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Nucleic Acids Research Jul 2022Human ribosomes have long been thought to be uniform factories with little regulatory function. Accumulating evidence emphasizes the heterogeneity of ribosomal protein...
Human ribosomes have long been thought to be uniform factories with little regulatory function. Accumulating evidence emphasizes the heterogeneity of ribosomal protein (RP) expression in specific cellular functions and development. However, a systematic understanding of functional relevance of RPs is lacking. Here, we surveyed translational and transcriptional changes after individual knockdown of 75 RPs, 44 from the large subunit (60S) and 31 from the small subunit (40S), by Ribo-seq and RNA-seq analyses. Deficiency of individual RPs altered specific subsets of genes transcriptionally and translationally. RP genes were under cotranslational regulation upon ribosomal stress, and deficiency of the 60S RPs and the 40S RPs had opposite effects. RP deficiency altered the expression of genes related to eight major functional classes, including the cell cycle, cellular metabolism, signal transduction and development. 60S RP deficiency led to greater inhibitory effects on cell growth than did 40S RP deficiency, through P53 signaling. Particularly, we showed that eS8/RPS8 deficiency stimulated apoptosis while eL13/RPL13 or eL18/RPL18 deficiency promoted senescence. We also validated the phenotypic impacts of uL5/RPL11 and eL15/RPL15 deficiency on retina development and angiogenesis, respectively. Overall, our study provides a valuable resource for and novel insights into ribosome regulation in cellular activities, development and diseases.
Topics: Gene Knockdown Techniques; Humans; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic; Transcription, Genetic
PubMed: 35137207
DOI: 10.1093/nar/gkac053 -
Nature Reviews. Molecular Cell Biology Feb 2019In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and... (Review)
Review
In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and have generated models for the mechanisms driving the assembly. More recently, the publication of numerous cryo-electron microscopy structures of yeast ribosome assembly intermediates has provided near-atomic resolution snapshots of ribosome precursor particles. Satisfyingly, these structural data support the genetic and biochemical models and provide additional mechanistic insight into ribosome assembly. In this Review, we discuss the mechanisms of assembly of the yeast small ribosomal subunit and large ribosomal subunit in the nucleolus, nucleus and cytoplasm. Particular emphasis is placed on concepts such as the mechanisms of RNA compaction, the functions of molecular switches and molecular mimicry, the irreversibility of assembly checkpoints and the roles of structural and functional proofreading of pre-ribosomal particles.
Topics: Animals; Cell Nucleus; Cryoelectron Microscopy; Cytoplasm; Humans; RNA; Ribosome Subunits
PubMed: 30467428
DOI: 10.1038/s41580-018-0078-y -
The Journal of Antimicrobial... Apr 2020This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we... (Review)
Review
This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we have examined the effect of 52 different antibiotics on ribosomal subunit formation in six different microorganisms. Most of the antimicrobials we have studied are specific, preventing the formation of only the subunit to which they bind. A few interesting exceptions have also been observed. Forty-one research publications and a book chapter have resulted from this investigation. This review will describe the methodology we used and the fit of our results to a hypothetical model. The model predicts that inhibition of subunit assembly and translation are equivalent targets for most of the antibiotics we have investigated.
Topics: Anti-Bacterial Agents; Bacteria; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits; Ribosomes
PubMed: 31942624
DOI: 10.1093/jac/dkz544 -
Nature Jun 2022Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small...
Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small mitoribosomal subunit (SSU) intermediates in complex with auxiliary factors, revealing a sequential assembly mechanism. The methyltransferase TFB1M binds to partially unfolded rRNA h45 that is promoted by RBFA, while the mRNA channel is blocked. This enables binding of METTL15 that promotes further rRNA maturation and a large conformational change of RBFA. The new conformation allows initiation factor mtIF3 to already occupy the subunit interface during the assembly. Finally, the mitochondria-specific ribosomal protein mS37 (ref. ) outcompetes RBFA to complete the assembly with the SSU-mS37-mtIF3 complex that proceeds towards mtIF2 binding and translation initiation. Our results explain how the action of step-specific factors modulate the dynamic assembly of the SSU, and adaptation of a unique protein, mS37, links the assembly to initiation to establish the catalytic human mitoribosome.
Topics: Humans; Cryoelectron Microscopy; DNA-Binding Proteins; Eukaryotic Initiation Factors; Mitochondria; Mitochondrial Proteins; Mitochondrial Ribosomes; Ribosomal Proteins; Ribosome Subunits, Small; RNA, Ribosomal; RNA-Binding Proteins; Transcription Factors
PubMed: 35676484
DOI: 10.1038/s41586-022-04795-x -
Molecules (Basel, Switzerland) Nov 2016Ribosome-inactivating proteins (RIPs) including ricin, Shiga toxin, and trichosanthin, are RNA -glycosidases that depurinate a specific adenine residue (A-4324 in rat... (Review)
Review
Ribosome-inactivating proteins (RIPs) including ricin, Shiga toxin, and trichosanthin, are RNA -glycosidases that depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. RIPs are grouped into three types according to the number of subunits and the organization of the precursor sequences. RIPs are two-domain proteins, with the active site located in the cleft between the N- and C-terminal domains. It has been found that the basic surface residues of the RIPs promote rapid and specific targeting to the ribosome and a number of RIPs have been shown to interact with the C-terminal regions of the P proteins of the ribosome. At present, the structural basis for the interaction of trichosanthin and ricin-A chain toward P2 peptide is known. This review surveys the structural features of the representative RIPs and discusses how they approach and interact with the ribosome.
Topics: Catalytic Domain; Models, Molecular; Molecular Structure; Protein Binding; Protein Interaction Domains and Motifs; Ribosome Inactivating Proteins; Ribosome Subunits; Ribosomes; Structure-Activity Relationship; Substrate Specificity
PubMed: 27879643
DOI: 10.3390/molecules21111588 -
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 -
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 -
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 -
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 -
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