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Nature Communications Mar 2022Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both...
Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome - license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.
Topics: Animals; Leukemia; Mammals; Mice; Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosomes; Shwachman-Diamond Syndrome
PubMed: 35322020
DOI: 10.1038/s41467-022-29214-7 -
Molecular Biology of the Cell Mar 2009Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes,...
Large cargoes require multiple receptors for efficient transport through the nuclear pore complex. The 60S ribosomal subunit is one of the bulkiest transport cargoes, and in yeast three different receptors, Crm1, Mex67/Mtr2, and Arx1, collaborate in its export. However, only Crm1, recruited by the adapter Nmd3, appears to be conserved for 60S export in higher eukaryotes. We asked if export of the large subunit requires specific receptors. We made protein fusions between mutant Nmd3 and various export receptors. Surprisingly, fusions of Mex67, the tRNA exportin Los1, Mtr2, Cse1, or Msn5 to Nmd3, lacking its Crm1-dependent nuclear export signal (NES), all functioned in export. Furthermore, these chimeric proteins supported 60S export even in the presence of the Crm1 inhibitor leptomycin B, indicating that export was now independent of Crm1. These results suggest that there is not a requirement for a specific export receptor for the large subunit, as recruitment of any receptor will suffice. Finally we show that the addition of an NES directly to the 60S ribosomal subunit protein Rpl3 promotes export. These results imply remarkable flexibility in the export pathway for the 60S subunit and help explain how different export receptors could have evolved in different eukaryotic lineages.
Topics: Fatty Acids, Unsaturated; GTP-Binding Proteins; Karyopherins; Models, Molecular; Nuclear Export Signals; Nuclear Proteins; Nucleocytoplasmic Transport Proteins; Protein Transport; RNA-Binding Proteins; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Exportin 1 Protein
PubMed: 19144820
DOI: 10.1091/mbc.e08-10-1000 -
Current Opinion in Chemical Biology Dec 2008Protein synthesis is inherently a dynamic process, requiring both small-scale and large-scale movements of tRNA and mRNA. It has long been suspected that these movements... (Review)
Review
Protein synthesis is inherently a dynamic process, requiring both small-scale and large-scale movements of tRNA and mRNA. It has long been suspected that these movements might be coupled to conformational changes in the ribosome, and in its RNA moieties in particular. Recently, the nature of ribosome structural dynamics has begun to emerge from a combination of approaches, most notably cryo-EM, X-ray crystallography, and FRET. Ribosome movement occurs both on a grand scale, as in the intersubunit rotational movements that are coupled to tRNA-mRNA translocation, and in intricate localized rearrangements such as those that accompany codon-anticodon recognition and peptide bond formation. In spite of much progress, our understanding of the mechanics of translation is now beset with countless new questions, reflecting the vast molecular architecture of the ribosome itself.
Topics: Movement; Peptidyl Transferases; RNA, Ribosomal; Ribosome Subunits, Small; Ribosomes
PubMed: 18848900
DOI: 10.1016/j.cbpa.2008.08.037 -
Nucleic Acids Research Jan 2023Protein synthesis by the ribosome requires large-scale rearrangements of the 'small' subunit (SSU; ∼1 MDa), including inter- and intra-subunit rotational motions....
Protein synthesis by the ribosome requires large-scale rearrangements of the 'small' subunit (SSU; ∼1 MDa), including inter- and intra-subunit rotational motions. However, with nearly 2000 structures of ribosomes and ribosomal subunits now publicly available, it is exceedingly difficult to design experiments based on analysis of all known rotation states. To overcome this, we developed an approach where the orientation of each SSU head and body is described in terms of three angular coordinates (rotation, tilt and tilt direction) and a single translation. By considering the entire RCSB PDB database, we describe 1208 fully-assembled ribosome complexes and 334 isolated small subunits, which span >50 species. This reveals aspects of subunit rearrangements that are universal, and others that are organism/domain-specific. For example, we show that tilt-like rearrangements of the SSU body (i.e. 'rolling') are pervasive in both prokaryotic and eukaryotic (cytosolic and mitochondrial) ribosomes. As another example, domain orientations associated with frameshifting in bacteria are similar to those found in eukaryotic ribosomes. Together, this study establishes a common foundation with which structural, simulation, single-molecule and biochemical efforts can more precisely interrogate the dynamics of this prototypical molecular machine.
Topics: Eukaryota; Protein Biosynthesis; Ribosome Subunits; Ribosomes; Rotation; Prokaryotic Cells; Biomechanical Phenomena
PubMed: 36583339
DOI: 10.1093/nar/gkac1211 -
The Biochemical Journal Jan 2017Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In... (Review)
Review
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.
