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1
Moraleva, Anastasia A., Alexander S. Deryabin, Yury P. Rubtsov, Maria P. Rubtsova, and Olga A. Dontsova. "Eukaryotic Ribosome Biogenesis: The 60S Subunit." Acta Naturae 14, no. 2 (July 21, 2022): 39–49. http://dx.doi.org/10.32607/actanaturae.11541.
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Ribosome biogenesis is consecutive coordinated maturation of ribosomal precursors in the nucleolus, nucleoplasm, and cytoplasm. The formation of mature ribosomal subunits involves hundreds of ribosomal biogenesis factors that ensure ribosomal RNA processing, tertiary structure, and interaction with ribosomal proteins. Although the main features and stages of ribosome biogenesis are conservative among different groups of eukaryotes, this process in human cells has become more complicated due to the larger size of the ribosomes and pre-ribosomes and intricate regulatory pathways affecting their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. A previous part of this review summarized recent data on the processing of the primary rRNA transcript and compared the maturation of the small 40S subunit in yeast and human cells. This part of the review focuses on the biogenesis of the large 60S subunit of eukaryotic ribosomes.
2
Shayan, Ramtin, Dana Rinaldi, Natacha Larburu, Laura Plassart, Stéphanie Balor, David Bouyssié, Simon Lebaron, Julien Marcoux, Pierre-Emmanuel Gleizes, and Célia Plisson-Chastang. "Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy." Molecules 25, no. 5 (March 3, 2020): 1125. http://dx.doi.org/10.3390/molecules25051125.
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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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Yu, Ting, and Fuxing Zeng. "Chloramphenicol Interferes with 50S Ribosomal Subunit Maturation via Direct and Indirect Mechanisms." Biomolecules 14, no. 10 (September 27, 2024): 1225. http://dx.doi.org/10.3390/biom14101225.
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Chloramphenicol (CAM), a well-known broad-spectrum antibiotic, inhibits peptide bond formation in bacterial ribosomes. It has been reported to affect ribosome assembly mainly through disrupting the balance of ribosomal proteins. The present study investigates the multifaceted effects of CAM on the maturation of the 50S ribosomal subunit in Escherichia coli (E. coli). Using label-free quantitative mass spectrometry (LFQ-MS), we observed that CAM treatment also leads to the upregulation of assembly factors. Further cryo-electron microscopy (cryo-EM) analysis of the ribosomal precursors characterized the CAM-treatment-accumulated pre-50S intermediates. Heterogeneous reconstruction identified 26 distinct pre-50S intermediates, which were categorized into nine main states based on their structural features. Our structural analysis highlighted that CAM severely impedes the formation of the central protuberance (CP), H89, and H58 during 50S ribosomal subunit maturation. The ELISA assay further demonstrated the direct binding of CAM to the ribosomal precursors, suggesting that the interference with 50S maturation occurs through a combination of direct and indirect mechanisms. These findings provide new insights into the mechanism of the action of CAM and provide a foundation for a better understanding of the assembly landscapes of the ribosome.
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Moraleva, Anastasia A., Alexander S. Deryabin, Yury P. Rubtsov, Maria P. Rubtsova, and Olga A. Dontsova. "Eukaryotic Ribosome Biogenesis: The 40S Subunit." Acta Naturae 14, no. 1 (May 10, 2022): 14–30. http://dx.doi.org/10.32607/actanaturae.11540.
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The formation of eukaryotic ribosomes is a sequential process of ribosomal precursors maturation in the nucleolus, nucleoplasm, and cytoplasm. Hundreds of ribosomal biogenesis factors ensure the accurate processing and formation of the ribosomal RNAs tertiary structure, and they interact with ribosomal proteins. Most of what we know about the ribosome assembly has been derived from yeast cell studies, and the mechanisms of ribosome biogenesis in eukaryotes are considered quite conservative. Although the main stages of ribosome biogenesis are similar across different groups of eukaryotes, this process in humans is much more complicated owing to the larger size of the ribosomes and pre-ribosomes and the emergence of regulatory pathways that affect their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. This review addresses the key aspects of yeast and human ribosome biogenesis, using the 40S subunit as an example. The mechanisms underlying these differences are still not well understood, because, unlike yeast, there are no effective methods for characterizing pre-ribosomal complexes in humans. Understanding the mechanisms of human ribosome assembly would have an incidence on a growing number of genetic diseases (ribosomopathies) caused by mutations in the genes encoding ribosomal proteins and ribosome biogenesis factors. In addition, there is evidence that ribosome assembly is regulated by oncogenic signaling pathways, and that defects in the ribosome biogenesis are linked to the activation of tumor suppressors.
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Sleiman, Sophie, and Francois Dragon. "Recent Advances on the Structure and Function of RNA Acetyltransferase Kre33/NAT10." Cells 8, no. 9 (September 5, 2019): 1035. http://dx.doi.org/10.3390/cells8091035.
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Ribosome biogenesis is one of the most energy demanding processes in the cell. In eukaryotes, the main steps of this process occur in the nucleolus and include pre-ribosomal RNA (pre-rRNA) processing, post-transcriptional modifications, and assembly of many non-ribosomal factors and ribosomal proteins in order to form mature and functional ribosomes. In yeast and humans, the nucleolar RNA acetyltransferase Kre33/NAT10 participates in different maturation events, such as acetylation and processing of 18S rRNA, and assembly of the 40S ribosomal subunit. Here, we review the structural and functional features of Kre33/NAT10 RNA acetyltransferase, and we underscore the importance of this enzyme in ribosome biogenesis, as well as in acetylation of non-ribosomal targets. We also report on the role of human NAT10 in Hutchinson–Gilford progeria syndrome.
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Bikmullin, Aydar G., Bulat Fatkhullin, Artem Stetsenko, Azat Gabdulkhakov, Natalia Garaeva, Liliia Nurullina, Evelina Klochkova, et al. "Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus." International Journal of Molecular Sciences 24, no. 3 (January 20, 2023): 2118. http://dx.doi.org/10.3390/ijms24032118.
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Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins’ folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S–RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.
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Martinez-Seidel, Federico, Olga Beine-Golovchuk, Yin-Chen Hsieh, Kheloud El Eshraky, Michal Gorka, Bo-Eng Cheong, Erika V. Jimenez-Posada, et al. "Spatially Enriched Paralog Rearrangements Argue Functionally Diverse Ribosomes Arise during Cold Acclimation in Arabidopsis." International Journal of Molecular Sciences 22, no. 11 (June 7, 2021): 6160. http://dx.doi.org/10.3390/ijms22116160.
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Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.
8
Warren, Alan J. "Shwachman-Diamond Syndrome and the Quality Control of Ribosome Assembly." Blood 128, no. 22 (December 2, 2016): SCI—42—SCI—42. http://dx.doi.org/10.1182/blood.v128.22.sci-42.sci-42.
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Abstract The synthesis of new ribosomes is a fundamental conserved process in all cells. Ribosomes are pre-assembled in the nucleus and subsequently exported to the cytoplasm where they acquire functionality through a series of final maturation steps that include formation of the catalytic center, recruitment of the last remaining ribosomal proteins and the removal of inhibitory assembly factors. Surprisingly, a number of key factors (SBDS, DNAJC21, RPL10 (uL16)) involved in late cytoplasmic maturation of the large (60S) ribosomal subunit are mutated in both inherited and sporadic forms of leukemia. In particular, biallelic mutations in the SBDS gene cause Shwachman-Diamond syndrome (SDS), a recessive bone marrow failure disorder with significant predisposition to acute myeloid leukemia. By using the latest advances in single-particle cryo-electron microscopy to elucidate the function of the SBDS protein, we have uncovered an elegant mechanism that couples final maturation of the 60S subunit to a quality control assessment of the structural integrity of the active sites of the ribosome. Further molecular dissection of this pathway may inform novel therapeutic strategies for SDS and leukemia more generally. References: 1. Weis F, Giudice E, Churcher M,et al. Mechanism of eIF6 release from the nascent 60S ribosomal subunit. Nat Struct Mol Biol, (2015) Nov;22(11):914-9. 2. Wong CC, Traynor D, Basse N, et al. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 3. Finch AJ, Hilcenko C, Basse N, et al. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes Dev (2011) 25: 917-929. 4. Menne TM, Goyenechea B, Sánchez-Puig N, et al. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures No relevant conflicts of interest to declare.
