Статті в журналах: "Pre-60S particle"

1

Kressler, Dieter, Daniela Roser, Brigitte Pertschy, and Ed Hurt. "The AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles." Journal of Cell Biology 181, no. 6 (June 16, 2008): 935–44. http://dx.doi.org/10.1083/jcb.200801181.

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Ribosome biogenesis takes place successively in the nucleolar, nucleoplasmic, and cytoplasmic compartments. Numerous nonribosomal factors transiently associate with the nascent ribosomes, but the mechanisms driving ribosome formation are mostly unknown. Here, we show that an energy-consuming enzyme, the AAA-type (ATPases associated with various cellular activities) ATPase Rix7, restructures a novel pre-60S particle at the transition from the nucleolus to nucleoplasm. Rix7 interacts genetically with Nsa1 and is targeted to the Nsa1-defined preribosomal particle. In vivo, Nsa1 cannot dissociate from pre-60S particles in rix7 mutants, causing nucleolar Nsa1 to escape to the cytoplasm, where it remains associated with aberrant 60S subunits. Altogether, our data suggest that Rix7 is required for the release of Nsa1 from a discrete preribosomal particle, thereby triggering the progression of 60S ribosome biogenesis.

2

Hung, Nai-Jung, Kai-Yin Lo, Samir S. Patel, Kara Helmke, and Arlen W. Johnson. "Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast." Molecular Biology of the Cell 19, no. 2 (February 2008): 735–44. http://dx.doi.org/10.1091/mbc.e07-09-0968.

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We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

3

Gadal, Olivier, Daniela Strauss, Elisabeth Petfalski, Pierre-Emmanuel Gleizes, Nicole Gas, David Tollervey, and Ed Hurt. "Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing." Journal of Cell Biology 157, no. 6 (June 10, 2002): 941–52. http://dx.doi.org/10.1083/jcb.200111039.

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Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.

4

Yao, Y., E. Demoinet, C. Saveanu, P. Lenormand, A. Jacquier, and M. Fromont-Racine. "Ecm1 is a new pre-ribosomal factor involved in pre-60S particle export." RNA 16, no. 5 (March 26, 2010): 1007–17. http://dx.doi.org/10.1261/rna.2012310.

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5

Bagatelli, Felipe F. M., Francisca N. de Luna Vitorino, Julia P. C. da Cunha, and Carla C. Oliveira. "The ribosome assembly factor Nop53 has a structural role in the formation of nuclear pre-60S intermediates, affecting late maturation events." Nucleic Acids Research 49, no. 12 (June 14, 2021): 7053–74. http://dx.doi.org/10.1093/nar/gkab494.

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Abstract Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S ‘foot’. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.

6

Fatica, Alessandro, Andrew D. Cronshaw, Mensur Dlakić, and David Tollervey. "Ssf1p Prevents Premature Processing of an Early Pre-60S Ribosomal Particle." Molecular Cell 9, no. 2 (February 2002): 341–51. http://dx.doi.org/10.1016/s1097-2765(02)00458-6.

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7

Dechampesme, Anne-Marie, Olga Koroleva, Isabelle Leger-Silvestre, Nicole Gas, and Sylvie Camier. "Assembly of 5S Ribosomal RNA Is Required at a Specific Step of the Pre-rRNA Processing Pathway." Journal of Cell Biology 145, no. 7 (June 28, 1999): 1369–80. http://dx.doi.org/10.1083/jcb.145.7.1369.

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A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.

8

Prohaska, Kimberly, and Noreen Williams. "Assembly of the Trypanosoma brucei 60S Ribosomal Subunit Nuclear Export Complex Requires Trypanosome-Specific Proteins P34 and P37." Eukaryotic Cell 8, no. 1 (August 22, 2008): 77–87. http://dx.doi.org/10.1128/ec.00234-08.

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ABSTRACT We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.

9

Castle, Christopher D., Erica K. Cassimere, and Catherine Denicourt. "LAS1L interacts with the mammalian Rix1 complex to regulate ribosome biogenesis." Molecular Biology of the Cell 23, no. 4 (February 15, 2012): 716–28. http://dx.doi.org/10.1091/mbc.e11-06-0530.

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The coordination of RNA polymerase I transcription with pre-rRNA processing, preribosomal particle assembly, and nuclear export is a finely tuned process requiring the concerted actions of a number of accessory factors. However, the exact functions of some of these proteins and how they assemble in subcomplexes remain poorly defined. LAS1L was first described as a nucleolar protein required for maturation of the 60S preribosomal subunit. In this paper, we demonstrate that LAS1L interacts with PELP1, TEX10, and WDR18, the mammalian homologues of the budding yeast Rix1 complex, along with NOL9 and SENP3, to form a novel nucleolar complex that cofractionates with the 60S preribosomal subunit. Depletion of LAS1L-associated proteins results in a p53-dependent G1 arrest and leads to defects in processing of the pre-rRNA internal transcribed spacer 2 region. We further show that the nucleolar localization of this complex requires active RNA polymerase I transcription and the small ubiquitin-like modifier–specific protease SENP3. Taken together, our data identify a novel mammalian complex required for 60S ribosomal subunit synthesis, providing further insight into the intricate, yet poorly described, process of ribosome biogenesis in higher eukaryotes.

