scholarly journals Nop53p is a novel nucleolar 60S ribosomal subunit biogenesis protein

2005 ◽  
Vol 388 (3) ◽  
pp. 819-826 ◽  
Author(s):  
Yaroslav SYDORSKYY ◽  
David J. DILWORTH ◽  
Brendan HALLORAN ◽  
Eugene C. YI ◽  
Taras MAKHNEVYCH ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Rrna Genes ◽  
Ribosomal Subunits ◽  
Nuclear Compartment ◽  
Growth Defects ◽  
Polysome Profiling ◽  
Rrna Maturation ◽  
Polymerase I

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.

Control points in eucaryotic ribosome biogenesis

1991 ◽  
Vol 69 (1) ◽  
pp. 5-22 ◽  
Author(s):  
D. E. Larson ◽  
P. Zahradka ◽  
B. H. Sells
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Rna Polymerase Iii ◽  
Ribosomal Subunits ◽  
Rrna Species ◽  
Polymerase I ◽  
Ribosomal Precursor ◽  
Precursor Molecules

Ribosome biogenesis in eucaryotic cells involves the coordinated synthesis of four rRNA species, transcribed by RNA polymerase I (18S, 28S, 5.8S) and RNA polymerase III (5S), and approximately 80 ribosomal proteins translated from mRNAs synthesized by RNA polymerase II. Assembly of the ribosomal subunits in the nucleolus, the site of 45S rRNA precursor gene transcription, requires the movement of 5S rRNA and ribosomal proteins from the nucleoplasm and cytoplasm, respectively, to this structure. To integrate these events and ensure the balanced production of individual ribosomal components, different strategies have been developed by eucaryotic organisms in response to a variety of physiological changes. This review presents an overview of the mechanisms modulating the production of ribosomal precursor molecules and the rate of ribosome biogenesis in various biological systems.Key words: rRNA, ribosomal proteins, nucleolus, ribosome.

scholarly journals Why Dom34 Stimulates Growth of Cells with Defects of 40S Ribosomal Subunit Biosynthesis

2010 ◽  
Vol 30 (23) ◽  
pp. 5562-5571 ◽  
Author(s):  
Arpita Bhattacharya ◽  
Kerri B. McIntosh ◽  
Ian M. Willis ◽  
Jonathan R. Warner
Keyword(s):  
Translation Initiation ◽  
Ribosomal Proteins ◽  
Slow Growth ◽  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Growth Defects ◽  
Structural Problems ◽  
Genome Wide ◽  
40S Ribosomal Subunit ◽  
Severe Shortage

ABSTRACT A set of genome-wide screens for proteins whose absence exacerbates growth defects due to pseudo-haploinsufficiency of ribosomal proteins in Saccharomyces cerevisiae identified Dom34 as being particularly important for cell growth when there is a deficit of 40S ribosomal subunits. In contrast, strains with a deficit of 60S ribosomal proteins were largely insensitive to the loss of Dom34. The slow growth of cells lacking Dom34 and haploinsufficient for a protein of the 40S subunit is caused by a severe shortage of 40S subunits available for translation initiation due to a combination of three effects: (i) the natural deficiency of 40S subunits due to defective synthesis, (ii) the sequestration of 40S subunits due to the large accumulation of free 60S subunits, and (iii) the accumulation of ribosomes “stuck” in a distinct 80S form, insensitive to the Mg2+ concentration, and at least temporarily unavailable for further translation. Our data suggest that these stuck ribosomes have neither mRNA nor tRNA. We postulate, based on our results and on previously published work, that the stuck ribosomes arise because of the lack of Dom34, which normally resolves a ribosome stalled due to insufficient tRNAs, to structural problems with its mRNA, or to a defect in the ribosome itself.

scholarly journals Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy

Molecules ◽  
2020 ◽  
Vol 25 (5) ◽  
pp. 1125 ◽  
Author(s):  
Ramtin Shayan ◽  
Dana Rinaldi ◽  
Natacha Larburu ◽  
Laura Plassart ◽  
Stéphanie Balor ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Particle Analysis ◽  
Ribosomal Subunits ◽  
Early Maturation ◽  
Cryo Electron Microscopy ◽  
Molecular Events ◽  
Final Maturation

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.

scholarly journals Prp43p Is a DEAH-Box Spliceosome Disassembly Factor Essential for Ribosome Biogenesis

2006 ◽  
Vol 26 (2) ◽  
pp. 523-534 ◽  
Author(s):  
D. Joshua Combs ◽  
Roland J. Nagel ◽  
Manuel Ares ◽  
Scott W. Stevens
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Splicing Defects ◽  
Essential Function ◽  
Polymerase I ◽  
Kinetics Of