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Graifer, Dmitri, and Galina Karpova. "Eukaryotic protein uS19: a component of the decoding site of ribosomes and a player in human diseases." Biochemical Journal 478, no. 5 (March 4, 2021): 997–1008. http://dx.doi.org/10.1042/bcj20200950.
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Proteins belonging to the universal ribosomal protein (rp) uS19 family are constituents of small ribosomal subunits, and their conserved globular parts are involved in the formation of the head of these subunits. The eukaryotic rp uS19 (previously known as S15) comprises a C-terminal extension that has no homology in the bacterial counterparts. This extension is directly implicated in the formation of the ribosomal decoding site and thereby affects translational fidelity in a manner that has no analogy in bacterial ribosomes. Another eukaryote-specific feature of rp uS19 is its essential participance in the 40S subunit maturation due to the interactions with the subunit assembly factors required for the nuclear exit of pre-40S particles. Beyond properties related to the translation machinery, eukaryotic rp uS19 has an extra-ribosomal function concerned with its direct involvement in the regulation of the activity of an important tumor suppressor p53 in the Mdm2/Mdmx-p53 pathway. Mutations in the RPS15 gene encoding rp uS19 are linked to diseases (Diamond Blackfan anemia, chronic lymphocytic leukemia and Parkinson's disease) caused either by defects in the ribosome biogenesis or disturbances in the functioning of ribosomes containing mutant rp uS19, likely due to the changed translational fidelity. Here, we review currently available data on the involvement of rp uS19 in the operation of the translational machinery and in the maturation of 40S subunits, on its extra-ribosomal function, and on relationships between mutations in the RPS15 gene and certain human diseases.
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Schedlbauer, Andreas, Idoia Iturrioz, Borja Ochoa-Lizarralde, Tammo Diercks, Jorge Pedro López-Alonso, José Luis Lavin, Tatsuya Kaminishi, et al. "A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit." Science Advances 7, no. 23 (June 2021): eabf7547. http://dx.doi.org/10.1126/sciadv.abf7547.
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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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Shetty, Sunil, and Umesh Varshney. "An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation." Proceedings of the National Academy of Sciences 113, no. 41 (October 3, 2016): E6126—E6134. http://dx.doi.org/10.1073/pnas.1609550113.
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Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3′ and 5′ ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.
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Lövgren, J. Mattias, and P. Mikael Wikström. "Hybrid Protein between Ribosomal Protein S16 and RimM of Escherichia coli Retains the Ribosome Maturation Function of Both Proteins." Journal of Bacteriology 183, no. 18 (September 15, 2001): 5352–57. http://dx.doi.org/10.1128/jb.183.18.5352-5357.2001.
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ABSTRACT The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of E. coli approximately threefold. Several faster-growing derivatives of the rimM amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the rimMmRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the rimM mRNA. Three other independently isolated suppressor mutations created a fusion betweenrpsP, encoding the ribosomal protein S16, andrimM on the chromosome as a result of mutations in therpsP stop codon preceding rimM. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.
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Baßler, Jochen, and Ed Hurt. "Eukaryotic Ribosome Assembly." Annual Review of Biochemistry 88, no. 1 (June 20, 2019): 281–306. http://dx.doi.org/10.1146/annurev-biochem-013118-110817.
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Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo–electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.
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Abetov, Danysh A., Vladimir S. Kiyan, Assylbek A. Zhylkibayev, Dilara A. Sarbassova, Sanzhar D. Alybayev, Eric Spooner, Min Sup Song, Rakhmetkazhy I. Bersimbaev, and Dos D. Sarbassov. "Formation of mammalian preribosomes proceeds from intermediate to composed state during ribosome maturation." Journal of Biological Chemistry 294, no. 28 (May 10, 2019): 10746–57. http://dx.doi.org/10.1074/jbc.ac119.008378.
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In eukaryotes, ribosome assembly is a rate-limiting step in ribosomal biogenesis that takes place in a distinctive subnuclear organelle, the nucleolus. How ribosomes get assembled at the nucleolar site by forming initial preribosomal complexes remains poorly characterized. In this study, using several human and murine cell lines, we developed a method for isolation of native mammalian preribosomal complexes by lysing cell nuclei through mild sonication. A sucrose gradient fractionation of the nuclear lysate resolved several ribonucleoprotein (RNP) complexes containing rRNAs and ribosomal proteins. Characterization of the RNP complexes with MS-based protein identification and Northern blotting–based rRNA detection approaches identified two types of preribosomes we named here as intermediate preribosomes (IPRibs) and composed preribosome (CPRib). IPRib complexes comprised large preribosomes (105S to 125S in size) containing the rRNA modification factors and premature rRNAs. We further observed that a distinctive CPRib complex consists of an 85S preribosome assembled with mature rRNAs and a ribosomal biogenesis factor, Ly1 antibody–reactive (LYAR), that does not associate with premature rRNAs and rRNA modification factors. rRNA-labeling experiments uncovered that IPRib assembly precedes CPRib complex formation. We also found that formation of the preribosomal complexes is nutrient-dependent because the abundances of IPRib and CPRib decreased substantially when cells were either deprived of amino acids or exposed to an mTOR kinase inhibitor. These findings indicate that preribosomes form via dynamic and nutrient-dependent processing events and progress from an intermediate to a composed state during ribosome maturation.
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Kim, Tae-Sung, Chang-Young Jang, Hag Dong Kim, Jae Yung Lee, Byung-Yoon Ahn, and Joon Kim. "Interaction of Hsp90 with Ribosomal Proteins Protects from Ubiquitination and Proteasome-dependent Degradation." Molecular Biology of the Cell 17, no. 2 (February 2006): 824–33. http://dx.doi.org/10.1091/mbc.e05-08-0713.
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Heat-shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of various transcription factors and protein kinases in signal transduction. Multifunctional ribosomal protein S3 (rpS3), a component of the ribosomal small subunit, is involved in DNA repair and apoptosis. Our data show that Hsp90 binds directly to rpS3 and the functional consequence of Hsp90-rpS3 interaction results in the prevention of the ubiquitination and the proteasome-dependent degradation of rpS3, subsequently retaining the function and the biogenesis of the ribosome. Interference of Hsp90 activity by Hsp90 inhibitors appears to dissociate rpS3 from Hsp90, associate the protein with Hsp70, and induce the degradation of free forms of rpS3. Furthermore, ribosomal protein S6 (rpS6) also interacted with Hsp90 and exhibited a similar effect upon treatment with Hsp90 inhibitors. Therefore, we conclude that Hsp90 regulates the function of ribosomes by maintaining the stability of 40S ribosomal proteins such as rpS3 and rpS6.
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Phan, Tamara, Fatima Khalid, and Sebastian Iben. "Nucleolar and Ribosomal Dysfunction—A Common Pathomechanism in Childhood Progerias?" Cells 8, no. 6 (June 4, 2019): 534. http://dx.doi.org/10.3390/cells8060534.