10

Warren, Alan J. "Shwachman-Diamond Syndrome and the Quality Control of Ribosome Assembly." Blood 128, no. 22 (December 2, 2016): SCI—42—SCI—42. http://dx.doi.org/10.1182/blood.v128.22.sci-42.sci-42.

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Abstract The synthesis of new ribosomes is a fundamental conserved process in all cells. Ribosomes are pre-assembled in the nucleus and subsequently exported to the cytoplasm where they acquire functionality through a series of final maturation steps that include formation of the catalytic center, recruitment of the last remaining ribosomal proteins and the removal of inhibitory assembly factors. Surprisingly, a number of key factors (SBDS, DNAJC21, RPL10 (uL16)) involved in late cytoplasmic maturation of the large (60S) ribosomal subunit are mutated in both inherited and sporadic forms of leukemia. In particular, biallelic mutations in the SBDS gene cause Shwachman-Diamond syndrome (SDS), a recessive bone marrow failure disorder with significant predisposition to acute myeloid leukemia. By using the latest advances in single-particle cryo-electron microscopy to elucidate the function of the SBDS protein, we have uncovered an elegant mechanism that couples final maturation of the 60S subunit to a quality control assessment of the structural integrity of the active sites of the ribosome. Further molecular dissection of this pathway may inform novel therapeutic strategies for SDS and leukemia more generally. References: 1. Weis F, Giudice E, Churcher M,et al. Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat Struct Mol Biol, (2015) Nov;22(11):914-9. 2. Wong CC, Traynor D, Basse N, et al. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev (2011) 25: 917-929. 4. Menne TM, Goyenechea B, Sánchez-Puig N, et al. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures No relevant conflicts of interest to declare.

11

Baudin-Baillieu, A., D. Tollervey, C. Cullin, and F. Lacroute. "Functional analysis of Rrp7p, an essential yeast protein involved in pre-rRNA processing and ribosome assembly." Molecular and Cellular Biology 17, no. 9 (September 1997): 5023–32. http://dx.doi.org/10.1128/mcb.17.9.5023.

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During the functional analysis of open reading frames (ORFs) identified during the sequencing of chromosome III of Saccharomyces cerevisiae, the previously uncharacterized ORF YCL031C (now designated RRP7) was deleted. RRP7 is essential for cell viability, and a conditional null allele was therefore constructed, by placing its expression under the control of a regulated GAL promoter. Genetic depletion of Rrp7p inhibited the pre-rRNA processing steps that lead to the production of the 20S pre-rRNA, resulting in reduced synthesis of the 18S rRNA and a reduced ratio of 40S to 60S ribosomal subunits. A screen for multicopy suppressors of the lethality of the GAL::rrp7 allele isolated the two genes encoding a previously unidentified ribosomal protein (r-protein) that is highly homologous to the rat r-protein S27. When present in multiple copies, either gene can suppress the lethality of an RRP7 deletion mutation and can partially restore the ribosomal subunit ratio in Rrp7p-depleted cells. Deletion of both r-protein genes is lethal; deletion of either single gene has an effect on pre-rRNA processing similar to that of Rrp7p depletion. We believe that Rrp7p is required for correct assembly of rpS27 into the preribosomal particle, with the inhibition of pre-rRNA processing appearing as a consequence of this defect.

12

Salih, Karzan Jalal, Owen Duncan, Lei Li, Josua Trösch, and A. Harvey Millar. "The composition and turnover of the Arabidopsis thaliana 80S cytosolic ribosome." Biochemical Journal 477, no. 16 (August 26, 2020): 3019–32. http://dx.doi.org/10.1042/bcj20200385.

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Cytosolic 80S ribosomes contain proteins of the mature cytosolic ribosome (r-proteins) as well as proteins with roles in ribosome biogenesis, protein folding or modification. Here, we refined the core r-protein composition in Arabidopsis thaliana by determining the abundance of different proteins during enrichment of ribosomes from cell cultures using peptide mass spectrometry. The turnover rates of 26 40S subunit r-proteins and 29 60S subunit r-proteins were also determined, showing that half of the ribosome population is replaced every 3–4 days. Three enriched proteins showed significantly shorter half-lives; a protein annotated as a ribosomal protein uL10 (RPP0D, At1g25260) with a half-life of 0.5 days and RACK1b and c with half-lives of 1–1.4 days. The At1g25260 protein is a homologue of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role in the 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins; 26 were enriched more than 10-fold during ribosome enrichment. Some of these enriched proteins have known roles in translation, while others are newly proposed ribosome-associated factors in plants. This analysis provides an improved understanding of A. thaliana ribosome protein content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and how protein turnover can help identify r-proteins involved in ribosome biogenesis or regulation in plants.

13

Kemmler, Stefan, Laura Occhipinti, Maria Veisu, and Vikram Govind Panse. "Yvh1 is required for a late maturation step in the 60S biogenesis pathway." Journal of Cell Biology 186, no. 6 (September 21, 2009): 863–80. http://dx.doi.org/10.1083/jcb.200904111.

