Making ribosomes

Microbial Cell, 2017

Genetics, 2013

Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structur...

Molecular Cell, 2001

. Yeast 35S pre-rRNA Donald F. Hunt, 3 and John L. Woolford, Jr. 1,5 is present in a 90S preribosomal particle (pre-rRNP), 1 Department of Biological Sciences which is converted to a 43S rRNP containing 20S pre-Carnegie Mellon University rRNA and a series of 66S rRNPs containing 27S or 25.5S Pittsburgh, Pennsylvania 15213 plus 7S pre-rRNAs (Trapman et al., 1975). The 43S rRNP 2 Laboratory of Cellular and Structural Biology is exported from the nucleolus to the cytoplasm, where The Rockefeller University it matures into a 40S ribosomal subunit containing 18S New York, New York 10021 rRNA (Udem and Warner, 1973; Trapman and Planta, 3 Departments of Chemistry and Pathology 1976). The 66S rRNPs are converted to 60S rRNPs con-University of Virginia taining 25S, 5.8S, and 5S rRNA through a series of matu-Charlottesville, Virginia 22901 ration steps in the nucleolus, nucleoplasm, and cyto-4 MDS Proteomics plasm (Eisinger et al., 1997; Kressler et al., 1999a; Ho Charlottesville, Virginia 22903 et al., 2000; Milkereit et al., 2001). Molecular genetic approaches in yeast have identified more than 70 different nonribosomal proteins involved in Summary ribosome biogenesis (reviewed in Kressler et al., 1999a; Venema and Tollervey , 1999). The roles of these proteins The pathway and complete collection of factors that in ribosome biogenesis are best characterized on the orchestrate ribosome assembly are not clear. To adbasis of their mutant phenotype with respect to RNA dress these problems, we affinity purified yeast preribometabolism (Venema and Tollervey, 1999). In many musomal particles containing the nucleolar protein Nop7p tants, processing of certain pre-rRNAs appears to be and developed means to separate their components. blocked, and those RNAs accumulate. In other mutants, Nop7p is associated primarily with 66S preribosomes rRNA-processing intermediates are made but rapidly containing either 27SB or 25.5S plus 7S pre-rRNAs. degraded, presumably due to aberrations in assembly Copurifying proteins identified by mass spectrometry of the rRNPs. While some proteins identified in these include ribosomal proteins, nonribosomal proteins mutant screens have clear functions in rRNA processing previously implicated in 60S ribosome biogenesis, and or modification, there is a large class of so-called "asproteins not known to be involved in ribosome producsembly factors" with no obvious functions. Although it tion. Analysis of strains mutant for eight of these prois presumed that most of these proteins associate with teins not previously implicated in ribosome biogenesis assembling ribosomes in pre-rRNPs, only a few proteins showed that they do participate in this pathway. These have been shown to cosediment on gradients with preresults demonstrate that proteomic approaches in ribosomes Zanchin et al., 1997; de la Cruz et al., 1998; concert with genetic tools provide powerful means to Si and Maitra, 1999; Billy et al., 2000; Bousquet-Antonelli purify and characterize ribosome assembly intermeet al., 2000; Milkereit et al., 2001). Many nonribosomal diates. proteins that might function in ribosome biogenesis also have been found in metazoan pre-rRNPs, although only a few have been studied in detail (reviewed in Piñ ol-Introduction Roma, 1999; Olson et al., 2000) . Most have not been identified, and the specificity of their association with Eukaryotic ribosome assembly is a complex process pre-rRNPs has not been established. occurring primarily in the nucleolus, a subcompartment Thus, we lack an understanding of the functions of of the nucleus where rRNA is transcribed, covalently most proteins involved in eukaryotic ribosome biogenemodified, processed, and assembled with ‫08ف‬ different sis and the specific ribosome maturation steps in which ribosomal proteins (Woolford and Warner, 1991; Venthey participate. Because many of these proteins may ema and Tollervey, 1999). In Saccharomyces cerevisiae, function in the context of preribosomal particles, it is three of the four rRNAs in mature ribosomal subunits, important to characterize the composition of these ribo-18S, 5.8S, and 25S rRNA, are derived from the 35S rRNA nucleoprotein complexes in detail. To achieve this goal, primary transcript, synthesized by RNA polymerase I. we affinity purified preribosomal particles containing 5S rRNA is transcribed separately by RNA polymerase molecules associated with yeast nucleolar protein III. After transcription, pre-rRNA undergoes multiple Nop7p (C. Adams et al., submitted) and identified their conserved modifications. External and internal tranprotein and RNA constituents. Purified Nop7p is associscribed spacer sequences are removed from 35S preated primarily with 27SB, 25.5S, and 7S pre-rRNAs, indicating that it is present in two consecutive 66S ribosome assembly intermediates in the pathway that produces 5 Correspondence: [email protected] 6 These authors contributed equally to this work. mature 60S ribosomal subunits. Proteins associated Affinity Purification and Identification of Proteins Associated Saccharomyces cerevisiae: DEAD-box proteins and related families. Trends Biochem. Sci. 24, 192-198. with Nop7p The TAP-tag cassette was PCR amplified from pBS1539 using two Deshmukh, M., Tsay, Y.