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The nucleolus organizes around the sites of transcription by RNA polymerase I (RNA Pol I). rDNA transcription by this enzyme is the key step of ribosome biogenesis and most of the assembly and maturation processes of the ribosome occur co-transcriptionally. Therefore, disturbances in rRNA transcription and processing translate to ribosomal malfunction. Nucleolar malfunction has recently been described in the classical progeria of childhood, Hutchinson–Gilford syndrome (HGPS), which is characterized by severe signs of premature aging, including atherosclerosis, alopecia, and osteoporosis. A deregulated ribosomal biogenesis with enlarged nucleoli is not only characteristic for HGPS patients, but it is also found in the fibroblasts of “normal” aging individuals. Cockayne syndrome (CS) is also characterized by signs of premature aging, including the loss of subcutaneous fat, alopecia, and cataracts. It has been shown that all genes in which a mutation causes CS, are involved in rDNA transcription by RNA Pol I. A disturbed ribosomal biogenesis affects mitochondria and translates into ribosomes with a reduced translational fidelity that causes endoplasmic reticulum (ER) stress and apoptosis. Therefore, it is speculated that disease-causing disturbances in the process of ribosomal biogenesis may be more common than hitherto anticipated.
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Albanèse, Véronique, Stefanie Reissmann, and Judith Frydman. "A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis." Journal of Cell Biology 189, no. 1 (April 5, 2010): 69–81. http://dx.doi.org/10.1083/jcb.201001054.
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Molecular chaperones assist cellular protein folding as well as oligomeric complex assembly. In eukaryotic cells, several chaperones termed chaperones linked to protein synthesis (CLIPS) are transcriptionally and physically linked to ribosomes and are implicated in protein biosynthesis. In this study, we show that a CLIPS network comprising two ribosome-anchored J-proteins, Jjj1 and Zuo1, function together with their partner Hsp70 proteins to mediate the biogenesis of ribosomes themselves. Jjj1 and Zuo1 have overlapping but distinct functions in this complex process involving the coordinated assembly and remodeling of dozens of proteins on the ribosomal RNA (rRNA). Both Jjj1 and Zuo1 associate with nuclear 60S ribosomal biogenesis intermediates and play an important role in nuclear rRNA processing, leading to mature 25S rRNA. In addition, Zuo1, acting together with its Hsp70 partner, SSB (stress 70 B), also participates in maturation of the 35S rRNA. Our results demonstrate that, in addition to their known cytoplasmic roles in de novo protein folding, some ribosome-anchored CLIPS chaperones play a critical role in nuclear steps of ribosome biogenesis.
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Zang, Hannah, Robert Shackelford, Alice Bewley, and Alexander E. Beeser. "Mutational Analyses of the Cysteine-Rich Domain of Yvh1, a Protein Required for Translational Competency in Yeast." Biology 11, no. 8 (August 22, 2022): 1246. http://dx.doi.org/10.3390/biology11081246.
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Ribosome assembly is a complex biological process facilitated by >200 trans-acting factors (TAFs) that function as scaffolds, place-holders or complex remodelers to promote efficient and directional ribosomal subunit assembly but are not themselves part of functional ribosomes. One such yeast TAF is encoded by Mrt4 which assembles onto pre-60S complexes in the nuclear compartment and remains bound to pre-60S complexes as they are exported into the cytoplasm. There, Mrt4 is displaced from pre-60S complexes facilitating the subsequent addition of the ribosomal stalk complex (P0/P1/P2). Ribosomal stalk proteins interact with translational GTPases (trGTPase) which facilitate and control protein synthesis on the ribosome. The rRNA-binding domain of Mrt4 is structurally similar to P0, with both proteins binding to the same interface of pre-60S subunits in a mutually exclusive manner; the addition of the ribosomal stalk therefore requires the displacement of Mrt4 from pre-60S subunits. Mrt4 removal requires the C-terminal cysteine-rich domain (CRD) of the dual-specificity phosphatase Yvh1. Unlike many other TAFs, yeast lacking Yvh1 are viable but retain Mrt4 on cytoplasmic pre-60S complexes precluding ribosomal stalk addition. Although Yvh1’s role in Mrt4 removal is well established, how Yvh1 accomplishes this is largely unknown. Here, we report an unbiased genetic screen to isolate Yvh1 variants that fail to displace Mrt4 from pre-60S ribosomes. Bioorthogonal non-canonical amino acid tagging (BONCAT) approaches demonstrate that these YVH1 loss-of-function variants also display defects in nascent protein production. The further characterization of one LOF variant, Yvh1F283L, establishes it as an expression-dependent, dominant-negative variant capable of interfering with endogenous Yvh1 function, and we describe how this Yvh1 variant can be used as a novel probe to better understand ribosome maturation and potentially ribosome heterogeneity in eukaryotes.
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Shankar, Vaishnavi, Robert Rauscher, Julia Reuther, Walid H. Gharib, Miriam Koch, and Norbert Polacek. "rRNA expansion segment 27Lb modulates the factor recruitment capacity of the yeast ribosome and shapes the proteome." Nucleic Acids Research 48, no. 6 (January 21, 2020): 3244–56. http://dx.doi.org/10.1093/nar/gkaa003.
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Abstract Fine-tuned regulation of protein biosynthesis is crucial for cellular fitness and became even more vital when cellular and organismal complexity increased during the course of evolution. In order to cope with this augmented demand for translation control, eukaryal ribosomes have gained extensions both at the ribosomal protein and rRNA levels. Here we analyze the functional role of ES27L, an rRNA expansion segment in the large ribosomal subunit of Saccharomyces cerevisiae. Deletion of the b-arm of this expansion segment, called ES27Lb, did not hamper growth during optimal conditions, thus demonstrating that this 25S rRNA segment is not inherently crucial for ribosome functioning. However, reductive stress results in retarded growth and rendered unique protein sets prone to aggregation. Lack of ES27Lb negatively affects ribosome-association of known co-translational N-terminal processing enzymes which in turn contributes to the observed protein aggregation. Likely as a compensatory response to these challenges, the truncated ribosomes showed re-adjusted translation of specific sets of mRNAs and thus fine-tune the translatome in order to re-establish proteostasis. Our study gives comprehensive insight into how a highly conserved eukaryal rRNA expansion segment defines ribosomal integrity, co-translational protein maturation events and consequently cellular fitness.
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Landry-Voyer, Anne-Marie, Sarah Bilodeau, Danny Bergeron, Kiersten L. Dionne, Sarah A. Port, Caroline Rouleau, François-Michel Boisvert, Ralph H. Kehlenbach, and François Bachand. "Human PDCD2L Is an Export Substrate of CRM1 That Associates with 40S Ribosomal Subunit Precursors." Molecular and Cellular Biology 36, no. 24 (October 3, 2016): 3019–32. http://dx.doi.org/10.1128/mcb.00303-16.
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Protein arginine methyltransferase 3 (PRMT3) forms a stable complex with 40S ribosomal protein S2 (RPS2) and contributes to ribosome biogenesis. However, the molecular mechanism by which PRMT3 influences ribosome biogenesis and/or function still remains unclear. Using quantitative proteomics, we identified human programmed cell death 2-like (PDCD2L) as a novel PRMT3-associated protein. Our data suggest that RPS2 promotes the formation of a conserved extraribosomal complex with PRMT3 and PDCD2L. We also show that PDCD2L associates with 40S subunit precursors that contain a 3′-extended form of the 18S rRNA (18S-E pre-rRNA) and several pre-40S maturation factors. PDCD2L shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner using a leucine-rich nuclear export signal that is sufficient to direct the export of a reporter protein. Although PDCD2L is not required for the biogenesis and export of 40S ribosomal subunits, we found that PDCD2L -null cells accumulate free 60S ribosomal subunits, which is indicative of a deficiency in 40S subunit availability. Our data also indicate that PDCD2L and its paralog, PDCD2, function redundantly in 40S ribosomal subunit production. Our findings uncover the existence of an extraribosomal complex consisting of PDCD2L, RPS2, and PRMT3 and support a role for PDCD2L in the late maturation of 40S ribosomal subunits.