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Before entering translation, preribosomal particles undergo sequential late maturation steps. In the case of pre-60S particles, these steps involve the release of shuttling maturation factors and transport receptors. In this study, we report a new maturation step in the 60S biogenesis pathway in budding yeast. We show that efficient release of the nucleolar/nuclear ribosomal-like protein Mrt4 (homologous to the acidic ribosomal P-protein Rpp0) from pre-60S particles requires the highly conserved protein Yvh1, which associates only with late pre-60S particles. Cell biological and biochemical analyses reveal that Mrt4 fails to dissociate from late pre-60S particles in yvh1Δ cells, inducing a delay in nuclear pre–ribosomal RNA processing and a pre-60S export defect in yvh1Δ cells. Moreover, we have isolated gain of function alleles of Mrt4 that specifically bypass the requirement for Yvh1 and rescue all yvh1Δ-associated phenotypes. Together, our data suggest that Yvh1-mediated release of Mrt4 precedes cytoplasmic loading of Rpp0 on pre-60S particles and is an obligatory late step toward construction of translation-competent 60S subunits.

14

Pertschy, Brigitte, Cosmin Saveanu, Gertrude Zisser, Alice Lebreton, Martin Tengg, Alain Jacquier, Eva Liebminger, et al. "Cytoplasmic Recycling of 60S Preribosomal Factors Depends on the AAA Protein Drg1." Molecular and Cellular Biology 27, no. 19 (July 23, 2007): 6581–92. http://dx.doi.org/10.1128/mcb.00668-07.

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ABSTRACT Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

15

Paternoga, Helge, Alexander Früh, Ruth Kunze, Bettina Bradatsch, Jochen Baßler, and Ed Hurt. "Mutational Analysis of the Nsa2 N-Terminus Reveals Its Essential Role in Ribosomal 60S Subunit Assembly." International Journal of Molecular Sciences 21, no. 23 (November 30, 2020): 9108. http://dx.doi.org/10.3390/ijms21239108.

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The ribosome assembly factor Nsa2 is part of the Rea1-Rsa4-Nsa2 interconnected relay on nuclear pre-60S particles that is essential for 60S ribosome biogenesis. Cryo-EM structures depict Nsa2 docked via its C-terminal β-barrel domain to nuclear pre-60S particles, whereas the extended N-terminus, consisting of three α-helical segments, meanders between various 25S rRNA helices with the extreme N-terminus in close vicinity to the Nog1 GTPase center. Here, we tested whether this unappreciated proximity between Nsa2 and Nog1 is of functional importance. Our findings demonstrate that a conservative mutation, Nsa2 Q3N, abolished cell growth and impaired 60S biogenesis. Subsequent genetic and biochemical analyses verified that the Nsa2 N-terminus is required to target Nsa2 to early pre-60S particles. However, overexpression of the Nsa2 N-terminus abolished cytoplasmic recycling of the Nog1 GTPase, and both Nog1 and the Nsa2-N (1-58) construct, but not the respective Nsa2-N (1-58) Q3N mutant, were found arrested on late cytoplasmic pre-60S particles. These findings point to specific roles of the different Nsa2 domains for 60S ribosome biogenesis.

16

Kappel, Lisa, Mathias Loibl, Gertrude Zisser, Isabella Klein, Gernot Fruhmann, Christof Gruber, Stefan Unterweger, Gerald Rechberger, Brigitte Pertschy, and Helmut Bergler. "Rlp24 activates the AAA-ATPase Drg1 to initiate cytoplasmic pre-60S maturation." Journal of Cell Biology 199, no. 5 (November 26, 2012): 771–82. http://dx.doi.org/10.1083/jcb.201205021.

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Formation of eukaryotic ribosomes is driven by energy-consuming enzymes. The AAA-ATPase Drg1 is essential for the release of several shuttling proteins from cytoplasmic pre-60S particles and the loading of late joining proteins. However, its exact role in ribosome biogenesis has been unknown. Here we show that the shuttling protein Rlp24 recruited Drg1 to pre-60S particles and stimulated its ATPase activity. ATP hydrolysis in the second AAA domain of Drg1 was required to release shuttling proteins. In vitro, Drg1 specifically and exclusively extracted Rlp24 from purified pre-60S particles. Rlp24 release required ATP and was promoted by the interaction of Drg1 with the nucleoporin Nup116. Subsequent ATP hydrolysis in the first AAA domain dissociated Drg1 from Rlp24, liberating both proteins for consecutive cycles of activity. Our results show that release of Rlp24 by Drg1 defines a key event in large subunit formation that is a prerequisite for progression of cytoplasmic pre-60S maturation.

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Wandrey, Franziska, Christian Montellese, Krisztian Koos, Lukas Badertscher, Lukas Bammert, Atlanta G. Cook, Ivo Zemp, Peter Horvath, and Ulrike Kutay. "The NF45/NF90 Heterodimer Contributes to the Biogenesis of 60S Ribosomal Subunits and Influences Nucleolar Morphology." Molecular and Cellular Biology 35, no. 20 (August 3, 2015): 3491–503. http://dx.doi.org/10.1128/mcb.00306-15.