-F., Paulovich, A.G., and Woolford, J.L., Jr. oligonucleotide primers that include sequences upstream and (1993). Yeast ribosomal protein L1 is required for the stability of downstream of the NOP7 stop codon. Amplified DNA was transnewly synthesized 5S rRNA and the assembly of 60S ribosomal formed into yeast; Trp ϩ transformants expressing Nop7p with the subunits. Mol. Cell. Biol. 13, 2835-2845. TAP tag at its carboxyl terminus were identified by immunoblotting Dunbar, D.A., Dragon, F., Lee, S.J., and Baserga, S.J. (2000). A and confirmed by genomic PCR. Cells expressing Nop7p-TAP were nucleolar protein related to ribosomal protein L7 is required for an grown in YEPGlu to 1 ϫ 10 8 cells/ml, and cell-free extracts were early step in large ribosomal subunit biogenesis. Proc. Natl. Acad. prepared as described in Zanchin and Goldfarb (1999), except that Sci. USA 97, 13027-13032. buffer A contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Eisinger, D.P., Dick, F.A., Denke, E., and Trumpower, B.L. (1997). EDTA (pH 8), and 0.1% NP-40. Affinity purification was performed Sqt1, which encodes an essential WD domain protein of Saccharoas described in Rigaut et al. (1999), except wash volumes were myces cerevisiae, suppress dominant-negative mutations of the riincreased 10-fold. Proteins were recovered from the final eluate bosomal protein gene QSR1. Mol. Cell. Biol. 17, 5146-5155. by precipitation in 10% TCA, dissolved in SDS sample buffer, and Fabian, G.R., and Hopper, A.K. (1987). RRP1, a Saccharomyces separated by SDS-PAGE on 4%-20% polyacrylamide NOVEX gels cerevisiae gene affecting rRNA processing and production of mature (Invitrogen). ribosomal subunits. J. Bacteriol. 169, 1571-1578. Affinity-purified proteins were separated by formic acid HPLC followed by SDS-PAGE. Each protein sample was dissolved in SDS Fath, S., Milkereit, P., Podtelejnikov, A.V., Bischler, N., Schultz, P., sample buffer and loaded onto a Vydac C4 column preequilibrated Bier, M., Mann, M., and Tschochner, H. (2000). Association of yeast with 20% solvent B (60% [v/v] formic acid in water), 80% solvent A RNA polymerase I with a nucleolar substructure active in rRNA syn-(60% [v/v] formic acid, 33% acetonitrile). A linear gradient of solvent thesis and processing. J. Cell Biol. 149, 575-589. B from 20% to 35% was run over 10 min, then to 100% was run Geerlings, T.H., Vos, J.C., and Raué , H.A. (2000). The final step in the over 270 min, and finally B was kept at 100% for an additional formation of 25S rRNA in Saccharomyces cerevisiae is performed by 20 min. Proteins in fractions were precipitated with TCA/sodium 5Ј → 3Ј exonucleases. RNA 6, 1698-1703. deoxycholate, suspended in SDS sample buffer, and separated by Hadjiolov, A. (1985). The nucleolus and ribosome biogenesis. Cell SDS-PAGE. Gel bands were excised, destained, and enzymatic di-Biol. Monogr. 12, 1-268. gestion was performed in-gel . Resultant peptides were extracted Ho, J.H.-N., Kallstrom, G., and Johnson, A.W. (2000). Nascent 60S and analyzed by mass spectrometry using a PerSeptive Biosystems ribosomal subunits enter the free pool bound by Nmd3p. RNA 6, MALDI-TOF Voyager DE-RP Mass Spectrometer. The search engine 1625-1634. PROWL was used for database search (http://prowl.rockefeller.edu). Huber, M.D., Dworet, J.H., Shire, K., Frappier, L., and McAlear, M.S. (2000). The budding yeast homolog of the human EBNA1-binding Acknowledgments protein 2 (Ebp2p) is an essential nucleolar protein required for pre-rRNA processing. J. Biol. Chem. 275, 28764-28773. We thank N. Zanchin and D. Goldfarb for antibodies versus Nip7p; B. Seraphin for TAP plasmid pBS1539; and L. Visomirski-Robic, P. Jacobs-Anderson, J.S., and Parker, R. (1998). The 3Ј to 5Ј degrada-Antunez de Mayolo, J. Brodsky, J. Lopez, T.G. Kinzy, and J. Warner tion of yeast mRNAs is a general mechanism for mRNA turnover for critical reading of the manuscript. This work was supported by that requires the SK12 DEVH box protein and 3Ј to 5Ј exonucleases grants from the Rita Allen, Sinsheimer,and Hirschl Foundations and of the exosome complex. EMBO J. 17, 1497-1506. the Rockefeller University to M.R.; NIH grant GM28301 to J.L.W.; Kressler, D., Linder, P., and de la Cruz, J. (1999a). Protein trans-NIH grant GM18708 to J.R.; NIH grant GM37537 to D.F.H.; NIH grant acting factors involved in ribosome biogenesis in Saccharomyces GM19937 to E.H.; and by funds from the government of Thailand cerevisiae. Mol. Cell. Biol. 19, 7897-7912. provided to P.H. Kressler, D., Rojo, M., Linder, P., and de la Cruz, J. (1999b). Spb1p is a putative methyltransferase required for 60S ribosomal subunit .H., and the yeast protein similar to the RNA 3Ј-phosphate cyclase, associ-Chartrand, P....