21
Ji, Xinhua. "Structural insights into cell cycle control by essential GTPase Era." Postępy Biochemii 62, no. 3 (November 18, 2016): 335–42. http://dx.doi.org/10.18388/pb.2016_33.
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Era (Escherichia coli Ras-like protein), essential for bacterial cell viability, is composed of an N-terminal GTPase domain and a C-terminal KH domain. In bacteria, it is required for the processing of 16S ribosomal RNA (rRNA) and maturation of 30S (small) ribosomal subunit. Era recognizes 10 nucleotides (1530GAUCACCUCC1539) near the 3â end of 16S rRNA and interacts with helix 45 (h45, nucleotides 1506-1529). GTP binding enables Era to bind RNA, RNA binding stimulates Eraâs GTP-hydrolyzing activity, and GTP hydrolysis releases Era from matured 30S ribosomal subunit. As such, Era controls cell growth rate via regulating the maturation of the 30S ribosomal subunit. Ribosomes manufacture proteins in all living organisms. The GAUCA sequence and h45 are highly conserved in all three kingdoms of life. Homologues of Era are present in eukaryotic cells. Hence, the mechanism of bacterial Era action also sheds light on the cell cycle control of eukaryotes.
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Flygare, Johan, Anna Aspesi, Joshua C. Bailey, Koichi Miyake, Jacqueline M. Caffrey, Stefan Karlsson, and Steven R. Ellis. "Human RPS19, the gene mutated in Diamond-Blackfan anemia, encodes a ribosomal protein required for the maturation of 40S ribosomal subunits." Blood 109, no. 3 (September 21, 2006): 980–86. http://dx.doi.org/10.1182/blood-2006-07-038232.
Full textAbstract:
Abstract Diamond-Blackfan anemia (DBA) typically presents with red blood cell aplasia that usually manifests in the first year of life. The only gene currently known to be mutated in DBA encodes ribosomal protein S19 (RPS19). Previous studies have shown that the yeast RPS19 protein is required for a specific step in the maturation of 40S ribosomal subunits. Our objective here was to determine whether the human RPS19 protein functions at a similar step in 40S subunit maturation. Studies where RPS19 expression is reduced by siRNA in the hematopoietic cell line, TF-1, show that human RPS19 is also required for a specific step in the maturation of 40S ribosomal subunits. This maturation defect can be monitored by studying rRNA-processing intermediates along the ribosome synthesis pathway. Analysis of these intermediates in CD34− cells from the bone marrow of patients with DBA harboring mutations in RPS19 revealed a pre-rRNA–processing defect similar to that observed in TF-1 cells where RPS19 expression was reduced. This defect was observed to a lesser extent in CD34+ cells from patients with DBA who have mutations in RPS19.
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Cottilli, Patrick, Borja Belda-Palazón, Charith Raj Adkar-Purushothama, Jean-Pierre Perreault, Enrico Schleiff, Ismael Rodrigo, Alejandro Ferrando, and Purificación Lisón. "Citrus exocortis viroid causes ribosomal stress in tomato plants." Nucleic Acids Research 47, no. 16 (August 8, 2019): 8649–61. http://dx.doi.org/10.1093/nar/gkz679.
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Abstract Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress.
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Warren, Alan John. "Linking Defective Ribosome Maturation to Shwachman-Diamond Syndrome." Blood 122, no. 21 (November 15, 2013): SCI—36—SCI—36. http://dx.doi.org/10.1182/blood.v122.21.sci-36.sci-36.
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Abstract Ribosomes are RNA-protein machines that translate the genetic information encoded by the mRNA template in all living cells. Recent high-resolution structures of the ribosome have revolutionized our understanding of protein translation. However, the mechanisms of ribosome assembly and the surveillance mechanisms that monitor this process and couple it to growth are poorly understood. Causative mutations and deletions of genes involved in ribosome biogenesis define an emerging group of disorders known as the ribosomopathies. Recent work from my laboratory strongly supports the hypothesis that Shwachman-Diamond syndrome (SDS) is a ribosomopathy caused by defective maturation of the large ribosomal subunit. Elucidation of the specific function of the SBDS protein that is deficient in SDS is revealing unexpected new insights that extend our understanding of the mechanisms underlying the late cytoplasmic steps of ribosome assembly and the quality control surveillance pathways that monitor 60S maturation. Genetic dissection of this pathway may inform novel therapeutic strategies for SDS. 1. Wong C.C., Traynor D., Basse N., Kay R.R., Warren A.J. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 2. Finch A.J., Hilcenko C., Basse N., Drynan L.F., Goyenechea B., Menne T.F., González Fernández Á., Simpson P., D’Santos C.S., Arends M.J., Donadieu J., Bellanné-Chantelot C., Costanzo M., Boone C., McKenzie A.N., Freund S.M., Warren A.J. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes and Development (2011) 25: 917-929. 3. Menne T.M., Goyenechea B., Sánchez-Puig N., Wong C.C., Tonkin L.M., Ancliff P., Brost R.L., Costanzo M., Boone C. and Warren A.J. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures: No relevant conflicts of interest to declare.
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Baudin-Baillieu, Agnès, and Olivier Namy. "Saccharomyces cerevisiae, a Powerful Model for Studying rRNA Modifications and Their Effects on Translation Fidelity." International Journal of Molecular Sciences 22, no. 14 (July 10, 2021): 7419. http://dx.doi.org/10.3390/ijms22147419.
Full textAbstract:
Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.
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Caesar, Stefanie, Markus Greiner, and Gabriel Schlenstedt. "Kap120 Functions as a Nuclear Import Receptor for Ribosome Assembly Factor Rpf1 in Yeast." Molecular and Cellular Biology 26, no. 8 (April 15, 2006): 3170–80. http://dx.doi.org/10.1128/mcb.26.8.3170-3180.2006.
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ABSTRACT The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin β protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.
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Lebreton, Alice, Cosmin Saveanu, Laurence Decourty, Jean-Christophe Rain, Alain Jacquier, and Micheline Fromont-Racine. "A functional network involved in the recycling of nucleocytoplasmic pre-60S factors." Journal of Cell Biology 173, no. 3 (May 1, 2006): 349–60. http://dx.doi.org/10.1083/jcb.200510080.
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Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation.
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Chierchia, Loredana, Margherita Tussellino, Domenico Guarino, Rosa Carotenuto, Nadia DeMarco, Chiara Campanella, Stefano Biffo, and Maria Carmela Vaccaro. "Cytoskeletal proteins associate with components of the ribosomal maturation and translation apparatus in Xenopus stage I oocytes." Zygote 23, no. 5 (September 17, 2014): 669–82. http://dx.doi.org/10.1017/s0967199414000409.