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The interleukin enhancer binding factors ILF2 (NF45) and ILF3 (NF90/NF110) have been implicated in various cellular pathways, such as transcription, microRNA (miRNA) processing, DNA repair, and translation, in mammalian cells. Using tandem affinity purification, we identified human NF45 and NF90 as components of precursors to 60S (pre-60S) ribosomal subunits. NF45 and NF90 are enriched in nucleoli and cosediment with pre-60S ribosomal particles in density gradient analysis. We show that association of the NF45/NF90 heterodimer with pre-60S ribosomal particles requires the double-stranded RNA binding domains of NF90, while depletion of NF45 and NF90 by RNA interference leads to a defect in 60S biogenesis. Nucleoli of cells depleted of NF45 and NF90 have altered morphology and display a characteristic spherical shape. These effects are not due to impaired rRNA transcription or processing of the precursors to 28S rRNA. Consistent with a role of the NF45/NF90 heterodimer in nucleolar steps of 60S subunit biogenesis, downregulation of NF45 and NF90 leads to a p53 response, accompanied by induction of the cyclin-dependent kinase inhibitor p21/CIP1, which can be counteracted by depletion of RPL11. Together, these data indicate that NF45 and NF90 are novel higher-eukaryote-specific factors required for the maturation of 60S ribosomal subunits.

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Rosado, Iván V., Christophe Dez, Simon Lebaron, Michèle Caizergues-Ferrer, Yves Henry, and Jesús de la Cruz. "Characterization of Saccharomyces cerevisiae Npa2p (Urb2p) Reveals a Low-Molecular-Mass Complex Containing Dbp6p, Npa1p (Urb1p), Nop8p, and Rsa3p Involved in Early Steps of 60S Ribosomal Subunit Biogenesis." Molecular and Cellular Biology 27, no. 4 (December 4, 2006): 1207–21. http://dx.doi.org/10.1128/mcb.01523-06.

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ABSTRACT We report the characterization of the yeast Npa2p (Urb2p) protein, which is essential for 60S ribosomal subunit biogenesis. We identified this protein in a synthetic lethal screening with the rsa3 null allele. Rsa3p is a genetic partner of the putative RNA helicase Dbp6p. Mutation or depletion of Npa2p leads to a net deficit in 60S subunits and a decrease in the levels all 27S pre-rRNAs and mature 25S and 5.8S rRNAs. This is likely due to instability of early pre-60S particles. Consistent with a role of Npa2p in 60S subunit biogenesis, green fluorescent protein-tagged Npa2p localizes predominantly to the nucleolus and TAP-tagged Npa2p sediments with large complexes in sucrose gradients and is associated mainly with 27SA2 pre-rRNA-containing preribosomal particles. In addition, we reveal a genetic synthetic interaction between Npa2p, several factors required for early steps of 60S subunit biogenesis (Dbp6p, Dbp7p, Dbp9p, Npa1p, Nop8p, and Rsa3p), and the 60S protein Rpl3p. Furthermore, coimmunoprecipitation and gel filtration analyses demonstrated that at least Npa2p, Dbp6p, Npa1p, Nop8p, and Rsa3p are present together in a subcomplex of low molecular mass whose integrity is independent of RNA. Our results support the idea that these five factors work in concert during the early steps of 60S subunit biogenesis.

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Saveanu, Cosmin, Abdelkader Namane, Pierre-Emmanuel Gleizes, Alice Lebreton, Jean-Claude Rousselle, Jacqueline Noaillac-Depeyre, Nicole Gas, Alain Jacquier, and Micheline Fromont-Racine. "Sequential Protein Association with Nascent 60S Ribosomal Particles." Molecular and Cellular Biology 23, no. 13 (July 1, 2003): 4449–60. http://dx.doi.org/10.1128/mcb.23.13.4449-4460.2003.

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ABSTRACT Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.

20

Thomson, Emma, and David Tollervey. "The Final Step in 5.8S rRNA Processing Is Cytoplasmic in Saccharomyces cerevisiae." Molecular and Cellular Biology 30, no. 4 (December 14, 2009): 976–84. http://dx.doi.org/10.1128/mcb.01359-09.

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ABSTRACT The 18S rRNA component of yeast (Saccharomyces cerevisiae) 40S ribosomes undergoes cytoplasmic 3′ cleavage following nuclear export, whereas exported pre-60S subunits were believed to contain only mature 5.8S and 25S rRNAs. However, in situ hybridization detected 3′-extended forms of 5.8S rRNA in the cytoplasm, which were lost when Crm1-dependent preribosome export was blocked by treatment with leptomycin B (LMB). LMB treatment rapidly blocked processing of 6S pre-rRNA to 5.8S rRNA, leading to TRAMP-dependent pre-rRNA degradation. The 6S pre-rRNA was coprecipitated with the 60S export adapter Nmd3 and cytoplasmic 60S synthesis factor Lsg1. The longer 5.8S+30 pre-rRNA (a form of 5.8S rRNA 3′ extended by ∼30 nucleotides) is processed to 6S by the nuclear exonuclease Rrp6, and nuclear pre-rRNA accumulated in the absence of Rrp6. In contrast, 6S to 5.8S processing requires the cytoplasmic exonuclease Ngl2, and cytoplasmic pre-rRNA accumulated in strains lacking Ngl2. We conclude that nuclear pre-60S particles containing the 6S pre-rRNA bind Nmd3 and Crm1 and are exported to the cytoplasm prior to final maturation by Ngl2.

