scholarly journals Cryo-EM structure of a late pre-40S ribosomal subunit from Saccharomyces cerevisiae

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
André Heuer ◽  
Emma Thomson ◽  
Christian Schmidt ◽  
Otto Berninghausen ◽  
Thomas Becker ◽  
...  
Keyword(s):  
Active Sites ◽  
18S Rrna ◽  
Structural Integrity ◽  
Ribosomal Subunit ◽  
Structural Knowledge ◽  
40S Ribosomal Subunit ◽  
Final Maturation ◽  
Assembly Factors ◽  
Mrna Binding ◽  
Ribosome Formation

Mechanistic understanding of eukaryotic ribosome formation requires a detailed structural knowledge of the numerous assembly intermediates, generated along a complex pathway. Here, we present the structure of a late pre-40S particle at 3.6 Å resolution, revealing in molecular detail how assembly factors regulate the timely folding of pre-18S rRNA. The structure shows that, rather than sterically blocking 40S translational active sites, the associated assembly factors Tsr1, Enp1, Rio2 and Pno1 collectively preclude their final maturation, thereby preventing untimely tRNA and mRNA binding and error prone translation. Moreover, the structure explains how Pno1 coordinates the 3’end cleavage of the 18S rRNA by Nob1 and how the late factor’s removal in the cytoplasm ensures the structural integrity of the maturing 40S subunit.

scholarly journals Structure of the eukaryotic cytoplasmic pre-40S ribosomal subunit

2017 ◽  
Author(s):  
Alain Scaiola ◽  
Cohue Peña ◽  
Melanie Weisser ◽  
Daniel Böhringer ◽  
Marc Leibundgut ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Rna ◽  
Ribosomal Subunit ◽  
Mature Form ◽  
Structural Framework ◽  
Cryo Electron Microscopy ◽  
40S Ribosomal Subunit ◽  
Final Maturation ◽  
Assembly Factors ◽  
Conformational Rearrangements

AbstractFinal maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA. Using cryo-electron microscopy (cryo-EM), we have determined the structure of a cytoplasmic pre-40S particle poised to initiate final maturation at a resolution of 3.4 Å. The structure reveals the extent of conformational rearrangements of the 3’ major and 3’ minor domains of the ribosomal RNA that take place during maturation, as well as the roles of the assembly factors Enp1, Ltv1, Rio2, Tsr1, and Pno1 in the process. Altogether, we provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle towards the mature form capable of engaging in translation.

Shwachman-Diamond Syndrome and the Quality Control of Ribosome Assembly

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-42-SCI-42
Author(s):  
Alan J. Warren
Keyword(s):  
Quality Control ◽  
Ribosomal Proteins ◽  
Active Sites ◽  
Structural Integrity ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Final Maturation ◽  
Shwachman Diamond Syndrome

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.

scholarly journals USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Christian Montellese ◽  
Jasmin van den Heuvel ◽  
Caroline Ashiono ◽  
Kerstin Dörner ◽  
André Melnik ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Synthesis ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Novel Proteins ◽  
40S Ribosomal Subunit ◽  
Final Maturation

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.

scholarly journals gar2 is a nucleolar protein fromSchizosaccharomyces pomberequired for 18S rRNA and 40S ribosomal subunit accumulation

1995 ◽  
Vol 23 (11) ◽  
pp. 1912-1918 ◽  
Author(s):  
Marie-Pierre Gulli ◽  
Jean-Philippe Girard ◽  
Dan Zabetakis ◽  
Bruno Lapeyre ◽  
Teri Melese ◽  
...  
Keyword(s):  
18S Rrna ◽  
Ribosomal Subunit ◽  
Nucleolar Protein ◽  
40S Ribosomal Subunit

scholarly journals The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

2020 ◽  
Author(s):  
Laura Plassart ◽  
Ramtin Shayan ◽  
Christian Montellese ◽  
Dana Rinaldi ◽  
Natacha Larburu ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Inactive Form ◽  
Final Maturation ◽  
Translation Apparatus ◽  
Translation Accuracy ◽  
Functional Analyses

Preventing premature interaction of preribosomes with the translation apparatus is essential to translation accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATPloaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

scholarly journals yRACK1/Asc1 proxiOMICs—Towards Illuminating Ships Passing in the Night

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1384 ◽  
Author(s):  
Kerstin Schmitt ◽  
Oliver Valerius
Keyword(s):  
Quality Control ◽  
Binding Proteins ◽  
Ribosomal Subunit ◽  
Stress Factors ◽  
Head Region ◽  
Ubiquitin E3 Ligase ◽  
Mrna Binding Proteins ◽  
40S Ribosomal Subunit ◽  
Mrna Binding

