and to gain clues for deciphering the internal 90S particle architecture.
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MATERIALS AND METHODS |
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Sucrose gradient analysis. Polysome analysis and fractionation of lysates through 7 to 50% sucrose gradients were performed as described previously (6). Extracts equivalent to 15 absorption units at the 260-nm wavelength were layered onto each gradient. After centrifugation, fractions were collected and subjected to either Western or Northern blot analyses.
Coimmunoprecipitation experiments.
Cell cultures were grown to an A600 of between 0.8 and 1.0. Cells equivalent to 20 A600 units were harvested and disrupted using glass beads (Sigma) in IP2 buffer (20 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 150 mM potassium acetate, 1 mM dithiothreitol, and 0.2% Triton X-100) supplemented with both a vanadyl ribonucleoside complex (5 mM; New England Biolabs) and a mixture of protease inhibitors (C
mplete; Roche Applied Science). Precleared lysates were incubated with anti-MYC antibodies (Roche Applied Science) at 4°C for 2 h under gentle rotation in the presence of RNasin (400 U/ml; Promega). After incubation with Gammabind Sepharose beads (GE Healthcare), immunoprecipitates were washed five times with IP2 buffer at 4°C. For protein analyses, half of the immunoprecipitated material was resuspended in 80 µl of sodium dodecyl sulfate (SDS) loading buffer, boiled, fractionated on 8% acrylamide gels, and analyzed by Western blotting. For RNA analyses, the other half of the sample was resuspended in 400 µl of 50 mM sodium acetate, 10 mM EDTA (pH 5.2), and 1% SDS and processed for RNA extraction.
RNA analysis. RNAs from total cellular lysates, gradient fractions or coimmunoprecipitations were prepared by the hot-phenol method (1). After ethanol precipitation, RNAs were resuspended in either formaldehyde loading buffer (to detect high-molecular-weight RNAs) or acrylamide-urea loading buffer (to detect snoRNAs), separated electrophoretically on either 1.2% agarose-formaldehyde (in the case of high-molecular-weight pre-rRNA precursors) or 8% acrylamide-8 M urea (in the case of snoRNAs) gels, and analyzed by Northern blotting. We used the following 32P-labeled oligonucleotides to detect the 35S pre-rRNA precursor and other downstream pre-rRNA intermediaries: probe 4 (region D-A2), 5'-TTAAGCGCAGGCCCGGCT-3'; probe 5 (region A2-A3), 5'-TGTTACCTCTGGGCC-3'; and probe 8 (region E-C2), 5'-GGCCAGCA ATTTCAAGTTA-3'. These regions of the 35S pre-rRNA molecule are depicted in Fig. S1 in the supplemental material. The U3 snoRNA was detected using a labeled 5'-GGATTGCGGACCAAGCTAA-3' probe. Oligonucleotide labeling, Northern blotting, and filter hybridizations were performed as previously described (6).
Mass spectrometry analysis.
Purifications of Pwp2p-MYC-, Rrp7p-MYC, Nan1p-MYC-, and Utp4p-MYC-containing complexes in conditional mutant strains were performed using a large-scale anti-MYC coimmunoprecipitation approach, exactly as indicated before (6). All protein bands observed after staining of the gels with Sypro Ruby (Molecular Probes) were processed and analyzed for their identification by mass spectrometry in the Genomics and Proteomics Unit of the Centro de Investigación del Cáncer of Salamanca, using an Ultraflex matrix-assisted laser desorption ionization-time-of-flight apparatus (Bruker). Complexes were purified in several independent experiments, and the patterns of proteins associated with each bait protein, in each experimental condition, were found to be the same every time. A protein was considered positively associated with a given bait protein when it was reproducibly detected as a strongly stained band in the corresponding gels of the immunoprecipitations performed with that bait. If so, it was included in Fig. 5 below. A protein was considered "not present" in the bait immunocomplexes when one of the following occurred: (i) it was not detected in the Sypro Ruby-stained SDS-polyacrylamide gels, (ii) the electrophoretic band identified by mass spectrometry did not fit the mass spectrometer fingerprint of the missing protein (these two cases occurred in 81% of the "not-present" proteins reported in Fig. 5), or (iii) the relative amount of that protein was drastically reduced in the bait immunoprecipitates compared to the levels of coimmunoprecipitation detected for other bait's partners (this occurred in 19% of the negative cases reported in this work). In addition to those cases, we also detected some strongly stained protein bands that could not be identified by mass spectrometry (6% of all stained bands analyzed). Due to their abundance and molecular size, we regarded those proteins as mixtures of degradation products or denatured immunoglobulin G chains released from the anti-MYC beads after the boiling step. These are nonidentifiable bands because the default parameters set up for the mass spectrometer protein identification software filters them out automatically. Finally, we identified proteins by mass spectrometry that had reported functions totally unrelated to ribosomal biogenesis. These proteins, representing 14% of all stained bands, always showed up associated to the chosen bait regardless of the experimental context and/or genetic and culture conditions used, suggesting that they likely represent contaminants carried over by the immunocomplexes. The identities of these bands will be provided upon request.