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Dez, Christophe, Carine Froment, Jacqueline Noaillac-Depeyre, Bernard Monsarrat, Michèle Caizergues-Ferrer, and Yves Henry. "Npa1p, a Component of Very Early Pre-60S Ribosomal Particles, Associates with a Subset of Small Nucleolar RNPs Required for Peptidyl Transferase Center Modification." Molecular and Cellular Biology 24, no. 14 (July 15, 2004): 6324–37. http://dx.doi.org/10.1128/mcb.24.14.6324-6337.2004.

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ABSTRACT We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p. In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely. Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles. By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor. Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center. Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.

22

Pratte, Dagmar, Ujjwala Singh, Guillaume Murat, and Dieter Kressler. "Mak5 and Ebp2 Act Together on Early Pre-60S Particles and Their Reduced Functionality Bypasses the Requirement for the Essential Pre-60S Factor Nsa1." PLoS ONE 8, no. 12 (December 2, 2013): e82741. http://dx.doi.org/10.1371/journal.pone.0082741.

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23

Lapik, Yevgeniya R., Julia M. Misra, Lester F. Lau, and Dimitri G. Pestov. "Restricting Conformational Flexibility of the Switch II Region Creates a Dominant-Inhibitory Phenotype in Obg GTPase Nog1." Molecular and Cellular Biology 27, no. 21 (September 4, 2007): 7735–44. http://dx.doi.org/10.1128/mcb.01161-07.

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ABSTRACT Nog1 is a conserved eukaryotic GTPase of the Obg family involved in the biogenesis of 60S ribosomal subunits. Here we report the unique dominant-inhibitory properties of a point mutation in the switch II region of mouse Nog1; this mutation is predicted to restrict conformational mobility of the GTP-binding domain. We show that although the mutation does not significantly affect GTP binding, ectopic expression of the mutant in mouse cells disrupts productive assembly of pre-60S subunits and arrests cell proliferation. The mutant impairs processing of multiple pre-rRNA intermediates, resulting in the degradation of the newly synthesized 5.8S/28S rRNA precursors. Sedimentation analysis of nucleolar preribosomes indicates that defective Nog1 function inhibits the conversion of 32S pre-rRNA-containing complexes to a smaller form, resulting in a drastic accumulation of enlarged pre-60S particles in the nucleolus. These results suggest that conformational changes in the switch II element of Nog1 have a critical importance for the dissociation of preribosome-bound factors during intranucleolar maturation and thereby strongly influence the overall efficiency of the assembly process.

24

Gleizes, Pierre-Emmanuel, Jacqueline Noaillac-Depeyre, Isabelle Léger-Silvestre, Frédéric Teulières, Jean-Yves Dauxois, Denys Pommet, Marie-Claude Azum-Gelade, and Nicole Gas. "Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex." Journal of Cell Biology 155, no. 6 (December 10, 2001): 923–36. http://dx.doi.org/10.1083/jcb.200108142.

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To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus. This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus. Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p–Nup159p–Nsp1p complex. In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome. Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export. Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport. Thus, the Nup82p–Nup159p–Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

25

Hung, Nai-Jung, and Arlen W. Johnson. "Nuclear Recycling of the Pre-60S Ribosomal Subunit-Associated Factor Arx1 Depends on Rei1 in Saccharomyces cerevisiae." Molecular and Cellular Biology 26, no. 10 (May 15, 2006): 3718–27. http://dx.doi.org/10.1128/mcb.26.10.3718-3727.2006.

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ABSTRACT Arx1 and Rei1 are found on late pre-60S ribosomal particles containing the export adaptor Nmd3. Arx1 is related to methionine aminopeptidases (MetAPs), and Rei1 is a C2H2 zinc finger protein whose function in ribosome biogenesis has not been previously characterized. Arx1 and Rei1 localized predominately to the nucleus and cytoplasm, respectively, but could be coimmunoprecipitated, suggesting that they are transiently in the same 60S complex. arx1Δ mutants showed a modest accumulation of 60S subunits in the nucleus, suggesting that Arx1 enhances 60S export. Deletion of REI1 led to cold sensitivity and redistribution of Arx1 to the cytoplasm, where it remained bound to free 60S subunits. However, deletion of ARX1 or the fusion of enhanced GFP (eGFP) to Rpl25 suppressed the cold sensitivity of an rei1Δ mutant. The presence of eGFP on Rpl25 or its neighboring protein Rpl35 reduced the binding of Arx1 to 60S subunits, suggesting that Arx1 binds to 60S subunits in the vicinity of the exit tunnel. Mutations in Arx1 that disrupted its binding to 60S also suppressed an rei1Δ mutant and restored the normal nuclear localization of Arx1. These results indicate that the cold sensitivity of rei1Δ cells is due to the persistence of Arx1 on 60S subunits in the cytoplasm. Furthermore, these results suggest that Rei1 is needed for release of Arx1 from nascent 60S subunits after export to the cytoplasm but not for the subsequent nuclear import of Arx1.