Journal of Biological Chemistry, 2012

Background: The contribution of ribosomal proteins to ribosome assembly and function is often not well understood. Results: L40 assembles within the cytoplasm into pre-60 S subunits and is required for Nmd3 and Rlp24 recycling. Conclusion: L40 contributes to formation of 60 S subunits competent for subunit joining and translation elongation. Significance: Our analysis of L40 function reveals an additional step during cytoplasmic pre-60 S maturation events.

RNA (New York, N.Y.), 2016

In higher eukaryotes, pre-rRNA processing occurs almost exclusively post-transcriptionally. This is not the case in rapidly dividing yeast, as the majority of nascent pre-rRNAs are processed cotranscriptionally, with cleavage at the A2site first releasing a pre-40S ribosomal subunit followed by release of a pre-60S ribosomal subunit upon transcription termination. Ribosome assembly is driven in part by hierarchical association of assembly factors and r-proteins. Groups of proteins are thought to associate with pre-ribosomes cotranscriptionally during early assembly steps, whereas others associate later, after transcription is completed. Here we describe a previously uncharacterized phenotype observed upon disruption of ribosome assembly, in which normally late-binding proteins associate earlier, with pre-ribosomes containing 35S pre-rRNA. As previously observed by many other groups, we show that disruption of 60S subunit biogenesis results in increased amounts of 35S pre-rRNA, sugge...

PLOS ONE, 2015

Cellular production of ribosomes involves the formation of highly defined interactions between ribosomal proteins (r-proteins) and ribosomal RNAs (rRNAs). Moreover in eukaryotic cells, efficient ribosome maturation requires the transient association of a large number of ribosome biogenesis factors (RBFs) with newly forming ribosomal subunits. Here, we investigated how r-protein assembly events in the large ribosomal subunit (LSU) rRNA domain II are coordinated with each other and with the association of RBFs in early LSU precursors of the yeast Saccharomyces cerevisiae. Specific effects on the pre-ribosomal association of RBFs could be observed in yeast mutants blocked in LSU rRNA domain II assembly. Moreover, formation of a cluster of r-proteins was identified as a downstream event in LSU rRNA domain II assembly. We analyzed in more detail the functional relevance of eukaryote specific bridges established by this r-protein cluster between LSU rRNA domain II and VI and discuss how they can support the stabilization and efficient processing of yeast early LSU precursor RNAs.

Molecular and Cellular Biology, 2006

Maintaining the appropriate balance between the small and large ribosomal subunits is critical for translation and cell growth. We previously identified the 40S ribosomal protein S2 (rpS2) as a substrate of the protein arginine methyltransferase 3 (RMT3) and reported a misregulation of the 40S/60S ratio in rmt3 deletion mutants of Schizosaccharomyces pombe. For this study, using DNA microarrays, we have investigated the genome-wide biological response of rmt3-null cells to this ribosomal subunit imbalance. Whereas little change was observed at the transcriptional level, a number of genes showed significant alterations in their polysomal-to-monosomal ratios in rmt3⌬ mutants. Importantly, nearly all of the 40S ribosomal proteinencoding mRNAs showed increased ribosome density in rmt3 disruptants. Sucrose gradient analysis also revealed that the ribosomal subunit imbalance detected in rmt3-null cells is due to a deficit in small-subunit levels and can be rescued by rpS2 overexpression. Our results indicate that rmt3-null fission yeast compensate for the reduced levels of small ribosomal subunits by increasing the ribosome density, and likely the translation efficiency, of 40S ribosomal protein-encoding mRNAs. Our findings support the existence of autoregulatory mechanisms that control ribosome biosynthesis and translation as an important layer of gene regulation.

Nucleic Acids Research, 2010

Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks.

Journal of bacteriology, 1979

Centrifugal elutriation was used to separate yeast cells by their cell cycle position. The rate of synthesis of ribosomal proteins showed a constant exponential increase through the cell cycle.

Rna, 2002