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RESULTS |
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15 to 20S sedimentation monoparticles and in an 90S complex (Fig. 2A). The 90S-associated Rrp7p-MYC fraction cofractionated with the 35S pre-rRNA and other 90S particle components such as the U3 snoRNA (Fig. 2A). A similar distribution of Rrp7p-MYC was observed in cells expressing wild-type levels of Pwp2p (see Fig. S2A in the supplemental material), indicating that this bimodal sedimentation profile is not due to HA-Pwp2p overexpression. The elimination of Pwp2p expression led to the expected blockage in the processing of the 35S pre-RNA, as evidenced by the accumulation of the primary transcript of the precursor (Fig. 2A and B), the disappearance of the transitional 27SA2 RNA generated upon cleavage of the 35S pre-rRNA at the A2 site (Fig. 2A; see Fig. S1 in the supplemental material), the concentration of the U3 snoRNA in small-sedimentation-coefficient fractions (Fig. 2A), and the lack of 40S ribosomal subunits (Fig. 2A). Despite the dramatic effects of the Pwp2p depletion on ribosomal biosynthesis, we observed that the sedimentation profile of Rrp7p-MYC was very similar to that observed in both wild-type and Pwp2p-overexpressing cells (compare Fig. 2A and Fig. S2A in the supplemental material), suggesting that the Rrp7 subunit could bind the 35S pre-rRNA in the absence of Pwp2p and the U3 snoRNP. In agreement with this, coimmunoprecipitation experiments showed that Rrp7p associated with the 35S pre-rRNA independently of Pwp2p (Fig. 2B). In contrast, and consistent with the accumulation of the U3 snoRNA in free monoparticles in Pwp2p-depleted cells (Fig. 2A), the detection of the U3 snoRNA in the Rrp7p immunoprecipitates was lost upon Pwp2p elimination (Fig. 2B). Control experiments confirmed the specificity of these RNA-protein interactions (Fig. 2B).
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The UTP-A/t-UTP component Nan1p is required for the association of the Pwp2p/UTP-B element Pwp2p with the 35S pre-rRNA but not vice versa.
We next analyzed the relationship between proteins of the UTP-A/t-UTP and Pwp2p/UTP-B subcomplexes for their docking onto the primary pre-rRNA precursor. We observed that Nan1p-MYC, a component of the UTP-A/t-UTP subcomplex, cofractionated almost exclusively with the 35S pre-rRNA and large U3 snoRNA-containing complexes under conditions of Pwp2p overexpression (Fig. 3A). In contrast, no low-sedimentation-coefficient monoparticles containing Nan1p were observed. This is probably due to the overexpression of HA-Pwp2p, because Nan1p-MYC is detected in 15 to 20S monoparticles in cells expressing wild-type levels of Pwp2p (see Fig. S2C in the supplemental material). Under conditions of Pwp2p depletion, Nan1p-MYC showed a sedimentation profile similar to that observed in wild-type cells (Fig. 3A). Parallel analyses confirmed that the biosynthesis of 40S ribosomal subunits (Fig. 3A) and the processing of the 35S pre-rRNA (Fig. 3B) were blocked upon Pwp2p elimination. We could not confirm in this case the physical association between Nan1p and the 35S pre-rRNA in the absence of Pwp2p, because Nan1p immunoprecipitates were consistently inefficient in pulling down that precursor (Fig. 3B, see also Fig. 4B below). This may be a reflection of either a rather feeble/indirect interaction or, alternatively, a conformation of the complex that does not protect the 35S pre-RNA from degradation during the immunoprecipitation step. However, we do not consider that this could be an artifactual problem derived from the inclusion of the C-terminal MYC epitope, because cells expressing this protein showed normal cleavage of the 35S pre-rRNA at A0-A2 sites and optimal levels of 40S ribosomal subunits (Fig. 3A; see Fig. 4A and see Fig. S2C in the supplemental material). Despite the lack of confirmation by coimmunoprecipitation, the sedimentation analysis of Nan1p indicates that its binding to the 35S pre-rRNA occurs independently of both Pwp2p and the U3 snoRNA. This is consistent with previous results indicating that t-UTP proteins can bind the primary pre-rRNA precursor in a U3 snoRNA-independent manner (14).