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SummaryActin-based cytoskeleton (CSK) and microtubules may bind to RNAs and related molecules implicated in translation. However, many questions remain to be answered regarding the role of cytoskeletal components in supporting the proteins involved in steps in the maturation and translation processes. Here, we performed co-immunoprecipitation and immunofluorescence to examine the association between spectrins, keratins and tubulin and proteins involved in 60S ribosomal maturation and translation in Xenopus stage I oocytes, including ribosomal rpl10, eukaryotic initiation factor 6 (Eif6), thesaurins A/B, homologs of the eEF1α elongation factor, and P0, the ribosomal stalk protein. We found that rpl10 and eif6 cross-reacted with the actin-based CSK and with tubulin. rpl10 co-localizes with spectrin, particularly in the perinuclear region. eif6 is similarly localized. Given that upon ribosomal maturation, the insertion of rpl10 into the 60S subunit occurs simultaneously with the release of eif6, one can hypothesise that actin-based CSK and microtubules provide the necessary scaffold for the insertion/release of these two molecules and, subsequently, for eif6 transport and binding to the mature 60S subunit. P0 and thesaurins cross-reacted with only spectrin and cytokeratins. Thesaurins aggregated at the oocyte periphery, rendering this a territory favourable site for protein synthesis; the CSK may support the interaction between thesaurins and sites of the translating ribosome. Moreover, given that the assembly of the ribosome stalk, where P0 is located, to the 60S subunit is essential for the release of eif6, it can be hypothesised that the CSK can facilitate the binding of the stalk to the 60S.
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Allam, Ramanjaneyulu, Vijaykumar Chennupati, Diogo F. T. Veiga, Kendle M. Maslowski, Aubry Tardivel, Manfredo Quadroni, Michel Duchosal, et al. "An Unexpected Role for Ribonuclease Inhibitor (RNH1) in Erythropoiesis." Blood 124, no. 21 (December 6, 2014): 244. http://dx.doi.org/10.1182/blood.v124.21.244.244.
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Abstract Ribonuclease Inhibitor (RNH1) is a ubiquitously expressed leucine-rich repeat protein. The human RNH1 gene evolved via gene duplication and is conserved among mammalian species. RNH1 binds to and inhibits pancreatic type ribonucleases. Further, RNH1 contains numerous cysteine residues whose sulfhydryl groups might play key structural roles and protect from oxidative damage (Dickson et al Prog. Nucleic Acid Res. Mol. Biol 2005). Despite of all these observations, the precise biological role of RNH1in vivo remains unexplored. Here, we describe an essential role for Rnh1 in the regulation of erythropoiesis by controlling erythroid differentiation. To understand the biological function of Rnh1, Rnh1-deficient (Rnh1-/-) mice were generated. Rnh1-/- embryos die between embryonic days E8.5 to E10 due to severe decrease in erythroid cells. Similar percentages of c-Kit+CD41+ cells (Hematopoietic stem/progenitor cells) were present in Rnh1-/- yolk sacs compared to control genotypes, however differentiation of mature erythroid cells was impaired. Rnh1 is expressed in erythroid cells and its expression coincides with the site of primitive erythropoiesis in the yolk sac. Gene expression studies revealed that levels of hematopoietic transcription factors (TF) in Rnh1-deficient yolk sacs were normal, but their target genes were down-regulated. These results indicate that a post-transcriptional mechanism that affects TF gene function. Supporting this, protein levels of the erythroid transcription factor GATA1 and PPARγ, previously shown to control the proliferation and differentiation of erythroid progenitors, were selectively impaired. Whereas myeloid transcription factors C/EBPa and C/EBPb were not affected in Rnh1-/- embryos, suggesting that Rnh1 deficiency specifically affects the translation of erythroid transcription factors. At the molecular level, using the human erythroid K562 cell line, we show that RNH1 is recruited to the ribosome complex and binds to the ribosomal proteins. RNH1-deficiency decreased polysome formation and conversely its overexpression increased polysome formation. Increased expression of RNH1 also increased globin gene expression in K562 cells. These results suggest that RNH1 associates with ribosomes and regulates the translation of erythroid-specific genes, which are necessary for erythroid differentiation. Furthermore, Rnh1 haploinsufficiency leads to decreased erythropoiesis in the spleen of adult mice. Ribosomal haploinsufficiency in several ribosomal genes is known to impair ribosome function and cause macrocytic anemia in Diamond–Blackfan anemia (DBA), a congenital bone marrow failure syndrome, and the 5q- syndrome, a subtype of myelodysplastic syndrome (Narla et al Int. J. Hematol 2011). Recently it has been shown that ribosomal haploinsufficiency can specifically cause a decrease in GATA1 mRNA translation (Ludwig et al Nature Med 2014). Similar to these ribosomal genes, we demonstrate that Rnh1 associates with ribosomes and its deficiency impairs the translation of Gata1 and other erythroid-specific transcription factors, which leads to arrest in erythroid maturation. Collectively our results unravel the important biological function of Rnh1 in the regulation of erythropoiesis, and point to novel therapeutic targets for disorders of erythropoiesis involving ribosomal defects. Summary Figure: RNH1 is recruited to ribosomal complex and is involved in translation of erythroid specific transcription factors (TF) e.g.GATA1. These TFs are necessary for differentiation of progenitor cells in to erythroid cells. RNH1 deficiency impairs the translation of GATA1 and other erythroid-specific transcription factors, which leads to arrest in erythroid maturation. Summary Figure:. RNH1 is recruited to ribosomal complex and is involved in translation of erythroid specific transcription factors (TF) e.g.GATA1. These TFs are necessary for differentiation of progenitor cells in to erythroid cells. RNH1 deficiency impairs the translation of GATA1 and other erythroid-specific transcription factors, which leads to arrest in erythroid maturation. Summary Figure: RNH1 is recruited to ribosomal complex and is involved in translation of erythroid specific transcription factors (TF) e.g.GATA1. These TFs are necessary for differentiation of progenitor cells in to erythroid cells. RNH1 deficiency impairs the translation of GATA1 and other erythroid-specific transcription factors, which leads to arrest in erythroid maturation. Disclosures No relevant conflicts of interest to declare.
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Matsuzaki, Yusuke, Yutaka Naito, Nami Miura, Taisuke Mori, Yukio Watabe, Seiichi Yoshimoto, Takahiko Shibahara, Masayuki Takano, and Kazufumi Honda. "RIOK2 Contributes to Cell Growth and Protein Synthesis in Human Oral Squamous Cell Carcinoma." Current Oncology 30, no. 1 (December 26, 2022): 381–91. http://dx.doi.org/10.3390/curroncol30010031.
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Ribosomes are responsible for the protein synthesis that maintains cellular homeostasis and is required for the rapid cellular division of cancer cells. However, the role of ribosome biogenesis mediators in the malignant behavior of tongue squamous cell carcinoma (TSCC) is unknown. In this study, we found that the expression of RIOK2, a key enzyme involved in the maturation steps of the pre-40S ribosomal complex, was significantly associated with poorer overall survival in patients with TSCC. Further, multivariate analysis revealed that RIOK2 is an independent prognostic factor (hazard ratio, 3.53; 95% confidence interval, 1.19–10.91). Inhibition of RIOK2 expression by siRNA decreased cell growth and S6 ribosomal protein expression in oral squamous cell carcinoma cell lines. RIOK2 knockdown also led to a significant decrease in the protein synthesis in cancer cells. RIOK2 has potential application as a novel therapeutic target for TSCC treatment.
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Prattes, Lo, Bergler, and Stanley. "Shaping the Nascent Ribosome: AAA-ATPases in Eukaryotic Ribosome Biogenesis." Biomolecules 9, no. 11 (November 7, 2019): 715. http://dx.doi.org/10.3390/biom9110715.
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AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors.
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Soufari, Heddy, Florent Waltz, Camila Parrot, Stéphanie Durrieu-Gaillard, Anthony Bochler, Lauriane Kuhn, Marie Sissler, and Yaser Hashem. "Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis." Proceedings of the National Academy of Sciences 117, no. 47 (November 9, 2020): 29851–61. http://dx.doi.org/10.1073/pnas.2011301117.
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Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed inTrypanosoma bruceialready highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoansLeishmania tarentolaeandTrypanosoma cruzimitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.
