RbfA Is Involved in Two Important Stages of 30S Subunit Assembly: Formation of the Central Pseudoknot and Docking of Helix 44 to the Decoding Center

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.

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Abstract P-27: The 30S Ribosomal Subunit Assembly Factor Rbfa Plays a Key Role in the Formation of the Central Pseudoknot and in the Correct Docking of Helix 44 of the Decoding Center

International Journal of Biomedicine ◽

10.21103/ijbm.11.suppl_1.p27 ◽

2021 ◽

Vol 11 (Suppl_1) ◽

pp. S23-S24

Author(s):

Elena Maksimova ◽

Alexey Korepanov ◽

Olesya Kravchenko ◽

Timur Baymukhametov ◽

Alexander Myasnikov ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Coli Strain ◽

Assembly Process ◽

Subunit Assembly ◽

Head Domain ◽

Multi Stage ◽

30S Subunit ◽

Assembly Factor

Background: Ribosome biogenesis is a complicated multi-stage process. In the cell, 30S ribosomal subunit assembly is fast and efficient, proceeding with the help of numerous assembly protein factors. The exact role of most assembly factors and mechanistic details of their operation remain unclear. The combination of genetic modification with cryo-EM analysis is widely used to identify the role of protein factors in assisting specific steps of the ribosome assembly process. The strain with knockout of a single assembly factor gene accumulates immature ribosomal particles which structural characterization reveals the information about the reactions catalyzed by the corresponding factor. Methods: We isolated the immature 30S subunits (pre-30S subunits) from the Escherichia coli strain lacking the rbfA gene (ΔrbfA) and characterized them by cryo-electron microscopy (cryo-EM). Results: Deletion of the assembly factor RbfA caused a substantial distortion of the structure of an important central pseudoknot which connects three major domains of 30S subunit and is necessary for ribosome stability. It was shown that the relative order of the assembly of the 3′ head domain and the docking of the functionally important helix 44 depends on the presence of RbfA. The formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. The cryo-EM maps for pre-30S subunits were divided into the classes corresponding to consecutive assembly intermediates: from the particles with completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and with partially distorted helix 44. Cryo-EM analysis of ΔrbfA 30S particles revealing the accumulation of two predominant classes of early and late intermediates (obtained at 2.7 Å resolutions) allowed us to suggest that RbfA participate in two stages of the 30S subunit assembly and is deeper involved in the maturation process than previously thought. Conclusion: In summary, RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including the folding of the head, and positioning of helix 44 in the decoding center at a later stage. An update to the model of factor-dependent 30S maturation was proposed, suggesting that RfbA is involved in most of the subunit assembly process.

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Assembly and early maturation of large subunit precursors

10.1101/486225 ◽

2018 ◽

Author(s):

Malik Chaker-Margot ◽

Sebastian Klinge

Keyword(s):

Large Subunit ◽

Ribosomal Subunit ◽

Subunit Assembly ◽

Early Maturation ◽

Complex Process ◽

Assembly Factors ◽

Early Recruitment ◽

Initial Assembly ◽

Shed Light

The eukaryotic ribosome is assembled through a complex process involving more than 200 factors. As pre-ribosomal RNA is transcribed, assembly factors bind the nascent pre-rRNA and guide its correct folding, modification and cleavage. While these early events in the assembly of the small ribosomal subunit have been relatively well-characterized, assembly of the large subunit precursors, or pre-60S, is less well understood. Recent structures of nucleolar intermediates of large subunit assembly have shed light on the role of many early large subunit assembly factors but how these particles emerge is still unknown. Here, we use the overexpression and purification of truncated pre-rRNAs to examine the initial assembly of pre-60S particles. Using this approach, we can recapitulate the early recruitment of large subunit assembly factors mainly to the domains I, II and VI of the assembling 25S rRNA.

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Ubiquitin and Ubiquitin-Like Proteins and Domains in Ribosome Production and Function: Chance or Necessity?

