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Nature Jul 2022Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases. A key commitment step is when the...
Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.
Topics: Cryoelectron Microscopy; Eukaryotic Initiation Factor-1; Eukaryotic Initiation Factors; Humans; RNA, Transfer, Met; Ribosome Subunits; Single Molecule Imaging
PubMed: 35732735
DOI: 10.1038/s41586-022-04858-z -
Nature Structural & Molecular Biology May 2023During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large...
During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.
Topics: Cryoelectron Microscopy; RNA, Ribosomal; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ribosomal Proteins; Ribosome Subunits, Large; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic
PubMed: 37037974
DOI: 10.1038/s41594-023-00947-3 -
Cell Reports Oct 2023Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in...
Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.
Topics: Cell Nucleolus; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Ribosomes; RNA, Ribosomal, 18S; Humans; Animals; Rats; DNA-Binding Proteins; Transcription Factors; Protein Biosynthesis
PubMed: 37851577
DOI: 10.1016/j.celrep.2023.113280 -
Cell Reports Aug 2021Reversible monoubiquitination of small subunit ribosomal proteins RPS2/uS5 and RPS3/uS3 has been noted to occur on ribosomes involved in ZNF598-dependent mRNA...
Reversible monoubiquitination of small subunit ribosomal proteins RPS2/uS5 and RPS3/uS3 has been noted to occur on ribosomes involved in ZNF598-dependent mRNA surveillance. Subsequent deubiquitination of RPS2 and RPS3 by USP10 is critical for recycling of stalled ribosomes in a process known as ribosome-associated quality control. Here, we identify and characterize the RPS2- and RPS3-specific E3 ligase Really Interesting New Gene (RING) finger protein 10 (RNF10) and its role in translation. Overexpression of RNF10 increases 40S ribosomal subunit degradation similarly to the knockout of USP10. Although a substantial fraction of RNF10-mediated RPS2 and RPS3 monoubiquitination results from ZNF598-dependent sensing of collided ribosomes, ZNF598-independent impairment of translation initiation and elongation also contributes to RPS2 and RPS3 monoubiquitination. RNF10 photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies crosslinked mRNAs, tRNAs, and 18S rRNAs, indicating recruitment of RNF10 to ribosomes stalled in translation. These impeded ribosomes are tagged by ubiquitin at their 40S subunit for subsequent programmed degradation unless rescued by USP10.
Topics: Carrier Proteins; Cross-Linking Reagents; HEK293 Cells; Humans; Models, Biological; Mutation; Peptides; Protein Biosynthesis; Protein Domains; RNA, Messenger; RNA, Transfer; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Ubiquitin Thiolesterase; Ubiquitination
PubMed: 34348161
DOI: 10.1016/j.celrep.2021.109468 -
Nature Feb 2023Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. Thus,...
Mitochondrial ribosomes (mitoribosomes) synthesize proteins encoded within the mitochondrial genome that are assembled into oxidative phosphorylation complexes. Thus, mitoribosome biogenesis is essential for ATP production and cellular metabolism. Here we used cryo-electron microscopy to determine nine structures of native yeast and human mitoribosomal small subunit assembly intermediates, illuminating the mechanistic basis for how GTPases are used to control early steps of decoding centre formation, how initial rRNA folding and processing events are mediated, and how mitoribosomal proteins have active roles during assembly. Furthermore, this series of intermediates from two species with divergent mitoribosomal architecture uncovers both conserved principles and species-specific adaptations that govern the maturation of mitoribosomal small subunits in eukaryotes. By revealing the dynamic interplay between assembly factors, mitoribosomal proteins and rRNA that are required to generate functional subunits, our structural analysis provides a vignette for how molecular complexity and diversity can evolve in large ribonucleoprotein assemblies.
Topics: Humans; Cryoelectron Microscopy; Mitochondrial Proteins; Mitochondrial Ribosomes; Ribosomal Proteins; Saccharomyces cerevisiae; RNA, Ribosomal; GTP Phosphohydrolases; Ribonucleoproteins; Fungal Proteins; Ribosome Subunits, Small
PubMed: 36482135
DOI: 10.1038/s41586-022-05621-0 -
Nucleic Acids Research Jun 2022NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is...
NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10's essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation 'machinery' that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.
Topics: Acetylation; Animals; Eukaryota; Humans; RNA, Ribosomal; RNA, Ribosomal, 18S; RNA, Small Nucleolar; Ribosome Subunits, Small
PubMed: 35648437
DOI: 10.1093/nar/gkac404 -
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 -
RNA (New York, N.Y.) Jul 2018The eukaryotic ribosome is made of four intricately folded ribosomal RNAs and 79 proteins. During rapid growth, yeast cells produce an incredible 2000 ribosomes every... (Review)
Review
The eukaryotic ribosome is made of four intricately folded ribosomal RNAs and 79 proteins. During rapid growth, yeast cells produce an incredible 2000 ribosomes every minute. Ribosome assembly involves more than 200 -acting factors, intervening from the transcription of the preribosomal RNA in the nucleolus to late maturation events in the cytoplasm. The biogenesis of the small ribosomal subunit, or 40S, is especially intricate, requiring more than four times the mass of the small subunit in assembly factors for its full maturation. Recent studies have provided new insights into the complex assembly of the 40S subunit. These data from cryo-electron microscopy, X-ray crystallography, and other biochemical and molecular biology methods, have elucidated the role of many factors required in small subunit maturation. Mechanisms of the regulation of ribosome assembly have also emerged from this body of work. This review aims to integrate these new results into an updated view of small subunit biogenesis and its regulation, in yeast, from transcription to the formation of the mature small subunit.
Topics: Biological Transport; Cell Cycle; Cytoplasm; Organelle Biogenesis; Ribosome Subunits, Small, Eukaryotic; Saccharomyces cerevisiae; TOR Serine-Threonine Kinases; Transcription, Genetic
PubMed: 29712726
DOI: 10.1261/rna.066985.118 -
Journal of Bacteriology Apr 2020When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to...
When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to favorable conditions. Here, we show that the ribosome dimerization protein hibernation-promoting factor (HPF) functions to protect essential ribosomal proteins. Ribosomes isolated from strains lacking HPF (Δ) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located directly at the ribosome dimer interface. We used single-particle cryo-electron microscopy (cryo-EM) to further characterize these ribosomes and observed that a high percentage of ribosomes were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function. HPF is almost universally conserved in bacteria, and HPF deletions in diverse species exhibit decreased viability during starvation. Our data provide mechanistic insight into this phenotype and establish a mechanism for how HPF protects ribosomes during quiescence. The formation of ribosome dimers during periods of dormancy is widespread among bacteria. Dimerization is typically mediated by a single protein, hibernation-promoting factor (HPF). Bacteria lacking HPF exhibit strong defects in viability and pathogenesis and, in some species, extreme loss of rRNA. The mechanistic basis of these phenotypes has not been determined. Here, we report that HPF from the Gram-positive bacterium preserves ribosomes by preventing the loss of essential ribosomal proteins at the dimer interface. This protection may explain phenotypes associated with the loss of HPF, since ribosome protection would aid survival during nutrient limitation and impart a strong selective advantage when the bacterial cell rapidly reinitiates growth in the presence of sufficient nutrients.
Topics: Bacillus subtilis; Bacterial Proteins; Cryoelectron Microscopy; Dimerization; Ribosome Subunits, Small; Ribosomes
PubMed: 32123037
DOI: 10.1128/JB.00009-20 -
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