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The UTP-A/t-UTP component Nan1p is required for binding of Rrp7p to the 35S pre-rRNA but not vice versa. To finish the initial part of this work, we evaluated the assembly relationships established between UTP-A/t-UTP and UTP-C components. The repression of the UTP-C-specific Rrp7p resulted in no substantial modifications in the sedimentation profile of Nan1p-MYC (Fig. 4A). In addition, and consistent with our previous results (Fig. 2C), we observed that the U3 snoRNA was also detected in 90S sedimentation fractions, indicating that the binding of the U3 snoRNP to the 35S pre-rRNA is Rrp7p independent (Fig. 4A). To further confirm these results, we assessed the physical association of Nan1p-MYC with specific RNA molecules of the 40S ribosomal subunit biosynthetic route. Unlike the results obtained under conditions of Rrp7 expression (Fig. 3B and 4B), we did find in this case significant amounts of both 35S and 23S pre-rRNAs bound to Nan1p when Rrp7p was absent from cells (Fig. 4B). These results confirm that Nan1p assembles onto the 35S pre-rRNA in an Rrp7-independent manner and, at the same time, suggest that Rrp7p conditions the type of structure established between the primary pre-rRNA precursor and some of its associated protein complexes. The cleavage of the 35S pre-rRNA at the A2 site (Fig. 4B) and the biosynthesis of the mature 40S ribosomal subunit (Fig. 4A) were both impaired under conditions of Rrp7p depletion, indicating that the presence of Nan1p protein in 90S fractions in the absence of Rrp7p was not due to the inefficient elimination of that protein. In contrast to the above results, we found in reciprocal experiments that the depletion of Nan1p led to the accumulation of Rrp7p in monoparticles with small sedimentation coefficients and to a lack of association of Rrp7p with both the 35S pre-rRNA and the U3 snoRNA (Fig. 4C and D).
Taken together, these results indicate that individual constituents of the UTP-A/t-UTP, Pwp2p/UTP-B, and UTP-C subcomplexes associate with the 35S pre-rRNA in three distinct assembly steps. Nan1p, a component of the UTP-A/t-UTP subcomplex, interacts with the 35S pre-rRNA independently of Pwp2p and Rrp7p, two proteins belonging to the Pwp2p/UTP-B and UTP-C subcomplexes, respectively. In addition, Pwp2p and Rrp7p depend on Nan1p but do not require each other to associate with the 35S pre-rRNA. These findings suggest that a subset of factors, which include the protein Nan1, form an assembly module that is required for the subsequent incorporation of two independent sets of proteins: one set includes Rrp7p and associated proteins, and the other set encompasses Pwp2p- and U3 snoRNA-containing complexes.