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Mageeney, Catherine M., and Vassie C. Ware. "Specialized eRpL22 paralogue-specific ribosomes regulate specific mRNA translation in spermatogenesis in Drosophila melanogaster." Molecular Biology of the Cell 30, no. 17 (August 2019): 2240–53. http://dx.doi.org/10.1091/mbc.e19-02-0086.
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The functional significance of ribosome heterogeneity in development and differentiation is relatively unexplored. We present the first in vivo evidence of ribosome heterogeneity playing a role in specific mRNA translation in a multicellular eukaryote. Eukaryotic-specific ribosomal protein paralogues eRpL22 and eRpL22-like are essential in development and required for sperm maturation and fertility in Drosophila. eRpL22 and eRpL22-like roles in spermatogenesis are not completely interchangeable. Flies depleted of eRpL22 and rescued by eRpL22-like overexpression have reduced fertility, confirming that eRpL22-like cannot substitute fully for eRpL22 function, and that paralogues have functionally distinct roles, not yet defined. We investigated the hypothesis that specific RNAs differentially associate with eRpL22 or eRpL22-like ribosomes, thereby establishing distinct ribosomal roles. RNA-seq identified 12,051 transcripts (mRNAs/noncoding RNAs) with 50% being enriched on specific polysome types. Analysis of ∼10% of the most abundant mRNAs suggests ribosome specialization for translating groups of mRNAs expressed at specific stages of spermatogenesis. Further, we show enrichment of “model” eRpL22-like polysome-associated testis mRNAs can occur outside the germline within S2 cells transfected with eRpL22-like, indicating that germline-specific factors are not required for selective translation. This study reveals specialized roles in translation for eRpL22 and eRpL22-like ribosomes in germline differentiation.
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McLeod, Tina, Akilu Abdullahi, Min Li, and Saverio Brogna. "Recent studies implicate the nucleolus as the major site of nuclear translation." Biochemical Society Transactions 42, no. 4 (August 1, 2014): 1224–28. http://dx.doi.org/10.1042/bst20140062.
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The nucleolus is the most prominent morphological feature within the nucleus of eukaryotic cells and is best known for its role in ribosome biogenesis. It forms around highly transcribed ribosomal RNA gene repeats which yield precursor rRNAs that are co-transcriptionally processed, folded and, while still within the nucleolus, associate with most of the ribosomal proteins. The nucleolus is therefore often thought of as a factory for making ribosomal subunits, which are exported as inactive precursors to the cytoplasm where late maturation makes them capable of mRNA binding and translation initiation. However, recent studies have shown substantial evidence for the presence of functional, translation competent ribosomal subunits within the nucleus, particularly in the nucleolus. These observations raise the intriguing possibility that the nucleolus, as well as being a ribosome factory, is also an important nuclear protein-synthesis plant.
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Delprato, Anna, Yasmine Al Kadri, Natacha Pérébaskine, Cécile Monfoulet, Yves Henry, Anthony K. Henras, and Sébastien Fribourg. "Crucial role of the Rcl1p–Bms1p interaction for yeast pre-ribosomal RNA processing." Nucleic Acids Research 42, no. 15 (July 26, 2014): 10161–72. http://dx.doi.org/10.1093/nar/gku682.
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Abstract The essential Rcl1p and Bms1p proteins form a complex required for 40S ribosomal subunit maturation. Bms1p is a GTPase and Rcl1p has been proposed to catalyse the endonucleolytic cleavage at site A2 separating the pre-40S and pre-60S maturation pathways. We determined the 2.0 Å crystal structure of Bms1p associated with Rcl1p. We demonstrate that Rcl1p nuclear import depends on Bms1p and that the two proteins are loaded into pre-ribosomes at a similar stage of the maturation pathway and remain present within pre-ribosomes after cleavage at A2. Importantly, GTP binding to Bms1p is not required for the import in the nucleus nor for the incorporation of Rcl1p into pre-ribosomes, but is essential for early pre-rRNA processing. We propose that GTP binding to Bms1p and/or GTP hydrolysis may induce conformational rearrangements within the Bms1p-Rcl1p complex allowing the interaction of Rcl1p with its RNA substrate.
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Kasai, Hide, Daita Nadano, Eiko Hidaka, Kayoko Higuchi, Masatomo Kawakubo, Taka-Aki Sato, and Jun Nakayama. "Differential Expression of Ribosomal Proteins in Human Normal and Neoplastic Colorectum." Journal of Histochemistry & Cytochemistry 51, no. 5 (May 2003): 567–73. http://dx.doi.org/10.1177/002215540305100502.
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Ribosomal proteins are a major component of ribosomes and play critical roles in protein biosynthesis. Recently it has been shown that the ribosomal proteins also function during various cellular processes that are independent of protein biosynthesis therefore called extraribosomal functions. In this study we have, for the first time, determined the expression profile of 12 ribosomal proteins (Sa, S8, S11, S12, S18, S24, L7, L13a, L18, L28, L32, and L35a) in normal epithelia of human colorectal mucosa using immunohistochemistry (IHC) and then compared their expression patterns with those of colorectal cancer. In the normal mucosa, ribosomal proteins were largely associated with the ribosomes of mucosal epithelia, and the expression level of ribosomal proteins, except for S11 and L7 proteins, was markedly increased in associated with maturation of the mucosal cells. On the other hand, these ribosomal proteins were markedly decreased in colorectal cancer compared with the normal mucosa. By contrast, S11 and L7 ribosomal proteins were rarely associated with the ribosomes of colorectal epithlia except immature mucosal cells, whereas their expression levels were significantly enchanced in colorectal cancer cells. In addition, L7 ribosomal protien was detected in the secretory granules of the enterochromaffin cells in the colorectal mucosa and in carcinoma cells expressing chromogranin A. These results indicate that the expression of ribosomal proteins is differentially regulated not only in normal mucosa but also in carcinoma of human colorectum, and suggest an extraribosomal function of L7 ribosomal protein in neuroendocrine function.
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Choesmel, Valérie, Daniel Bacqueville, Jacques Rouquette, Jacqueline Noaillac-Depeyre, Sébastien Fribourg, Aurore Crétien, Thierry Leblanc, Gil Tchernia, Lydie Da Costa, and Pierre-Emmanuel Gleizes. "Impaired ribosome biogenesis in Diamond-Blackfan anemia." Blood 109, no. 3 (October 19, 2006): 1275–83. http://dx.doi.org/10.1182/blood-2006-07-038372.
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Abstract The gene encoding the ribosomal protein S19 (RPS19) is frequently mutated in Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. The consequence of these mutations on the onset of the disease remains obscure. Here, we show that RPS19 plays an essential role in biogenesis of the 40S small ribosomal subunit in human cells. Knockdown of RPS19 expression by siRNAs impairs 18S rRNA synthesis and formation of 40S subunits and induces apoptosis in HeLa cells. Pre-rRNA processing is altered, which leads to an arrest in the maturation of precursors to the 18S rRNA. Under these conditions, pre-40S particles are not exported to the cytoplasm and accumulate in the nucleoplasm of the cells in perinuclear dots. Consistently, we find that ribosome biogenesis and nucleolar organization is altered in skin fibroblasts from DBA patients bearing mutations in the RPS19 gene. In addition, maturation of the 18S rRNA is also perturbed in cells from a patient bearing no RPS19-related mutation. These results support the hypothesis that DBA is directly related to a defect in ribosome biogenesis and indicate that yet to be discovered DBA-related genes may be involved in the synthesis of the ribosomal subunits.
