Faculty Opinions recommendation of The roles of ribosomal proteins in the structure assembly, and evolution of the large ribosomal subunit.

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

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Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast

Nature Communications ◽

10.1038/s41467-021-26207-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Mariam Jaafar ◽

Julia Contreras ◽

Carine Dominique ◽

Sara Martín-Villanueva ◽

Régine Capeyrou ◽

...

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Rna Helicase ◽

Genetic Interactions ◽

Large Ribosomal Subunit ◽

Base Pairing ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center

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

Biochemical Journal ◽

10.1042/bcj20160516 ◽

2017 ◽

Vol 474 (2) ◽

pp. 195-214 ◽

Cited By ~ 44

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.

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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 ◽

10.1093/nar/gkz1079 ◽

2019 ◽

Cited By ~ 1

Author(s):

Christina M Braun ◽

Philipp Hackert ◽

Catharina E Schmid ◽

Markus T Bohnsack ◽

Katherine E Bohnsack ◽

...

Keyword(s):

Binding Sites ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Small Subunit ◽

Large Ribosomal Subunit ◽

Rrna Sequence ◽

Ribosomal Subunits ◽

External Transcribed Spacer ◽

Exit Tunnel ◽

Domain Iii

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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Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors

PLoS ONE ◽

10.1371/journal.pone.0252497 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0252497

Author(s):

Gisela Pöll ◽

Michael Pilsl ◽

Joachim Griesenbeck ◽

Herbert Tschochner ◽

Philipp Milkereit

Keyword(s):

Electron Microscopy ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Protein Assembly ◽

Functional Coupling ◽

Large Ribosomal Subunit ◽

Cryo Electron Microscopy ◽

Intrinsic Quality ◽

Domain Arrangement ◽

The Stability

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

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Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half assembled intermediate

10.1101/242446 ◽

2018 ◽

Author(s):

Dejian Zhou ◽

Xing Zhu ◽

Sanduo Zheng ◽

Dan Tan ◽

Meng-Qiu Dong ◽

...

Keyword(s):

Electron Microscopy ◽

Ribosomal Proteins ◽

Large Subunit ◽

Ribosomal Subunit ◽

Large Ribosomal Subunit ◽

Ribosomal Subunits ◽

Cryo Electron Microscopy ◽

5.8S Rrna ◽

Early Processing ◽

Large Subunit Rrna

AbstractAssembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. How the highly intertwined structure of 60S large ribosomal subunits is established is unknown. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half assembled subunit. Domains I, II and VI of 25S/5.8S rRNA tightly pack into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to the establishment of the global architecture of 60S subunits.

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The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly

Journal of Bacteriology ◽

10.1128/jb.00444-06 ◽

2006 ◽

Vol 188 (19) ◽

pp. 6757-6770 ◽

Cited By ~ 98

Author(s):

Mengxi Jiang ◽

Kaustuv Datta ◽

Angela Walker ◽

John Strahler ◽

Pia Bagamasbad ◽

...

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Large Subunit ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Macromolecular Complex ◽

Large Ribosomal Subunit ◽

Proteomic Approach ◽

Temporal Relationships

ABSTRACT The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtAE, in late steps of large ribosomal subunit biogenesis. CgtAE belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtAE cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtAE mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtAE is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtAE. We also show that purified CgtAE associates with purified ribosomal particles in the GTP-bound form. Finally, CgtAE cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.

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Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis

Nucleic Acids Research ◽

10.1093/nar/gkaa893 ◽

2020 ◽

Vol 48 (20) ◽

pp. 11750-11761

Author(s):

Donna Matzov ◽

Masato Taoka ◽

Yuko Nobe ◽

Yoshio Yamauchi ◽

Yehuda Halfon ◽

...

Keyword(s):

Ribosomal Rna ◽

Euglena Gracilis ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Catalytic Rna ◽

Central Component ◽

Large Ribosomal Subunit ◽

Eukaryotic Alga ◽

Cytoplasmic Ribosome ◽

Exit Tunnel

Abstract Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.

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