Characterization of partially assembled preribosomal particles. The above studies suggested the presence of discrete building blocks of the 90S preribosomal particle that can be detected as free small complexes in vivo and/or forming part of both partially assembled and fully assembled 90S particles. However, our experiments could not identify unambiguously the components of each subunit or its physical interactions with proteins of other 90S particle subunits. Due to this, we decided to conduct a proteomic-based approach to identify the proteins associated with Pwp2p-MYC, Rrp7p-MYC, and Nan1p-MYC. To make this analysis more comprehensive, we also included in these experiments the identification of proteins bound to Utp4p-MYC, another component of the previously described UTP-A/t-UTP subcomplex (14, 22). We expected that, by comparing the spectra of proteins bound to these proteins under the experimental conditions used for the above sections (i.e., presence or absence of other 90S particle proteins), these experiments could shed light on the intrinsic components of each subunit and, at the same time, reveal close interactions with neighboring subunits when in the context of partially assembled preribosomal particles. Consistent with this hypothesis, we found that the selected bait proteins associated with different sets of proteins depending on the type of protein eliminated in the original yeast strain. In the case of Pwp2p, we observed that it associated with intrinsic components of the Pwp2p/UTP-B subunit and an integral component of the Mpp10p complex (Mpp10p) in the absence of Nan1p (Fig. 5; see Fig. S4A in the supplemental material). By contrast, in the absence of Rrp7p, Pwp2p was found associated with additional 90S proteins, including the UTP-A/t-UTP complex (Fig. 5; see Fig. S4B in the supplemental material). This differential proteomic pattern is fully consistent with our prior experiments showing that Pwp2p is present as both free and 35S pre-RNA-associated complexes in the absence of Rrp7p, but it is only detected as free 15 to 20S monoparticles in the absence of Nan1p (Fig. 2C and D and 3C and D). A similar script was found with Rrp7, since this protein bound primarily to UTP-C components in the absence of Nan1p (Fig. 5; see Fig. S4C in the supplemental material) but also associated with UTP-A/t-UTP components and three additional 90S particle proteins when Pwp2p was the protein missing in the lysates (Fig. 5; see Fig. S4D in the supplemental material). Very importantly, UTP-A/t-UTP components, together with Rrp5p, were the only proteins commonly found associated with Rrp7p and Pwp2p when these proteins were isolated from Pwp2p- and Rrp7p-deficient cells, respectively, further confirming that these two subunits bind independently to the UTP-A/t-UTP·35S pre-RNA complex (Fig. 5). In agreement with the idea of two independent assembling branches for the generation of the 90S particle, the spectra of Nan1p-binding proteins were also different depending on whether Nan1p was immunopurified from Pwp2p- or Rrp7p-deficient cells. In the former case, we found that Nan1p could bind only other UTP-A/t-UTP proteins and Rrp5p (Fig. 5; see Fig. S4E in the supplemental material). However, we did not detect the UTP-A-specific Pol5p, indicating that the Nan1p subunit that we are detecting is probably the t-UTP subcomplex (Fig. 5; see Fig. S4E in the supplemental material). Surprisingly, we did not find any UTP-C protein associated with Nan1p in these immunopurifications, suggesting that some Nan1p-associated proteins might be lost during the purification procedure. Consistent with this, we previously observed that Nan1p immunoprecipitates are very inefficient in pulling the 35S pre-rRNA precursor (Fig. 3B and 4B). When we performed the same set of experiments with another UTP-A/t-UTP component (Utp4p), we found the same set of Nan1p-binding proteins, but, in addition, we could recover substoichiometric amounts of Utp22p, an integral component of the UTP-C complex (Fig. 5; see Fig. S4F in the supplemental material). In the absence of Rrp7, we found that Nan1p associated with its t-UTP complex counterparts and, additionally, with a wider collection of factors that included Pwp2p/UTP-B and other 90S particle components (Fig. 5; see Fig. S4G in the supplemental material), a result consistent with the previous observations indicating that Nan1p, Pwp2p, and the U3 RNA remained associated with the 35S pre-rRNA upon Rrp7p elimination (Fig. 2C and D and 4A and B). Taken collectively, these results allowed us to correlate unequivocally the 90S particle subunits detected in our gradient fractionation experiments with the previously reported UTP complexes, to confirm the rules of engagement of those subunits in the context of the 90S particle, and to elucidate the compositions of two multisubunit intermediaries during the assembly of that preribosomal particle.
Rrp5p is another building block of the 90S particle required for the docking of UTP-C components onto the primary pre-RNA transcript. An intriguing result derived from the aforementioned proteomic experiments was the observation that Rrp5p, a protein not previously assigned to any of the reported 90S particle subcomplexes, was a recurrent binding partner for proteins of the three UTP complexes when they were present in partially assembled 90S particle structures (Fig. 5; see Fig. S4 in the supplemental material). Rrp5p is a 90S particle component essential for the cleavage of the 35S pre-rRNA precursor at the A0, A1, A2, and A3 sites and, consequently, for the production of both 18S and 5.8S pre-RNAs (35) (see Fig. S1 in the supplemental material). We used a strategy similar to that described in previous sections to evaluate the effect of the Rrp5p depletion on the association of the identified 90S particle subunits with the 35S pre-rRNA. We observed that the expression of Rrp5p was essential for the interaction of Rrp7p with the 35S pre-rRNA (Fig. 6A). In contrast, this protein was irrelevant for the proper assembly of the U3 snoRNA (Fig. 6A; see Fig. S5A and B in the supplemental material), Nan1p (see Fig. S5A in the supplemental material), and Pwp2p (see Fig. S5B in the supplemental material) onto large preribosomal particles. Consistent with these results, we observed that the spectra of binding partners of Pwp2p and Nan1p were highly similar when they were immunopurified from either Rrp7p- or Rrp5p-depleted cells (Fig. 5 and 6B and C; see Fig. S4 in the supplemental material). In contrast, Rrp7p could bind only to UTP-C components and not to UTP-A/t-UTP or Pwp2p/UTP-B proteins in the absence of Rrp5p expression (Fig. 5 and 6D). These results indicate that the UTP-C complex requires Rrp5p for its incorporation into early preribosomal particles and, in addition, further confirm the UTP-C-independent association of the Pwp2p/UTP-B-U3 snoRNA branch with the primary pre-rRNA precursor.