26

DE MARCHIS, M. L. "Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation." RNA 11, no. 4 (April 1, 2005): 495–502. http://dx.doi.org/10.1261/rna.7200205.

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27

Lee, W. C., D. Zabetakis, and T. Mélèse. "NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits." Molecular and Cellular Biology 12, no. 9 (September 1992): 3865–71. http://dx.doi.org/10.1128/mcb.12.9.3865.

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NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.

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Lee, W. C., D. Zabetakis, and T. Mélèse. "NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits." Molecular and Cellular Biology 12, no. 9 (September 1992): 3865–71. http://dx.doi.org/10.1128/mcb.12.9.3865-3871.1992.

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NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.

29

Sydorskyy, Y., D. J. Dilworth, E. C. Yi, D. R. Goodlett, R. W. Wozniak, and J. D. Aitchison. "Intersection of the Kap123p-Mediated Nuclear Import and Ribosome Export Pathways." Molecular and Cellular Biology 23, no. 6 (March 15, 2003): 2042–54. http://dx.doi.org/10.1128/mcb.23.6.2042-2054.2003.

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ABSTRACT Kap123p is a yeast β-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a ΔRAI1-induced decrease in the overall biogenesis efficiency.

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Prattes, Lo, Bergler, and Stanley. "Shaping the Nascent Ribosome: AAA-ATPases in Eukaryotic Ribosome Biogenesis." Biomolecules 9, no. 11 (November 7, 2019): 715. http://dx.doi.org/10.3390/biom9110715.

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AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors.

31

Ma, Chengying, Shan Wu, Ningning Li, Yan Chen, Kaige Yan, Zhifei Li, Lvqin Zheng, Jianlin Lei, John L. Woolford, and Ning Gao. "Structural snapshot of cytoplasmic pre-60S ribosomal particles bound by Nmd3, Lsg1, Tif6 and Reh1." Nature Structural & Molecular Biology 24, no. 3 (January 23, 2017): 214–20. http://dx.doi.org/10.1038/nsmb.3364.

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32

Kressler, Dieter, Jesús de la Cruz, Manuel Rojo, and Patrick Linder. "Dbp6p Is an Essential Putative ATP-Dependent RNA Helicase Required for 60S-Ribosomal-Subunit Assembly inSaccharomyces cerevisiae." Molecular and Cellular Biology 18, no. 4 (April 1, 1998): 1855–65. http://dx.doi.org/10.1128/mcb.18.4.1855.

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A previously uncharacterizedSaccharomyces cerevisiaeopen reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore namedDBP6(DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.

33

Kressler, Dieter, Ed Hurt, Helmut Bergler, and Jochen Baßler. "The power of AAA-ATPases on the road of pre-60S ribosome maturation — Molecular machines that strip pre-ribosomal particles." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1823, no. 1 (January 2012): 92–100. http://dx.doi.org/10.1016/j.bbamcr.2011.06.017.

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34

Saveanu, Cosmin, Jean-Claude Rousselle, Pascal Lenormand, Abdelkader Namane, Alain Jacquier, and Micheline Fromont-Racine. "The p21-Activated Protein Kinase Inhibitor Skb15 and Its Budding Yeast Homologue Are 60S Ribosome Assembly Factors." Molecular and Cellular Biology 27, no. 8 (February 16, 2007): 2897–909. http://dx.doi.org/10.1128/mcb.00064-07.

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ABSTRACT Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G1, and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.

35

Lebaron, Simon, Carine Froment, Micheline Fromont-Racine, Jean-Christophe Rain, Bernard Monsarrat, Michèle Caizergues-Ferrer, and Yves Henry. "The Splicing ATPase Prp43p Is a Component of Multiple Preribosomal Particles." Molecular and Cellular Biology 25, no. 21 (November 1, 2005): 9269–82. http://dx.doi.org/10.1128/mcb.25.21.9269-9282.2005.

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ABSTRACT Prp43p is a putative helicase of the DEAH family which is required for the release of the lariat intron from the spliceosome. Prp43p could also play a role in ribosome synthesis, since it accumulates in the nucleolus. Consistent with this hypothesis, we find that depletion of Prp43p leads to accumulation of 35S pre-rRNA and strongly reduces levels of all downstream pre-rRNA processing intermediates. As a result, the steady-state levels of mature rRNAs are greatly diminished following Prp43p depletion. We present data arguing that such effects are unlikely to be solely due to splicing defects. Moreover, we demonstrate by a combination of a comprehensive two-hybrid screen, tandem-affinity purification followed by mass spectrometry, and Northern analyses that Prp43p is associated with 90S, pre-60S, and pre-40S ribosomal particles. Prp43p seems preferentially associated with Pfa1p, a novel specific component of pre-40S ribosomal particles. In addition, Prp43p interacts with components of the RNA polymerase I (Pol I) transcription machinery and with mature 18S and 25S rRNAs. Hence, Prp43p might be delivered to nascent 90S ribosomal particles during pre-rRNA transcription and remain associated with preribosomal particles until their final maturation steps in the cytoplasm. Our data also suggest that the ATPase activity of Prp43p is required for early steps of pre-rRNA processing and normal accumulation of mature rRNAs.

