scholarly journals The KRR1 gene encodes a protein required for 18S rRNA synthesis and 40S ribosomal subunit assembly in Saccharomyces cerevisiae.

2000 ◽  
Vol 47 (4) ◽  
pp. 993-1005 ◽  
Author(s):  
R Gromadka ◽  
J Rytka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Synthesis ◽  
Drastic Reduction ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Dividing Cells

The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability. Krr1p contains a motif of clustered basic amino acids highly conserved in the evolutionarly distant species from yeast to human. We demonstrate that Krr1p is localized in the nucleolus. The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. In vivo depletion of Krr1p leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis. We propose that Krr1p is required for proper processing of pre-rRNA and the assembly of preribosomal 40S subunits.

scholarly journals Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (12) ◽  
pp. 7283-7294 ◽  
Author(s):  
D Kressler ◽  
J de la Cruz ◽  
M Rojo ◽  
P Linder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
18S Rrna ◽  
Amino Acid Level ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Northern Hybridization ◽  
Ribosomal Subunits ◽  
Dead Box

A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.

scholarly journals The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

2020 ◽  
Author(s):  
Laura Plassart ◽  
Ramtin Shayan ◽  
Christian Montellese ◽  
Dana Rinaldi ◽  
Natacha Larburu ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Inactive Form ◽  
Final Maturation ◽  
Translation Apparatus ◽  
Translation Accuracy ◽  
Functional Analyses

Preventing premature interaction of preribosomes with the translation apparatus is essential to translation accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATPloaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

scholarly journals The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits

2012 ◽  
Vol 23 (21) ◽  
pp. 4313-4322 ◽  
Author(s):  
Richa Sardana ◽  
Arlen W. Johnson
Keyword(s):  
Slow Growth ◽  
Ribosomal Subunit ◽  
Adaptor Protein ◽  
Small Subunit ◽  
Stable Complex ◽  
Sedimentation Analysis ◽  
Ribosomal Subunits ◽  
Translation Factors ◽  
40S Ribosomal Subunit

We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates.

scholarly journals Impaired ribosome biogenesis in Diamond-Blackfan anemia

Blood ◽  
2006 ◽  
Vol 109 (3) ◽  
pp. 1275-1283 ◽  
Author(s):  
Valérie Choesmel ◽  
Daniel Bacqueville ◽  
Jacques Rouquette ◽  
Jacqueline Noaillac-Depeyre ◽  
Sébastien Fribourg ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Diamond Blackfan Anemia ◽  
Nucleolar Organization ◽  
Ribosomal Protein S19

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.

scholarly journals Deletion of the PAT1 Gene Affects Translation Initiation and Suppresses a PAB1 Gene Deletion in Yeast

2000 ◽  
Vol 20 (10) ◽  
pp. 3538-3549 ◽  
Author(s):  
Françoise Wyers ◽  
Michèle Minet ◽  
Marie Elisabeth Dufour ◽  
Le Thuy Anh Vo ◽  
François Lacroute
Keyword(s):  
Translation Initiation ◽  
Gene Deletion ◽  
Ribosomal Subunit ◽  
Large Decrease ◽  
Free System ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
Initiation Of Translation

ABSTRACT The yeast poly(A) binding protein Pab1p mediates the interactions between the 5′ cap structure and the 3′ poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. ThePAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

scholarly journals Dbp6p Is an Essential Putative ATP-Dependent RNA Helicase Required for 60S-Ribosomal-Subunit Assembly inSaccharomyces cerevisiae

1998 ◽  
Vol 18 (4) ◽  
pp. 1855-1865 ◽  
Author(s):  
Dieter Kressler ◽  
Jesús de la Cruz ◽  
Manuel Rojo ◽  
Patrick Linder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Subunit ◽  
Rna Helicase ◽  
Open Reading Frame ◽  
Northern Hybridization ◽  
Ribosomal Subunits ◽  
Steady State Analysis ◽  
Reading Frame ◽  
Dead Box

A previously uncharacterizedSaccharomyces cerevisiaeopen reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore namedDBP6(DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.

scholarly journals The Saccharomyces cerevisiae TIF6 Gene Encoding Translation Initiation Factor 6 Is Required for 60S Ribosomal Subunit Biogenesis

2001 ◽  
Vol 21 (5) ◽  
pp. 1453-1462 ◽  
Author(s):  
Uttiya Basu ◽  
Kausik Si ◽  
Jonathan R. Warner ◽  
Umadas Maitra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Translation Initiation ◽  
Essential Gene ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Cell Fractionation ◽  
Ribosomal Subunits

ABSTRACT Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. InSaccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.

Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide

1981 ◽  
Vol 1 (1) ◽  
pp. 51-57
Author(s):  
T L Helser ◽  
R A Baan ◽  
A E Dahlberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Subunit ◽  
Electrophoretic Analysis ◽  
Initiation Factor ◽  
Ribosomal Subunits ◽  
Messenger Ribonucleic Acid ◽  
Protein Gel ◽  
40S Ribosomal Subunit ◽  
Acid Protein

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.

scholarly journals Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide.

1981 ◽  
Vol 1 (1) ◽  
pp. 51-57 ◽  
Author(s):  
T L Helser ◽  
R A Baan ◽  
A E Dahlberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Subunit ◽  
Electrophoretic Analysis ◽  
Initiation Factor ◽  
Ribosomal Subunits ◽  
Messenger Ribonucleic Acid ◽  
Protein Gel ◽  
40S Ribosomal Subunit ◽  
Acid Protein

Under specific conditions cycloheximide treatment of Saccharomyces cerevisiae caused the accumulation of a type of polyribosome called "halfmer." Limited ribonuclease digestion of halfmers released particles from the polyribosomes identified as 40S ribosomal subunits. The data demonstrated that halfmers are polyribosomes containing an additional 40S ribosomal subunit attached to the messenger ribonucleic acid. Protein gel electrophoretic analysis of halfmers revealed numerous nonribosomal proteins. Two of these proteins comigrate with subunits of yeast initiation factor eIF2.

scholarly journals The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Laura Plassart ◽  
Ramtin Shayan ◽  
Christian Montellese ◽  
Dana Rinaldi ◽  
Natacha Larburu ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Ribosomal Subunits ◽  
Translational Accuracy ◽  
40S Ribosomal Subunit ◽  
Inactive Form ◽  
Final Maturation ◽  
Translation Apparatus ◽  
Functional Analyses

Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-EM analysis of late human pre-40S particles purified using a catalytically-inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

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