An in silico view of the 90S preribosomal particle and its constitutive building blocks. We finally resorted to a bioinformatic-based analysis to extract more information about the internal architecture and topology of the 90S preribosomal particle. To this end, we used several computational methods to generate a high-confidence network of the protein-protein interactions established among the 27 90S proteins identified in our proteomic analyses and, for the sake of completeness, of five additional ones not detected in our immunopurifications that belonged to the UTP-C (Cka2, Ckb1, and Ckb2), Pwp2p/UTP-B (Utp21p), and Mpp10p (Imp3) subcomplexes. With this strategy, we could probe bioinformatically over 60% (32 proteins) of all known 90S particle components. To strengthen the significance of the analysis, the network was built by compiling the protein-protein interaction data generated in the present report with additional information derived from previously published proteomic analyses (see Material and Methods). This approach led to the detection of 234 independent pairwise interactions (see Fig. S6 in the supplemental material), which were used subsequently to develop a final interactome network of 90S particle components (Fig. 7). This analysis pinpointed a subgroup of 15 heavily interconnected 90S proteins that included Utp22p (a UTP-C member), Utp20p, and all components of the t-UTP and Pwp2p/UTP-B subunits (Fig. 7A). Given the extensive number of interactions established by these first-order interactors, it is likely that they form a particle core that stabilizes the rest of the 90S particle components. The remaining set of analyzed proteins showed up in the analysis as second-order interactors, since they establish many fewer binary associations and are seen at the periphery of the 90S core structure (Fig. 7A). These peripheral molecules include five UTP-C proteins that are linked to the core elements through Utp22p (Fig. 7A) and, in addition, a heterogeneous subset of proteins connected to the particle core via associations with different first-order interactors (Fig. 7A). This subset of proteins includes, among others, the Mpp10p complex (Fig. 7A).
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Our results also indicate that the 90S preribosome is a ribonucleoprotein complex that assembles in a highly hierarchical and topologically defined manner (Fig. 8). The t-UTP subunit is at the pinnacle of this hierarchical process, since it is required for the subsequent assembly of the rest of identified subunits, including essential components of the 90S preribosome such as the U3 snoRNP. Because t-UTP does not depend on other subunits for the binding to the 35S pre-rRNA, we propose that its assembly is a very early and autonomous step (Fig. 8). The rest of the 90S particle modules bind to the t-UTP-primed 35S pre-rRNA following two different, and mutually independent, assembly branches (Fig. 8). In one of these branches, the U3 snoRNP and the Pwp2p/UTP-B subunit bind to the 35S pre-rRNA and the t-UTP subunit to form a highly stable intermediate of the 90S preribosomal particle (Fig. 8). This assembly branch contains additional proteins and/or subunits, because parallel proteomic experiments indicate that nine additional proteins (Mpp10p, Imp4p, Utp20p, Bms1p, Kre33p, Enp2p, Noc4p, Krr1p, and Nop1p) assemble onto preribosomes in conjunction with the Pwp2p/UTP-B subunit in an UTP-C- and Rrp5p-independent manner (Fig. 8). Although we still do not know whether this whole cohort of proteins incorporates onto the 35S pre-rRNA at the same time or in subsequent assembly steps, previous evidence suggests that at least some of them (Mpp10p and Imp4p) do assemble concurrently with the Pwp2p/UTP-B subunit and the U3 snoRNP (6, 15). In the other assembly branch, the growth of the particle requires the initial incorporation of Rrp5p that facilitates the subsequent binding of the UTP-C module (Fig. 8). Unlike the case of the coassembly of Pwp2p/UTP-B, U3 snoRNP, and the Mpp10p subunits, our proteomic experiments indicate that Rrp5p and UTP-C do not have to assemble simultaneously, because large amounts of Rrp5p are found in the partially assembled 90S particle intermediates formed in cells lacking the UTP-C component Rrp7p. As a consequence of this bifurcated incorporation of building blocks onto the t-UTP-primed 35S pre-rRNA precursor, we could identify intermediary assembly states of the 90S particle that contain either t-UTP·Pwp2p/UTP-B·U3 snoRNP·Mpp10p or, alternatively, the t-UTP·Rrp5p·UTP-C subunits bound to the 35S pre-rRNA. Importantly, components of these partially assembled complexes are readily detected in association with the full-length 35S pre-rRNA transcript, ruling out the possibility that they could represent either free preassembly stages or postcleavage 90S particle by-products. Although this hierarchical assembly evokes a temporal order of incorporation of 90S subunits onto the 35S pre-rRNA, it should be noted that our data are compatible with other scenarios. For example, it is possible that the upstream position of t-UTP in this assembly protocol is due not to an earlier incorporation of this subunit onto the 35S pre-rRNA but, rather, to the generation of a highly stable t-UTP/UTP-A·Pwp2p/UTP-B·UTP-C complex that maintains its integrity during the sedimentation and immunoprecipitation experiments.