ABSTRACT The known function of the DEXH/D-box protein Prp43p is the removal of the U2, U5, and U6 snRNPs from the postsplicing lariat-intron ribonucleoprotein complex. We demonstrate that affinity-purified Prp43p-associated material includes the expected spliceosomal components; however, we also identify several preribosomal complexes that are specifically purified with Prp43p. Conditional prp43 mutant alleles confer a 35S pre-rRNA processing defect, with subsequent depletion of 27S and 20S precursors. Upon a shift to a nonpermissive temperature, both large and small-ribosomal-subunit proteins accumulate in the nucleolus of prp43 mutants. Pulse-chase analysis demonstrates delayed kinetics of 35S, 27S, and 20S pre-rRNA processing with turnover of these intermediates. Microarray analysis of pre-mRNA splicing defects in prp43 mutants shows a very mild effect, similar to that of nonessential pre-mRNA splicing factors. Prp43p is the first DEXH/D-box protein shown to function in both RNA polymerase I and polymerase II transcript metabolism. Its essential function is in its newly characterized role in ribosome biogenesis of both ribosomal subunits, positioning Prp43p to regulate both pre-mRNA splicing and ribosome biogenesis.

scholarly journals Intersection of the Kap123p-Mediated Nuclear Import and Ribosome Export Pathways

2003 ◽  
Vol 23 (6) ◽  
pp. 2042-2054 ◽  
Author(s):  
Y. Sydorskyy ◽  
D. J. Dilworth ◽  
E. C. Yi ◽  
D. R. Goodlett ◽  
R. W. Wozniak ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Genetic Interaction ◽  
Ribosomal Subunit ◽  
Nucleocytoplasmic Transport ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Double Mutant Strain ◽  
Ribosome Export

ABSTRACT Kap123p is a yeast β-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a ΔRAI1-induced decrease in the overall biogenesis efficiency.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

scholarly journals The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1687-1699
Author(s):  
Jesús de la Cruz ◽  
Thierry Lacombe ◽  
Olivier Deloche ◽  
Patrick Linder ◽  
Dieter Kressler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosome Biogenesis ◽  
Null Allele ◽  
Ribosomal Subunit ◽  
Interaction Network ◽  
The Novel ◽  
Rna Helicases ◽  
Ribosomal Subunits ◽  
Synthetic Lethal ◽  
Synthetically Lethal

Abstract Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells

1983 ◽  
Vol 3 (2) ◽  
pp. 190-197
Author(s):  
J J Madjar ◽  
M Frahm ◽  
S McGill ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Resistance Mutations ◽  
Cho Cell ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
One Step

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

scholarly journals Drug-induced dispersal of transcribed rRNA genes and transcriptional products: immunolocalization and silver staining of different nucleolar components in rat cells treated with 5,6-dichloro-beta-D-ribofuranosylbenzimidazole.

1984 ◽  
Vol 99 (2) ◽  
pp. 672-679 ◽  
Author(s):  
U Scheer ◽  
B Hügle ◽  
R Hazan ◽  
K M Rose
Keyword(s):  
Rna Polymerase ◽  
Organizer Region ◽  
Nucleolar Organizer Region ◽  
Ribosomal Subunit ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Linear Distribution ◽  
Nucleolar Material ◽  
Polymerase I

Upon incubation of cultured rat cells with the adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), nucleoli reversibly dissociate into their substructures, disperse throughout the nuclear interior, and form nucleolar "necklaces". We have used this experimental system, which does not inhibit transcription of the rRNA genes, to study by immunocytochemistry the distribution of active rRNA genes and their transcriptional products during nucleolar dispersal and recovery to normal morphology. Antibodies to RNA polymerase I allow detection of template-engaged polymerase, and monoclonal antibodies to a ribosomal protein (S1) of the small ribosomal subunit permit localization of nucleolar preribosomal particles. The results show that, under the action of DRB transcribed rRNA, genes spread throughout the nucleoplasm and finally appear in the form of several rows, each containing several (up to 30) granules positive for RNA polymerase I and argyrophilic proteins. Nucleolar material containing preribosomal particles also appears in granular structures spread over the nucleoplasm but its distribution is distinct from that of rRNA gene-containing granules. We conclude that, although transcriptional units and preribosomal particles are both redistributed in response to DRB, these entities retain their individuality as functionally defined subunits. We further propose that each RNA polymerase-positive granular unit represents a single transcription unit and that each continuous array of granules ("string of nucleolar beads") reflects the linear distribution of rRNA genes along a nucleolar organizer region. Based on the total number of polymerase I-positive granules we estimate that a minimum of 60 rRNA genes are active during interphase of DRB-treated rat cells.

scholarly journals Late Cytoplasmic Maturation of the Small Ribosomal Subunit Requires RIO Proteins in Saccharomyces cerevisiae

2003 ◽  
Vol 23 (6) ◽  
pp. 2083-2095 ◽  
Author(s):  
Emmanuel Vanrobays ◽  
Jean-Paul Gelugne ◽  
Pierre-Emmanuel Gleizes ◽  
Michele Caizergues-Ferrer
Keyword(s):  
18S Rrna ◽  
Nuclear Protein ◽  
Essential Gene ◽  
Sequence Similarity ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Ribosomal Subunits ◽  
Rrna Maturation ◽  
Cytoplasmic Maturation

ABSTRACT Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.

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