36

de la Cruz, Jesús, Thierry Lacombe, Olivier Deloche, Patrick Linder, and Dieter Kressler. "The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae." Genetics 166, no. 4 (April 1, 2004): 1687–99. http://dx.doi.org/10.1093/genetics/166.4.1687.

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Abstract Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.

37

Cepeda, Leidy Paola P., Felipe F. M. Bagatelli, Renata M. Santos, Marlon D. M. Santos, Fabio C. S. Nogueira, and Carla C. Oliveira. "The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast." Journal of Biological Chemistry 294, no. 50 (October 29, 2019): 19365–80. http://dx.doi.org/10.1074/jbc.ra119.010193.

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38

Strezoska, Žaklina, Dimitri G. Pestov, and Lester F. Lau. "Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis." Molecular and Cellular Biology 20, no. 15 (August 1, 2000): 5516–28. http://dx.doi.org/10.1128/mcb.20.15.5516-5528.2000.

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ABSTRACT We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Δ, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Δ leads to cell growth arrest in the G1phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Δ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Δ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

39

Babiano, Reyes, Gwenael Badis, Cosmin Saveanu, Abdelkader Namane, Antonia Doyen, Antonio Díaz-Quintana, Alain Jacquier, Micheline Fromont-Racine, and Jesús de la Cruz. "Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles." Nucleic Acids Research 41, no. 20 (August 13, 2013): 9461–70. http://dx.doi.org/10.1093/nar/gkt726.

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40

Maser, R. L., and J. P. Calvet. "U3 small nuclear RNA can be psoralen-cross-linked in vivo to the 5' external transcribed spacer of pre-ribosomal-RNA." Proceedings of the National Academy of Sciences 86, no. 17 (September 1989): 6523–27. http://dx.doi.org/10.1073/pnas.86.17.6523.

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U3 small nuclear RNA is hydrogen-bonded to high molecular weight nucleolar RNA and can be isolated from greater than 60S pre-ribosomal ribonucleoprotein particles, suggesting that it is involved in processing of ribosomal RNA precursors (pre-rRNA) or in ribosome biogenesis. Here we have used in vivo psoralen cross-linking to identify the region of pre-rRNA interacting with U3 RNA. Quantitative hybridization selection/depletion experiments with clones of rRNA-encoding DNA (rDNA) and cross-linked nuclear RNA showed that all of the cross-linked U3 RNA was associated with a region that includes the external transcribed spacer (ETS) at the 5' end of the human rRNA precursor. To further identify the site of interaction within the approximately 3.7-kilobase ETS, Southern blots of rDNA clones were sandwich-hybridized with cross-linked RNA and then probed for cross-linked U3 RNA. These experiments showed that U3 RNA was cross-linked to a 258-base sequence between nucleotides +438 and +695, just downstream of the ETS early cleavage site (+414). The localization of U3 to this region of the rRNA precursor was not expected from previous models for a base-paired U3-rRNA interaction and suggests that U3 plays a role in the initial pre-rRNA processing event.

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Jaafar, Mariam, Julia Contreras, Carine Dominique, Sara Martín-Villanueva, Régine Capeyrou, Patrice Vitali, Olga Rodríguez-Galán, et al. "Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast." Nature Communications 12, no. 1 (October 22, 2021). http://dx.doi.org/10.1038/s41467-021-26207-w.

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AbstractSynthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

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Ruland, Jan Andreas, Annika Marie Krüger, Kerstin Dörner, Rohan Bhatia, Sabine Wirths, Daniel Poetes, Ulrike Kutay, Jan Peter Siebrasse, and Ulrich Kubitscheck. "Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time." Nature Communications 12, no. 1 (October 27, 2021). http://dx.doi.org/10.1038/s41467-021-26323-7.

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AbstractRibosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of ∼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s−1 in vivo.

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Kargas, Vasileios, Pablo Castro-Hartmann, Norberto Escudero-Urquijo, Kyle Dent, Christine Hilcenko, Carolin Sailer, Gertrude Zisser, et al. "Mechanism of completion of peptidyltransferase centre assembly in eukaryotes." eLife 8 (May 22, 2019). http://dx.doi.org/10.7554/elife.44904.

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During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.

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Rink, Constance, and Noreen Williams. "Unique Interactions of the Nuclear Export Receptors TbMex67 and TbMtr2 with Components of the 5S Ribonuclear Particle in Trypanosoma brucei." mSphere 4, no. 4 (August 14, 2019). http://dx.doi.org/10.1128/msphere.00471-19.

