scholarly journals Conformational switches control early maturation of the eukaryotic small ribosomal subunit

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mirjam Hunziker ◽  
Jonas Barandun ◽  
Olga Buzovetsky ◽  
Caitlin Steckler ◽  
Henrik Molina ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Early Maturation ◽  
External Transcribed Spacer ◽  
Assembly Factors

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

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.

Requirements for the nuclear export of the small ribosomal subunit

2002 ◽  
Vol 115 (14) ◽  
pp. 2985-2995 ◽  
Author(s):  
Terence I. Moy ◽  
Pamela A. Silver
Keyword(s):  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Large Scale ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Small Ribosomal Subunit ◽  
Factors Affecting ◽  
The Stability

Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants,we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.

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 complete structure of the small subunit processome

2017 ◽  
Author(s):  
Jonas Barandun ◽  
Malik Chaker-Margot ◽  
Mirjam Hunziker ◽  
Kelly R. Molloy ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
18S Rrna ◽  
Molecular Mimicry ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Complete Structure ◽  
Small Ribosomal Subunit ◽  
Rna Remodeling ◽  
Rrna Cleavage

The small subunit processome represents the earliest stable precursor of the eukaryotic small ribosomal subunit. Here we present the cryo-EM structure of the Saccharomyces cerevisiae small subunit processome at an overall resolution of 3.8 Å, which provides an essentially complete atomic model of this assembly. In this nucleolar superstructure, 51 ribosome assembly factors and two RNAs encapsulate the 18S rRNA precursor and 15 ribosomal proteins in a state that precedes pre-rRNA cleavage at site A1. Extended flexible proteins are employed to connect distant sites in this particle. Molecular mimicry, steric hindrance as well as protein-and RNA-mediated RNA remodeling are used in a concerted fashion to prevent the premature formation of the central pseudoknot and its surrounding elements within the small ribosomal subunit.

scholarly journals The DEAH-box RNA helicase Dhr1 contains a remarkable carboxyl terminal domain essential for small ribosomal subunit biogenesis

2019 ◽  
Vol 47 (14) ◽  
pp. 7548-7563 ◽  
Author(s):  
Amlan Roychowdhury ◽  
Clément Joret ◽  
Gabrielle Bourgeois ◽  
Valérie Heurgué-Hamard ◽  
Denis L J Lafontaine ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Rna Helicases ◽  
Range Interaction ◽  
Small Ribosomal Subunit ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Assembly Factors ◽  
Carboxyl Terminal

Abstract Ribosome biogenesis is an essential process in all living cells, which entails countless highly sequential and dynamic structural reorganization events. These include formation of dozens RNA helices through Watson-Crick base-pairing within ribosomal RNAs (rRNAs) and between rRNAs and small nucleolar RNAs (snoRNAs), transient association of hundreds of proteinaceous assembly factors to nascent precursor (pre-)ribosomes, and stable assembly of ribosomal proteins. Unsurprisingly, the largest group of ribosome assembly factors are energy-consuming proteins (NTPases) including 25 RNA helicases in budding yeast. Among these, the DEAH-box Dhr1 is essential to displace the box C/D snoRNA U3 from the pre-rRNAs where it is bound in order to prevent premature formation of the central pseudoknot, a dramatic irreversible long-range interaction essential to the overall folding of the small ribosomal subunit. Here, we report the crystal structure of the Dhr1 helicase module, revealing the presence of a remarkable carboxyl-terminal domain essential for Dhr1 function in ribosome biogenesis in vivo and important for its interaction with its coactivator Utp14 in vitro. Furthermore, we report the functional consequences on ribosome biogenesis of DHX37 (human Dhr1) mutations found in patients suffering from microcephaly and other neurological diseases.

scholarly journals Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

2017 ◽  
Vol 474 (2) ◽  
pp. 195-214 ◽  
Author(s):  
Salini Konikkat ◽  
John L. Woolford,
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Ribosome Assembly ◽  
Small Nucleolar Rnas ◽  
Large Ribosomal Subunit ◽  
Yeast Saccharomyces Cerevisiae ◽  
Subunit Assembly ◽  
Nucleolar Rnas

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.

scholarly journals A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

2014 ◽  
Vol 207 (4) ◽  
pp. 481-498 ◽  
Author(s):  
Jochen Baßler ◽  
Helge Paternoga ◽  
Iris Holdermann ◽  
Matthias Thoms ◽  
Sander Granneman ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Ribosome Assembly ◽  
Functional Importance ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Eukaryotic Ribosome ◽  
Assembly Factors

Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.

scholarly journals Structural Probing with MNase Tethered to Ribosome Assembly Factors Resolves Flexible RNA Regions within the Nascent Pre-Ribosomal RNA

2022 ◽  
Vol 8 (1) ◽  
pp. 1
Author(s):  
Tom Dielforder ◽  
Christina Maria Braun ◽  
Fabian Hölzgen ◽  
Shuang Li ◽  
Mona Thiele ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Dynamic Information ◽  
Nascent Transcript ◽  
Ribonucleoprotein Complexes ◽  
U3 Snorna ◽  
Assembly Factors ◽  
Compositional Changes ◽  
Ribosomal Precursor

The synthesis of ribosomes involves the correct folding of the pre-ribosomal RNA within pre-ribosomal particles. The first ribosomal precursor or small subunit processome assembles stepwise on the nascent transcript of the 35S gene. At the earlier stages, the pre-ribosomal particles undergo structural and compositional changes, resulting in heterogeneous populations of particles with highly flexible regions. Structural probing methods are suitable for resolving these structures and providing evidence about the architecture of ribonucleoprotein complexes. Our approach used MNase tethered to the assembly factors Nan1/Utp17, Utp10, Utp12, and Utp13, which among other factors, initiate the formation of the small subunit processome. Our results provide dynamic information about the folding of the pre-ribosomes by elucidating the relative organization of the 5′ETS and ITS1 regions within the 35S and U3 snoRNA around the C-terminal domains of Nan1/Utp17, Utp10, Utp12, and Utp13.

scholarly journals Hierarchical recruitment of ribosomal proteins and assembly factors remodels nucleolar pre-60S ribosomes

2018 ◽  
Vol 217 (7) ◽  
pp. 2503-2518 ◽  
Author(s):  
Stephanie Biedka ◽  
Jelena Micic ◽  
Daniel Wilson ◽  
Hailey Brown ◽  
Luke Diorio-Toth ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Ribosome Assembly ◽  
Rrna Processing ◽  
Endonucleolytic Cleavage ◽  
Cryo Electron Microscopy ◽  
External Transcribed Spacer ◽  
Assembly Factors

Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage and interpreted these results using cryo–electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps.

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