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Liu, Phillip C. C., and Dennis J. Thiele. "Novel Stress-responsive Genes EMG1 and NOP14 Encode Conserved, Interacting Proteins Required for 40S Ribosome Biogenesis." Molecular Biology of the Cell 12, no. 11 (November 2001): 3644–57. http://dx.doi.org/10.1091/mbc.12.11.3644.
Full textAbstract:
Under stressful conditions organisms adjust the synthesis, processing, and trafficking of molecules to allow survival from and recovery after stress. In baker's yeast Saccharomyces cerevisiae, the cellular production of ribosomes is tightly matched with environmental conditions and nutrient availability through coordinate transcriptional regulation of genes involved in ribosome biogenesis. On the basis of stress-responsive gene expression and functional studies, we have identified a novel, evolutionarily conserved gene, EMG1, that has similar stress-responsive gene expression patterns as ribosomal protein genes and is required for the biogenesis of the 40S ribosomal subunit. The Emg1 protein is distributed throughout the cell; however, its nuclear localization depends on physical interaction with a newly characterized nucleolar protein, Nop14. Yeast depleted of Nop14 or harboring a temperature-sensitive allele of emg1 have selectively reduced levels of the 20S pre-rRNA and mature18S rRNA and diminished cellular levels of the 40S ribosomal subunit. Neither Emg1 nor Nop14 contain any characterized functional motifs; however, isolation and functional analyses of mammalian orthologues of Emg1 and Nop14 suggest that these proteins are functionally conserved among eukaryotes. We conclude that Emg1 and Nop14 are novel proteins whose interaction is required for the maturation of the 18S rRNA and for 40S ribosome production.
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Deliu, Lisa Patricia, Michael Turingan, Deeshpaul Jadir, Byoungchun Lee, Abhishek Ghosh, and Savraj Singh Grewal. "Serotonergic neuron ribosomal proteins regulate the neuroendocrine control of Drosophila development." PLOS Genetics 18, no. 9 (September 1, 2022): e1010371. http://dx.doi.org/10.1371/journal.pgen.1010371.
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The regulation of ribosome function is a conserved mechanism of growth control. While studies in single cell systems have defined how ribosomes contribute to cell growth, the mechanisms that link ribosome function to organismal growth are less clear. Here we explore this issue using Drosophila Minutes, a class of heterozygous mutants for ribosomal proteins. These animals exhibit a delay in larval development caused by decreased production of the steroid hormone ecdysone, the main regulator of larval maturation. We found that this developmental delay is not caused by decreases in either global ribosome numbers or translation rates. Instead, we show that they are due in part to loss of Rp function specifically in a subset of serotonin (5-HT) neurons that innervate the prothoracic gland to control ecdysone production. We find that these effects do not occur due to altered protein synthesis or proteostasis, but that Minute animals have reduced expression of synaptotagmin, a synaptic vesicle protein, and that the Minute developmental delay can be partially reversed by overexpression of synaptic vesicle proteins in 5-HTergic cells. These results identify a 5-HT cell-specific role for ribosomal function in the neuroendocrine control of animal growth and development.
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Weidner, Stanisław, and Kazimierz Zalewski. "Changes in ribosomal proteins in wheat embryos in the course of grain development and maturation." Acta Societatis Botanicorum Poloniae 51, no. 2 (2014): 283–90. http://dx.doi.org/10.5586/asbp.1982.025.
Full textAbstract:
It was found, by comparing the densitometric profiles of ribosomal proteins of wheat embryos in milk and full grain ripeness, that in the process of development and ripening of caryopses the percentual proportion of low molecular weight proteins increases at the cost of those of high molecular weight. This concerns both acidic and basic proteins. In electrophoretic separation of ribosomal proteins from embryos of fully ripe seeds by the method of two-dimensional electrophoresis the appearance of three new low molecular weight proteins - an acidic one and two basic ones - was observed. These proteins were not found in the embryos of caryopses of milk ripeness. These results indicate that with development and ripening of wheat caryopses new low molecular weight ribosomal proteins are built into the ribosomes in the embryo. These changes are both quantitative and qualitative.
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Maksimova, Elena, Olesya Kravchenko, Alexey Korepanov, and Elena Stolboushkina. "Protein Assistants of Small Ribosomal Subunit Biogenesis in Bacteria." Microorganisms 10, no. 4 (March 30, 2022): 747. http://dx.doi.org/10.3390/microorganisms10040747.
Full textAbstract:
Ribosome biogenesis is a fundamental and multistage process. The basic steps of ribosome assembly are the transcription, processing, folding, and modification of rRNA; the translation, folding, and modification of r-proteins; and consecutive binding of ribosomal proteins to rRNAs. Ribosome maturation is facilitated by biogenesis factors that include a broad spectrum of proteins: GTPases, RNA helicases, endonucleases, modification enzymes, molecular chaperones, etc. The ribosome assembly factors assist proper rRNA folding and protein–RNA interactions and may sense the checkpoints during the assembly to ensure correct order of this process. Inactivation of these factors is accompanied by severe growth phenotypes and accumulation of immature ribosomal subunits containing unprocessed rRNA, which reduces overall translation efficiency and causes translational errors. In this review, we focus on the structural and biochemical analysis of the 30S ribosomal subunit assembly factors RbfA, YjeQ (RsgA), Era, KsgA (RsmA), RimJ, RimM, RimP, and Hfq, which take part in the decoding-center folding.
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Papagiannopoulos, Christos I., Nikoleta F. Theodoroula, and Ioannis S. Vizirianakis. "miR-16-5p Promotes Erythroid Maturation of Erythroleukemia Cells by Regulating Ribosome Biogenesis." Pharmaceuticals 14, no. 2 (February 9, 2021): 137. http://dx.doi.org/10.3390/ph14020137.
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miRNAs constitute a class of non-coding RNA that act as powerful epigenetic regulators in animal and plant cells. In order to identify putative tumor-suppressor miRNAs we profiled the expression of various miRNAs during differentiation of erythroleukemia cells. RNA was purified before and after differentiation induction and subjected to quantitative RT-PCR. The majority of the miRNAs tested were found upregulated in differentiated cells with miR-16-5p showing the most significant increase. Functional studies using gain- and loss-of-function constructs proposed that miR-16-5p has a role in promoting the erythroid differentiation program of murine erythroleukemia (MEL) cells. In order to identify the underlying mechanism of action, we utilized bioinformatic in-silico platforms that incorporate predictions for the genes targeted by miR-16-5p. Interestingly, ribosome constituents, as well as ribosome biogenesis factors, were overrepresented among the miR-16-5p predicted gene targets. Accordingly, biochemical experiments showed that, indeed, miR-16-5p could modulate the levels of independent ribosomal proteins, and the overall ribosomal levels in cultured cells. In conclusion, miR-16-5p is identified as a differentiation-promoting agent in erythroleukemia cells, demonstrating antiproliferative activity, likely as a result of its ability to target the ribosomal machinery and restore any imbalanced activity imposed by the malignancy and the blockade of differentiation.
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Saveanu, Cosmin, Abdelkader Namane, Pierre-Emmanuel Gleizes, Alice Lebreton, Jean-Claude Rousselle, Jacqueline Noaillac-Depeyre, Nicole Gas, Alain Jacquier, and Micheline Fromont-Racine. "Sequential Protein Association with Nascent 60S Ribosomal Particles." Molecular and Cellular Biology 23, no. 13 (July 1, 2003): 4449–60. http://dx.doi.org/10.1128/mcb.23.13.4449-4460.2003.
Full textAbstract:
ABSTRACT Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.
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Rozanska, Agata, Ricarda Richter-Dennerlein, Joanna Rorbach, Fei Gao, Richard J. Lewis, Zofia M. Chrzanowska-Lightowlers, and Robert N. Lightowlers. "The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome." Biochemical Journal 474, no. 13 (June 13, 2017): 2145–58. http://dx.doi.org/10.1042/bcj20170256.
