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.

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.

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 The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

2021 ◽  
Author(s):  
Haina Huang ◽  
Melissa Parker ◽  
Katrin Karbstein
Keyword(s):  
Quality Control ◽  
Binding Site ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Yeast Strains

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

scholarly journals Grc3 programs the essential endoribonuclease Las1 for specific RNA cleavage

2017 ◽  
Vol 114 (28) ◽  
pp. E5530-E5538 ◽  
Author(s):  
Monica C. Pillon ◽  
Mack Sobhany ◽  
Mario J. Borgnia ◽  
Jason G. Williams ◽  
Robin E. Stanley
Keyword(s):  
Ribosomal Rna ◽  
Rna Splicing ◽  
Kinase Activity ◽  
Rrna Processing ◽  
Binding Partner ◽  
Mechanistic Insight ◽  
Polynucleotide Kinase ◽  
And Function ◽  
Tetrameric Complex

Las1 is a recently discovered endoribonuclease that collaborates with Grc3–Rat1–Rai1 to process precursor ribosomal RNA (rRNA), yet its mechanism of action remains unknown. Disruption of the mammalian Las1 gene has been linked to congenital lethal motor neuron disease and X-linked intellectual disability disorders, thus highlighting the necessity to understand Las1 regulation and function. Here, we report that the essential Las1 endoribonuclease requires its binding partner, the polynucleotide kinase Grc3, for specific C2 cleavage. Our results establish that Grc3 drives Las1 endoribonuclease cleavage to its targeted C2 site both in vitro and in Saccharomyces cerevisiae. Moreover, we observed Las1-dependent activation of the Grc3 kinase activity exclusively toward single-stranded RNA. Together, Las1 and Grc3 assemble into a tetrameric complex that is required for competent rRNA processing. The tetrameric Grc3/Las1 cross talk draws unexpected parallels to endoribonucleases RNaseL and Ire1, and establishes Grc3/Las1 as a unique member of the RNaseL/Ire1 RNA splicing family. Together, our work provides mechanistic insight for the regulation of the Las1 endoribonuclease and identifies the tetrameric Grc3/Las1 complex as a unique example of a protein-guided programmable endoribonuclease.

scholarly journals Late Cytoplasmic Maturation of the Small Ribosomal Subunit Requires RIO Proteins in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (6) ◽  
pp. 2083-2095 ◽  
Author(s):  
Emmanuel Vanrobays ◽  
Jean-Paul Gelugne ◽  
Pierre-Emmanuel Gleizes ◽  
Michele Caizergues-Ferrer
Keyword(s):  
18S Rrna ◽  
Nuclear Protein ◽  
Essential Gene ◽  
Sequence Similarity ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Rrna Maturation ◽  
Cytoplasmic Maturation

ABSTRACT Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.

scholarly journals The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

RNA ◽  
2022 ◽  
pp. rna.078994.121
Author(s):  
Haina Huang ◽  
Melissa D Parker ◽  
Katrin Karbstein
Keyword(s):  
Quality Control ◽  
Binding Site ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Kinase Activity ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Yeast Strains

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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 Prp43p Is a DEAH-Box Spliceosome Disassembly Factor Essential for Ribosome Biogenesis

2006 ◽  
Vol 26 (2) ◽  
pp. 523-534 ◽  
Author(s):  
D. Joshua Combs ◽  
Roland J. Nagel ◽  
Manuel Ares ◽  
Scott W. Stevens
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Splicing Defects ◽  
Essential Function ◽  
Polymerase I ◽  
Kinetics Of

ABSTRACT The known function of the DEXH/D-box protein Prp43p is the removal of the U2, U5, and U6 snRNPs from the postsplicing lariat-intron ribonucleoprotein complex. We demonstrate that affinity-purified Prp43p-associated material includes the expected spliceosomal components; however, we also identify several preribosomal complexes that are specifically purified with Prp43p. Conditional prp43 mutant alleles confer a 35S pre-rRNA processing defect, with subsequent depletion of 27S and 20S precursors. Upon a shift to a nonpermissive temperature, both large and small-ribosomal-subunit proteins accumulate in the nucleolus of prp43 mutants. Pulse-chase analysis demonstrates delayed kinetics of 35S, 27S, and 20S pre-rRNA processing with turnover of these intermediates. Microarray analysis of pre-mRNA splicing defects in prp43 mutants shows a very mild effect, similar to that of nonessential pre-mRNA splicing factors. Prp43p is the first DEXH/D-box protein shown to function in both RNA polymerase I and polymerase II transcript metabolism. Its essential function is in its newly characterized role in ribosome biogenesis of both ribosomal subunits, positioning Prp43p to regulate both pre-mRNA splicing and ribosome biogenesis.

scholarly journals Fanconi anemia protein FANCI functions in ribosome biogenesis

2019 ◽  
Vol 116 (7) ◽  
pp. 2561-2570 ◽  
Author(s):  
Samuel B. Sondalle ◽  
Simonne Longerich ◽  
Lisa M. Ogawa ◽  
Patrick Sung ◽  
Susan J. Baserga
Keyword(s):  
Dna Repair ◽  
Fanconi Anemia ◽  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Bone Marrow Failure ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Large Ribosomal Subunit ◽  
Interstrand Cross Links ◽  
Cross Links

Fanconi anemia (FA) is a disease of DNA repair characterized by bone marrow failure and a reduced ability to remove DNA interstrand cross-links. Here, we provide evidence that the FA protein FANCI also functions in ribosome biogenesis, the process of making ribosomes that initiates in the nucleolus. We show that FANCI localizes to the nucleolus and is functionally and physically tied to the transcription of pre-ribosomal RNA (pre-rRNA) and to large ribosomal subunit (LSU) pre-rRNA processing independent of FANCD2. While FANCI is known to be monoubiquitinated when activated for DNA repair, we find that it is predominantly in the deubiquitinated state in the nucleolus, requiring the nucleoplasmic deubiquitinase (DUB) USP1 and the nucleolar DUB USP36. Our model suggests a possible dual pathophysiology for FA that includes defects in DNA repair and in ribosome biogenesis.

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