International Journal of Molecular Sciences ◽

10.3390/ijms22094359 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4359

Author(s):

Sara Martín-Villanueva ◽

Gabriel Gutiérrez ◽

Dieter Kressler ◽

Jesús de la Cruz

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosome Assembly ◽

Cellular Processes ◽

Substrate Protein ◽

Precursor Proteins ◽

Assembly Factors ◽

Ubiquitin Moiety ◽

And Function

Ubiquitin is a small protein that is highly conserved throughout eukaryotes. It operates as a reversible post-translational modifier through a process known as ubiquitination, which involves the addition of one or several ubiquitin moieties to a substrate protein. These modifications mark proteins for proteasome-dependent degradation or alter their localization or activity in a variety of cellular processes. In most eukaryotes, ubiquitin is generated by the proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor, or as a single N-terminal moiety to ribosomal proteins, which are practically invariably eL40 and eS31. Herein, we summarize the contribution of the ubiquitin moiety within precursors of ribosomal proteins to ribosome biogenesis and function and discuss the biological relevance of having maintained the explicit fusion to eL40 and eS31 during evolution. There are other ubiquitin-like proteins, which also work as post-translational modifiers, among them the small ubiquitin-like modifier (SUMO). Both ubiquitin and SUMO are able to modify ribosome assembly factors and ribosomal proteins to regulate ribosome biogenesis and function. Strikingly, ubiquitin-like domains are also found within two ribosome assembly factors; hence, the functional role of these proteins will also be highlighted.

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Cryo-EM structure of 90S small ribosomal subunit precursors in transition states

Science ◽

10.1126/science.aba9690 ◽

2020 ◽

Vol 369 (6510) ◽

pp. 1477-1481 ◽

Cited By ~ 3

Author(s):

Yifei Du ◽

Weidong An ◽

Xing Zhu ◽

Qi Sun ◽

Jia Qi ◽

...

Keyword(s):

Structural Changes ◽

Critical Role ◽

Ribosomal Subunit ◽

Rna Degradation ◽

Ribosomal Subunits ◽

Cryo Electron Microscopy ◽

Nuclear Exosome ◽

Assembly Intermediate ◽

Insight Into

The 90S preribosome is a large, early assembly intermediate of small ribosomal subunits that undergoes structural changes to give a pre-40S ribosome. Here, we gained insight into this transition by determining cryo–electron microscopy structures of Saccharomyces cerevisiae intermediates in the path from the 90S to the pre-40S. The full transition is blocked by deletion of RNA helicase Dhr1. A series of structural snapshots revealed that the excised 5′ external transcribed spacer (5′ ETS) is degraded within 90S, driving stepwise disassembly of assembly factors and ribosome maturation. The nuclear exosome, an RNA degradation machine, docks on the 90S through helicase Mtr4 and is primed to digest the 3′ end of the 5′ ETS. The structures resolved between 3.2- and 8.6-angstrom resolution reveal key intermediates and the critical role of 5′ ETS degradation in 90S progression.

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Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy

Molecules ◽

10.3390/molecules25051125 ◽

2020 ◽

Vol 25 (5) ◽

pp. 1125 ◽

Cited By ~ 3

Author(s):

Ramtin Shayan ◽

Dana Rinaldi ◽

Natacha Larburu ◽

Laura Plassart ◽

Stéphanie Balor ◽

...

Keyword(s):

Electron Microscopy ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Particle Analysis ◽

Ribosomal Subunits ◽

Early Maturation ◽

Cryo Electron Microscopy ◽

Molecular Events ◽

Final Maturation

Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the “resolution revolution” of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a “vibrating” or “wriggling” stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position.

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Molecular architecture of the 90S small subunit pre-ribosome

eLife ◽

10.7554/elife.22086 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 61

Author(s):

Qi Sun ◽

Xing Zhu ◽

Jia Qi ◽

Weidong An ◽

Pengfei Lan ◽

...

Keyword(s):

Small Subunit ◽

Molecular Architecture ◽

Ribosomal Subunits ◽

Cryo Electron Microscopy ◽

U3 Snorna ◽

Density Maps ◽

External Transcribed Spacer ◽

Assembly Factors ◽

Protein Components ◽

Insight Into

Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors.