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Our data showing the stepwise assembly of the 90S particle are reminiscent of the changes observed in the 90S preribosome in chromatin spreads by electron microscopy. Due to the megadalton size of 90S particles, these structures can be visualized using this technique as "knobs" present at the 5' ends of the nascent pre-rRNA transcripts (7, 26). Interestingly, it has been observed that such knobs are rather dynamic structures, because they change in size and shape as transcription of the 35S pre-rRNA proceeds (26). Although these changes are evocative of some of the assembly steps described here, it should be noted that there are important differences between the results obtained using these two experimental avenues that preclude any side-by-side comparisons. For instance, it has been shown that the early small knobs detected by electron microscopy disappear upon U3 snoRNP depletion (26), indicating that they cannot correspond to the initial t-UTP assembly stage proposed here. It is possible, therefore, that the putative Nan1p/t-UTP·35S pre-RNA complex is too small to be detected in electron micrographs or, more likely, that it lacks enough stability to survive the highly stringent conditions used for the preparation of the chromatin spreads. The future analysis of each of our conditional mutant strains using electron microscopy will clarify these discrepancies and, at the same time, will provide a more mechanistic interpretation of the dynamic changes undergone by the terminal knobs during 35S pre-rRNA transcription.
In addition to the obvious advantage of reducing the levels of freedom involved in the assembly of the numerous components that form part of the mature 90S preribosome, the subdivision of this particle into smaller building blocks offers further functional advantages. For instance, it is tempting to speculate that the stepwise assembly of the 90S subunits could be potentially used to separate in time and space different folding, maturation, or site-specific modification events that take place on the pre-rRNA. The existence of preassembled subunits can also facilitate the participation of specific 90S protein subsets in 90S particle-independent processes such as 60S ribosomal subunit biogenesis (as could be the case for the putative Rrp5p/Has1p complex found in this study) or rDNA-related transcriptional events (as is the case for the t-UTP subunit). These and other functional possibilities can be addressed in the future by further studying the preribosome subcomplexes and assembly intermediates described in this study.
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ACKNOWLEDGMENTS |
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M.D.'s work is supported by the Spanish Ministry of Education and Science (MEC) (BFU2005-06421) and the Castilla-León Autonomous Government (SA040/02). X.R.B.'s work is supported by grants from the U.S. NCI/NIH (5RO1-CA73735-10), MEC (SAF2003-00028), and Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/0001, Fondo de Investigaciones Sanitarias [FIS], Carlos III Institute, Spanish Ministry of Health). J.D.L.R.'s work is possible due to a grant from the Spanish Ministry of Health (PI030920). J.P.-F. was supported by a postdoctoral fellowship from the Spanish Cooperative Research Network of Cancer Centers (C03/10, Spanish Ministry of Health) and the RTICC (RD06/0020/0001). All Spanish funding is cosponsored by the European FEDER Program.
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FOOTNOTES |
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Published ahead of print on 21 May 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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REFERENCES |
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745 (optimal score = 1,000) are depicted as closed circles and identified by arabic numerals. The specific stability value of each branching point is indicated in parentheses.