Diverse signals and stress factors regulate the activity and homeostasis of ribosomes in all cells. The Saccharomyces cerevisiae protein Asc1/yRACK1 occupies an exposed site at the head region of the 40S ribosomal subunit (hr40S) and represents a central hub for signaling pathways. Asc1 strongly affects protein phosphorylation and is involved in quality control pathways induced by translation elongation arrest. Therefore, it is important to understand the dynamics of protein formations in the Asc1 microenvironment at the hr40S. We made use of the in vivo protein-proximity labeling technique Biotin IDentification (BioID). Unbiased proxiOMICs from two adjacent perspectives identified nucleocytoplasmic shuttling mRNA-binding proteins, the deubiquitinase complex Ubp3-Bre5, as well as the ubiquitin E3 ligase Hel2 as neighbors of Asc1. We observed Asc1-dependency of hr40S localization of mRNA-binding proteins and the Ubp3 co-factor Bre5. Hel2 and Ubp3-Bre5 are described to balance the mono-ubiquitination of Rps3 (uS3) during ribosome quality control. Here, we show that the absence of Asc1 resulted in massive exposure and accessibility of the C-terminal tail of its ribosomal neighbor Rps3 (uS3). Asc1 and some of its direct neighbors together might form a ribosomal decision tree that is tightly connected to close-by signaling modules.

scholarly journals Processing of the Ribosomal Ubiquitin-Like Fusion Protein FUBI-eS30/FAU is Required for 40S Maturation and Depends on USP36

2021 ◽  
Author(s):  
Jasmin van den Heuvel ◽  
Caroline Ashiono ◽  
Ludovic Gillet ◽  
Kerstin Doerner ◽  
Emanuel Wyler ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Affinity Purification ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Functional Importance ◽  
40S Ribosomal Subunit ◽  
Differential Affinity

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

scholarly journals Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jasmin van den Heuvel ◽  
Caroline Ashiono ◽  
Ludovic C Gillet ◽  
Kerstin Dörner ◽  
Emanuel Wyler ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Affinity Purification ◽  
Ribosomal Subunit ◽  
Wild Type ◽  
Functional Importance ◽  
40S Ribosomal Subunit ◽  
Differential Affinity

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

scholarly journals The KRR1 gene encodes a protein required for 18S rRNA synthesis and 40S ribosomal subunit assembly in Saccharomyces cerevisiae.

2000 ◽  
Vol 47 (4) ◽  
pp. 993-1005 ◽  
Author(s):  
R Gromadka ◽  
J Rytka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Synthesis ◽  
Drastic Reduction ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Dividing Cells

The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability. Krr1p contains a motif of clustered basic amino acids highly conserved in the evolutionarly distant species from yeast to human. We demonstrate that Krr1p is localized in the nucleolus. The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. In vivo depletion of Krr1p leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis. We propose that Krr1p is required for proper processing of pre-rRNA and the assembly of preribosomal 40S subunits.

scholarly journals Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2

2009 ◽  
Vol 185 (7) ◽  
pp. 1167-1180 ◽  
Author(s):  
Ivo Zemp ◽  
Thomas Wild ◽  
Marie-Françoise O'Donohue ◽  
Franziska Wandrey ◽  
Barbara Widmann ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ribosomal Rna ◽  
Kinase Activity ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
40S Ribosomal Subunit ◽  
Final Maturation ◽  
Physical Presence ◽  
Cytoplasmic Maturation ◽  
Kinetics Of

During their biogenesis, 40S ribosomal subunit precursors are exported from the nucleus to the cytoplasm, where final maturation occurs. In this study, we show that the protein kinase human Rio2 (hRio2) is part of a late 40S preribosomal particle in human cells. Using a novel 40S biogenesis and export assay, we analyzed the contribution of hRio2 to late 40S maturation. Although hRio2 is not absolutely required for pre-40S export, deletion of its binding site for the export receptor CRM1 decelerated the kinetics of this process. Moreover, in the absence of hRio2, final cytoplasmic 40S maturation is blocked because the recycling of several trans-acting factors and cytoplasmic 18S-E precursor ribosomal RNA (rRNA [pre-rRNA]) processing are defective. Intriguingly, the physical presence of hRio2 but not its kinase activity is necessary for the release of hEnp1 from cytoplasmic 40S precursors. In contrast, hRio2 kinase activity is essential for the recycling of hDim2, hLtv1, and hNob1 as well as for 18S-E pre-rRNA processing. Thus, hRio2 is involved in late 40S maturation at several distinct steps.

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