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Visualising formation of the ribosomal active site in mitochondria

10.1101/2021.03.19.436169 ◽

2021 ◽

Author(s):

Viswanathan Chandrasekaran ◽

Nirupa Desai ◽

Nicholas O. Burton ◽

Hanting Yang ◽

Jon Price ◽

...

Keyword(s):

Stress Responses ◽

Large Subunit ◽

Mitochondrial Protein ◽

Mitochondrial Stress ◽

Cryo Electron Microscopy ◽

Complex Process ◽

Mitochondrial Ribosome ◽

Peptidyl Transferase Centre

SummaryRibosome assembly is an essential and complex process that is regulated at each step by specific biogenesis factors. Using cryo-electron microscopy, we identify and order major steps in the formation of the highly conserved peptidyl transferase centre (PTC) and tRNA binding sites in the large subunit of the human mitochondrial ribosome (mitoribosome). The conserved GTPase GTPBP7 regulates the folding and incorporation of core 16S ribosomal RNA (rRNA) helices and the ribosomal protein bL36m, and ensures that the PTC base U3039 has been 2′-O-methylated. Additionally, GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which facilitate earlier steps by sequestering H68-71 of the 16S rRNA and allowing biogenesis factors to access the maturing PTC. Consistent with the central role of NSUN4•MTERF4 and GTPBP7 during mitoribosome biogenesis, in vivo mutagenesis designed to disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activates mitochondrial stress responses and results in severely reduced viability, developmental delays and sterility. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4 but not its enzymatic activity, is indispensable for mitochondrial protein synthesis in metazoans.

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SOD1 regulates ribosome biogenesis in KRAS mutant non-small cell lung cancer

Nature Communications ◽

10.1038/s41467-021-22480-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xiaowen Wang ◽

Hong Zhang ◽

Russell Sapio ◽

Jun Yang ◽

Justin Wong ◽

...

Keyword(s):

Lung Cancer ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Tumor Burden ◽

Cancer Cell Proliferation ◽

Lung Cancer Cell ◽

Ribosomal Subunits

AbstractSOD1 is known as the major cytoplasmic superoxide dismutase and an anticancer target. However, the role of SOD1 in cancer is not fully understood. Herein we describe the generation of an inducible Sod1 knockout in KRAS-driven NSCLC mouse model. Sod1 knockout markedly reduces tumor burden in vivo and blocks growth of KRAS mutant NSCLC cells in vitro. Intriguingly, SOD1 is enriched in the nucleus and notably in the nucleolus of NSCLC cells. The nuclear and nucleolar, not cytoplasmic, form of SOD1 is essential for lung cancer cell proliferation. Moreover, SOD1 interacts with PeBoW complex and controls its assembly necessary for pre-60S ribosomal subunit maturation. Mechanistically, SOD1 regulates co-localization of PeBoW with and processing of pre-rRNA, and maturation of cytoplasmic 60S ribosomal subunits in KRAS mutant lung cancer cells. Collectively, our study unravels a nuclear SOD1 function essential for ribosome biogenesis and proliferation in KRAS-driven lung cancer.

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A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit

Science Advances ◽

10.1126/sciadv.abf7547 ◽

2021 ◽

Vol 7 (23) ◽

pp. eabf7547

Author(s):

Andreas Schedlbauer ◽

Idoia Iturrioz ◽

Borja Ochoa-Lizarralde ◽

Tammo Diercks ◽

Jorge Pedro López-Alonso ◽

...

Keyword(s):

Molecular Mechanisms ◽

Structural Information ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Structural Description ◽

Cryo Electron Microscopy ◽

Bacterial Ribosome ◽

Core Set ◽

Assembly Factors ◽

Conformational Landscape

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria use an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo–electron microscopy to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S ribosomal RNA central to decoding site maturation and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes.

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

The Journal of Cell Biology ◽

10.1083/jcb.201711037 ◽

2018 ◽

Vol 217 (7) ◽

pp. 2503-2518 ◽

Cited By ~ 20

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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