Full textAbstract:
Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation.
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Lejars, Maxence, Asaki Kobayashi, and Eliane Hajnsdorf. "RNase III, Ribosome Biogenesis and Beyond." Microorganisms 9, no. 12 (December 17, 2021): 2608. http://dx.doi.org/10.3390/microorganisms9122608.
Full textAbstract:
The ribosome is the universal catalyst for protein synthesis. Despite extensive studies, the diversity of structures and functions of this ribonucleoprotein is yet to be fully understood. Deciphering the biogenesis of the ribosome in a step-by-step manner revealed that this complexity is achieved through a plethora of effectors involved in the maturation and assembly of ribosomal RNAs and proteins. Conserved from bacteria to eukaryotes, double-stranded specific RNase III enzymes play a large role in the regulation of gene expression and the processing of ribosomal RNAs. In this review, we describe the canonical role of RNase III in the biogenesis of the ribosome comparing conserved and unique features from bacteria to eukaryotes. Furthermore, we report additional roles in ribosome biogenesis re-enforcing the importance of RNase III.
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SYDORSKYY, Yaroslav, David J. DILWORTH, Brendan HALLORAN, Eugene C. YI, Taras MAKHNEVYCH, Richard W. WOZNIAK, and John D. AITCHISON. "Nop53p is a novel nucleolar 60S ribosomal subunit biogenesis protein." Biochemical Journal 388, no. 3 (June 7, 2005): 819–26. http://dx.doi.org/10.1042/bj20041297.
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Ribosome biogenesis in Saccharomyces cerevisiae occurs primarily in a specialized nuclear compartment termed the nucleolus within which the rRNA genes are transcribed by RNA polymerase I into a large 35 S rRNA precursor. The ensuing association/dissociation and catalytic activity of numerous trans-acting protein factors, RNAs and ribosomal proteins ultimately leads to the maturation of the precursor rRNAs into 25, 5.8 and 18 S rRNAs and the formation of mature cytoplasmic 40 and 60 S ribosomal subunits. Although many components involved in ribosome biogenesis have been identified, our understanding of this essential cellular process remains limited. In the present study we demonstrate a crucial role for the previously uncharacterized nucleolar protein Nop53p (Ypl146p) in ribosome biogenesis. Specifically, Nop53p appears to be most important for biogenesis of the 60 S subunit. It physically interacts with rRNA processing factors, notably Cbf5p and Nop2p, and co-fractionates specifically with pre-60 S particles on sucrose gradients. Deletion or mutations within NOP53 cause significant growth defects and display significant 60 S subunit deficiencies, an imbalance in the 40 S:60 S ratio, as revealed by polysome profiling, and defects in progression beyond the 27 S stage of 25 S rRNA maturation during 60 S biogenesis.
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Burwick, Nicholas, Scott A. Coats, Tomoka Nakamura, and Akiko Shimamura. "Impaired ribosomal subunit association in Shwachman-Diamond syndrome." Blood 120, no. 26 (December 20, 2012): 5143–52. http://dx.doi.org/10.1182/blood-2012-04-420166.
Full textAbstract:
Abstract Shwachman-Diamond syndrome (SDS) is an autosomal-recessive marrow failure syndrome with a predisposition to leukemia. SDS patients harbor biallelic mutations in the SBDS gene, resulting in low levels of SBDS protein. Data from nonhuman models demonstrate that the SBDS protein facilitates the release of eIF6, a factor that prevents ribosome joining. The complete abrogation of Sbds expression in these models results in severe cellular and lethal physiologic abnormalities that differ from the human disease phenotype. Because human SDS cells are characterized by partial rather than complete loss of SBDS expression, we interrogated SDS patient cells for defects in ribosomal assembly. SDS patient cells exhibit altered ribosomal profiles and impaired association of the 40S and 60S subunits. Introduction of a wild-type SBDS cDNA into SDS patient cells corrected the ribosomal association defect, while patient-derived SBDS point mutants only partially improved subunit association. Knockdown of eIF6 expression improved ribosomal subunit association but did not correct the hematopoietic defect of SBDS-deficient cells. In summary, we demonstrate an SBDS-dependent ribosome maturation defect in SDS patient cells. The role of ribosomal subunit joining in marrow failure warrants further investigation.
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Wong, Chi C., David Traynor, Nicolas Basse, Robert R. Kay, and Alan J. Warren. "Defective ribosome assembly in Shwachman-Diamond syndrome." Blood 118, no. 16 (October 20, 2011): 4305–12. http://dx.doi.org/10.1182/blood-2011-06-353938.
Full textAbstract:
AbstractShwachman-Diamond syndrome (SDS), a recessive leukemia predisposition disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, skeletal abnormalities and poor growth, is caused by mutations in the highly conserved SBDS gene. Here, we test the hypothesis that defective ribosome biogenesis underlies the pathogenesis of SDS. We create conditional mutants in the essential SBDS ortholog of the ancient eukaryote Dictyostelium discoideum using temperature-sensitive, self-splicing inteins, showing that mutant cells fail to grow at the restrictive temperature because ribosomal subunit joining is markedly impaired. Remarkably, wild type human SBDS complements the growth and ribosome assembly defects in mutant Dictyostelium cells, but disease-associated human SBDS variants are defective. SBDS directly interacts with the GTPase elongation factor-like 1 (EFL1) on nascent 60S subunits in vivo and together they catalyze eviction of the ribosome antiassociation factor eukaryotic initiation factor 6 (eIF6), a prerequisite for the translational activation of ribosomes. Importantly, lymphoblasts from SDS patients harbor a striking defect in ribosomal subunit joining whose magnitude is inversely proportional to the level of SBDS protein. These findings in Dictyostelium and SDS patient cells provide compelling support for the hypothesis that SDS is a ribosomopathy caused by corruption of an essential cytoplasmic step in 60S subunit maturation.
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Stephan, Niklas C., Anne B. Ries, Daniel Boehringer, and Nenad Ban. "Structural basis of successive adenosine modifications by the conserved ribosomal methyltransferase KsgA." Nucleic Acids Research 49, no. 11 (June 4, 2021): 6389–98. http://dx.doi.org/10.1093/nar/gkab430.
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Abstract Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.
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Tabb, Amy L., Takahiko Utsugi, Clavia R. Wooten-Kee, Takeshi Sasaki, Steven A. Edling, William Gump, Yoshiko Kikuchi, and Steven R. Ellis. "Genes Encoding Ribosomal Proteins Rps0A/B of Saccharomyces cerevisiae Interact With TOM1 Mutants Defective in Ribosome Synthesis." Genetics 157, no. 3 (March 1, 2001): 1107–16. http://dx.doi.org/10.1093/genetics/157.3.1107.
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Abstract The Saccharomyces cerevisiae RPS0A/B genes encode proteins of the 40S ribosomal subunit that are required for the maturation of 18S rRNA. We show here that the RPS0 genes interact genetically with TOM1. TOM1 encodes a member of the hect-domain-containing E3 ubiquitin-protein ligase family that is required for growth at elevated temperatures. Mutant alleles of the RPS0 and TOM1 genes have synergistic effects on cell growth at temperatures permissive for TOM1 mutants. Moreover, the growth arrest of TOM1 mutants at elevated temperatures is partially suppressed by overexpression of RPS0A/B. Strains with mutant alleles of TOM1 are defective in multiple steps in rRNA processing, and interactions between RPS0A/B and TOM1 stem, in part, from their roles in the maturation of ribosomal subunits. Ribosome synthesis is therefore included among the cellular processes governed by members of the hect-domain-containing E3 ubiquitin-protein ligase family.