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ABSTRACT Eukaryotic ribosome biogenesis is a complicated and highly conserved biological process. A critical step in ribosome biogenesis is the translocation of the immature ribosomal subunits from the nucleoplasm, across the nucleopore complex, to the cytoplasm where they undergo final maturation. Many nonribosomal proteins are needed to facilitate export of the ribosomal subunits, and one complex participating in export of the pre-60S in Saccharomyces cerevisiae is the heterodimer Mex67-Mtr2. In Trypanomsoma brucei, the process of ribosome biogenesis differs from the yeast process in key steps and is not yet fully characterized. However, our laboratory has previously identified the trypanosome-specific proteins P34/P37 and has shown that P34/P37 are necessary for the formation of the 5S ribonuclear particle (RNP) and for the nuclear export of the pre-60S subunit. We have also shown that loss of TbMex67 or TbMtr2 leads to aberrant ribosome formation, rRNA processing, and polysome formation in T. brucei. In this study, we characterize the interaction of TbMex67 and TbMtr2 with the components of the 5S RNP (P34/P37, L5 and 5S rRNA) of the 60S subunit. We demonstrate that TbMex67 directly interacts with P34 and L5 proteins as well as 5S rRNA, while TbMtr2 does not. Using protein sequence alignments and structure prediction modeling, we show that TbMex67 lacks the amino acids previously shown to be essential for binding to 5S rRNA in yeast and in general aligns more closely with the human orthologue (NXF1 or TAP). This work suggests that the T. brucei Mex67-Mtr2 binds ribosomal cargo differently from the yeast system. IMPORTANCE Trypanosoma brucei is the causative agent for both African sleeping sickness in humans and nagana in cattle. Ribosome biogenesis in these pathogens requires both conserved and trypanosome-specific proteins to coordinate in a complex pathway. We have previously shown that the trypanosome-speciﬁc proteins P34/P37 are essential to the interaction of the TbNmd3-TbXpoI export complex with the 60S ribosomal subunits, allowing their translocation across the nuclear envelope. Our recent studies show that the trypanosome orthologues of the auxiliary export proteins TbMex67-TbMtr2 are required for ribosome assembly, proper rRNA processing, and polysome formation. Here we show that TbMex67-TbMtr2 interact with members of the 60S ribosomal subunit 5S RNP. Although TbMex67 has a unique structure among the Mex67 orthologues and forms unique interactions with the 5S RNP, particularly with trypanosome-specific P34/P37, it performs a conserved function in ribosome assembly. These unique structures and parasite-specific interactions may provide new therapeutic targets against this important parasite.

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Aquino, Gerald Ryan R., Philipp Hackert, Nicolai Krogh, Kuan-Ting Pan, Mariam Jaafar, Anthony K. Henras, Henrik Nielsen, Henning Urlaub, Katherine E. Bohnsack, and Markus T. Bohnsack. "The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly." Nature Communications 12, no. 1 (October 22, 2021). http://dx.doi.org/10.1038/s41467-021-26208-9.

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AbstractEarly pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre-60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center (PTC) and is responsible for stabilizing interactions between the 5′ and 3′ ends of the 25S, an essential pre-requisite for subsequent pre-60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation.

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Sosnowski, Piotr, Linas Urnavicius, Andreas Boland, Robert Fagiewicz, Johan Busselez, Gabor Papai, and Helgo Schmidt. "The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1." eLife 7 (November 26, 2018). http://dx.doi.org/10.7554/elife.39163.

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The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

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Micic, Jelena, Olga Rodríguez-Galán, Reyes Babiano, Fiona Fitzgerald, José Fernández-Fernández, Yunyang Zhang, Ning Gao, John L. Woolford, and Jesús de la Cruz. "Ribosomal protein eL39 is important for maturation of the nascent polypeptide exit tunnel and proper protein folding during translation." Nucleic Acids Research, May 27, 2022. http://dx.doi.org/10.1093/nar/gkac366.

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Abstract During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

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Liang, Xiaomeng, Mei-Qing Zuo, Yunyang Zhang, Ningning Li, Chengying Ma, Meng-Qiu Dong, and Ning Gao. "Structural snapshots of human pre-60S ribosomal particles before and after nuclear export." Nature Communications 11, no. 1 (July 15, 2020). http://dx.doi.org/10.1038/s41467-020-17237-x.

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Braun, Christina M., Philipp Hackert, Catharina E. Schmid, Markus T. Bohnsack, Katherine E. Bohnsack, and Jorge Perez-Fernandez. "Pol5 is required for recycling of small subunit biogenesis factors and for formation of the peptide exit tunnel of the large ribosomal subunit." Nucleic Acids Research, November 20, 2019. http://dx.doi.org/10.1093/nar/gkz1079.

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Abstract More than 200 assembly factors (AFs) are required for the production of ribosomes in yeast. The stepwise association and dissociation of these AFs with the pre-ribosomal subunits occurs in a hierarchical manner to ensure correct maturation of the pre-rRNAs and assembly of the ribosomal proteins. Although decades of research have provided a wealth of insights into the functions of many AFs, others remain poorly characterized. Pol5 was initially classified with B-type DNA polymerases, however, several lines of evidence indicate the involvement of this protein in ribosome assembly. Here, we show that depletion of Pol5 affects the processing of pre-rRNAs destined for the both the large and small subunits. Furthermore, we identify binding sites for Pol5 in the 5′ external transcribed spacer and within domain III of the 25S rRNA sequence. Consistent with this, we reveal that Pol5 is required for recruitment of ribosomal proteins that form the polypeptide exit tunnel in the LSU and that depletion of Pol5 impairs the release of 5′ ETS fragments from early pre-40S particles. The dual functions of Pol5 in 60S assembly and recycling of pre-40S AFs suggest that this factor could contribute to ensuring the stoichiometric production of ribosomal subunits.

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