Topics: Active Transport, Cell Nucleus; Binding Sites; Cell Nucleolus; Cytoplasm; Models, Molecular; Organelle Biogenesis; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; RNA Precursors; RNA, Ribosomal; RNA, Small Nucleolar; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; Saccharomyces cerevisiae
PubMed: 28062837
DOI: 10.1042/BCJ20160516 -
Proceedings of the National Academy of... Jul 2022RtcB is involved in transfer RNA (tRNA) splicing in archaeal and eukaryotic organisms. However, most RtcBs are found in bacteria, whose tRNAs have no introns. Because...
RtcB is involved in transfer RNA (tRNA) splicing in archaeal and eukaryotic organisms. However, most RtcBs are found in bacteria, whose tRNAs have no introns. Because tRNAs are the substrates of archaeal and eukaryotic RtcB, it is assumed that bacterial RtcBs are for repair of damaged tRNAs. Here, we show that a subset of bacterial RtcB, denoted RtcB2 herein, specifically repair ribosomal damage in the decoding center. To access the damage site for repair, however, the damaged 70S ribosome needs to be dismantled first, and this is accomplished by bacterial PrfH. Peptide-release assays revealed that PrfH is only active with the damaged 70S ribosome but not with the intact one. A 2.55-Å cryo-electron microscopy structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into PrfH discriminating between the damaged and the intact ribosomes via specific recognition of the cleaved 3'-terminal nucleotide. RNA repair assays demonstrated that RtcB2 efficiently repairs the damaged 30S ribosomal subunit but not the damaged tRNAs. Cell-based assays showed that the RtcB2-PrfH pair reverse the damage inflicted by ribosome-specific ribotoxins in vivo. Thus, our combined biochemical, structural, and cell-based studies have uncovered a bacterial defense system specifically evolved to reverse the lethal ribosomal damage in the decoding center for cell survival.
Topics: Amino Acyl-tRNA Synthetases; Cryoelectron Microscopy; Escherichia coli Proteins; Protein Conformation; RNA Splicing; RNA, Transfer; Ribosome Subunits, Large, Bacterial
PubMed: 35858322
DOI: 10.1073/pnas.2202464119 -
Proceedings of the National Academy of... Dec 2015We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by...
We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.
Topics: Biocatalysis; Escherichia coli; Evolution, Molecular; Models, Molecular; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Messenger; RNA, Ribosomal; RNA, Transfer; Ribosome Subunits; Ribosomes
PubMed: 26621738
DOI: 10.1073/pnas.1509761112 -
Nature Communications May 2023In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal...
In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.
Topics: Cryoelectron Microscopy; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Ribosomes
PubMed: 37169754
DOI: 10.1038/s41467-023-38161-w -
Cellular and Molecular Life Sciences :... Feb 2009The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site... (Review)
Review
The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site and fMet-tRNA(fMet) to the ribosome, which results in the formation of the first codon-anticodon interaction and sets the reading frame for the decoding of the mRNA. This interaction takes place in the peptidyl site of the 30S ribosomal subunit and is controlled by the initiation factors IF1, IF2 and IF3 to form the 30S initiation complex. The binding of the 50S subunit and the ejection of the IFs mark the irreversible transition to the elongation phase. Visualization of these ligands on the ribosome has been achieved by cryo-electron microscopy and X-ray crystallography studies, which has helped to understand the mechanism of translation initiation at the molecular level. Conformational changes associated with different functional states provide a dynamic view of the initiation process and of its regulation.
Topics: Bacteria; Models, Molecular; Multiprotein Complexes; Nucleic Acid Conformation; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Protein Biosynthesis; Protein Conformation; RNA, Messenger; RNA, Transfer, Met; Ribosome Subunits, Small, Bacterial
PubMed: 19011758
DOI: 10.1007/s00018-008-8416-4 -
Nature Structural & Molecular Biology Oct 2023Ribosome assembly is orchestrated by many assembly factors, including ribosomal RNA methyltransferases, whose precise role is poorly understood. Here, we leverage the...
Ribosome assembly is orchestrated by many assembly factors, including ribosomal RNA methyltransferases, whose precise role is poorly understood. Here, we leverage the power of cryo-EM and machine learning to discover that the E. coli methyltransferase KsgA performs a 'proofreading' function in the assembly of the small ribosomal subunit by recognizing and partially disassembling particles that have matured but are not competent for translation. We propose that this activity allows inactive particles an opportunity to reassemble into an active state, thereby increasing overall assembly fidelity. Detailed structural quantifications in our datasets additionally enabled the expansion of the Nomura assembly map to highlight rRNA helix and r-protein interdependencies, detailing how the binding and docking of these elements are tightly coupled. These results have wide-ranging implications for our understanding of the quality-control mechanisms governing ribosome biogenesis and showcase the power of heterogeneity analysis in cryo-EM to unveil functionally relevant information in biological systems.
Topics: Escherichia coli; Ribosome Subunits, Small; Escherichia coli Proteins; RNA, Ribosomal; Ribosomal Proteins
PubMed: 37653244
DOI: 10.1038/s41594-023-01078-5