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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7283 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7283-7294 ◽

Cited By ~ 108

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.

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Glycerophosphocholine-dependent Growth Requires Gde1p (YPL110c) and Git1p in Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1074/jbc.m507051200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36110-36117 ◽

Cited By ~ 58

Author(s):

Edward Fisher ◽

Claudia Almaguer ◽

Roman Holic ◽

Peter Griac ◽

Jana Patton-Vogt

Keyword(s):

Saccharomyces Cerevisiae ◽

Wild Type Strain ◽

Open Reading Frame ◽

Reading Frame ◽

Intracellular Fraction ◽

Dependent Growth ◽

Phosphate Source ◽

Glycerophosphocholine Phosphodiesterase ◽

In Cells

Glycerophosphocholine is formed via the deacylation of the phospholipid phosphatidylcholine. The protein encoded by Saccharomyces cerevisiae open reading frame YPL110c effects glycerophosphocholine metabolism in vivo, most likely by acting as a glycerophosphocholine phosphodiesterase. Deletion of YPL110c causes an accumulation of glycerophosphocholine in cells prelabeled with [14C]choline. Correspondingly, overexpression of YPL110c results in reduced intracellular glycerophosphocholine in cells prelabeled with [14C]choline. Glycerophospho[3H]choline supplied in the growth medium accumulates to a much greater extent in the intracellular fraction of a YPL110Δ strain than in a wild type strain. Furthermore, glycerophospho[3H]choline accumulation requires the transporter encoded by GIT1, a known glycerophosphoinositol transporter. Growth on glycerophosphocholine as the sole phosphate source requires YPL110c and the Git1p permease. In contrast to glycerophosphocholine, glycerophosphoinositol metabolism is unaffected by deletion of YPL110c. The open reading frame YPL110c has been termed GDE1.

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

Acta Biochimica Polonica ◽

10.18388/abp.2000_3953 ◽

2000 ◽

Vol 47 (4) ◽

pp. 993-1005 ◽

Cited By ~ 7

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.

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Dbp3p, a putative RNA helicase in Saccharomyces cerevisiae, is required for efficient pre-rRNA processing predominantly at site A3.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1354 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1354-1365 ◽

Cited By ~ 69

Author(s):

P L Weaver ◽

C Sun ◽

T H Chang

Keyword(s):

Saccharomyces Cerevisiae ◽

Slow Growth ◽

Rna Helicase ◽

Amino Terminus ◽

Ribosomal Biogenesis ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Rnase Mrp ◽

Dead Box ◽

Evolutionarily Conserved

In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention.

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

Molecular and Cellular Biology ◽

10.1128/mcb.21.5.1453-1462.2001 ◽

2001 ◽

Vol 21 (5) ◽

pp. 1453-1462 ◽

Cited By ~ 115

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.

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GIT1, a Gene Encoding a Novel Transporter for Glycerophosphoinositol in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/149.4.1707 ◽

1998 ◽

Vol 149 (4) ◽

pp. 1707-1715 ◽

Cited By ~ 2

Author(s):

J L Patton-Vogt ◽

S A Henry

Keyword(s):

Saccharomyces Cerevisiae ◽

Regulatory Gene ◽

Sugar Transport ◽

Open Reading Frame ◽

Gene Encoding ◽

Reading Frame ◽

Membrane Spanning ◽

Membrane Spanning Domains ◽

Uptake And Metabolism ◽

Cells Cultured

Abstract Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git−) defective in the uptake and metabolism of GroPIns. One mutant was found to be affected in the gene encoding the transcription factor, SPT7. Mutants of the positive regulatory gene INO2, but not of its partner, INO4, also have the Git− phenotype. Another mutant was complemented by a single open reading frame (ORF) termed GIT1 (glycerophosphoinositol). This ORF consists of 1556 bp predicted to encode a polypeptide of 518 amino acids and 57.3 kD. The predicted Git1p has similarity to a variety of S. cerevisiae transporters, including a phosphate transporter (Pho84p), and both inositol transporters (Itr1p and Itr2p). Furthermore, Git1p contains a sugar transport motif and 12 potential membrane-spanning domains. Transport assays performed on a git1 mutant together with the above evidence indicate that the GIT1 gene encodes a permease involved in the uptake of GroPIns.

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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 ◽

10.1093/genetics/166.4.1687 ◽

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.

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Human Cytomegalovirus Open Reading Frame TRL11/IRL11 Encodes an Immunoglobulin G Fc-Binding Protein

Journal of Virology ◽

10.1128/jvi.75.22.11218-11221.2001 ◽

2001 ◽

Vol 75 (22) ◽

pp. 11218-11221 ◽

Cited By ~ 56

Author(s):

Brendan N. Lilley ◽

Hidde L. Ploegh ◽

Rebecca S. Tirabassi

Keyword(s):

Human Cytomegalovirus ◽

Immunoglobulin G ◽

Binding Protein ◽

Fc Receptors ◽

Binding Activity ◽

Open Reading Frame ◽

Membrane Glycoprotein ◽

Type I ◽

Reading Frame

ABSTRACT Several herpesviruses encode Fc receptors that may play a role in preventing antibody-mediated clearance of the virus in vivo. Human cytomegalovirus (HCMV) induces an Fc-binding activity in cells upon infection, but the gene that encodes this Fc-binding protein has not been identified. Here, we demonstrate that the HCMV AD169 open reading frame TRL11 and its identical copy, IRL11, encode a type I membrane glycoprotein that possesses IgG Fc-binding capabilities.

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Regulation of Metalloprotease Gene Expression in Vibrio vulnificus by a Vibrio harveyi LuxR Homologue

Journal of Bacteriology ◽

10.1128/jb.183.4.1369-1375.2001 ◽

2001 ◽

Vol 183 (4) ◽

pp. 1369-1375 ◽

Cited By ~ 74

Author(s):

Chung-Ping Shao ◽

Lien-I Hor

Keyword(s):

Parent Strain ◽

Vibrio Vulnificus ◽

Vibrio Harveyi ◽

Negative Regulation ◽

Open Reading Frame ◽

Protease Gene ◽

Reading Frame ◽

Allelic Exchange ◽

Exchange Technique

ABSTRACT Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyiLuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene ofV. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificusvirulence in mice.

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Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2835-2845.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2835-2845

Author(s):

M Deshmukh ◽

Y F Tsay ◽

A G Paulovich ◽

J L Woolford

Keyword(s):

Ribosomal Protein ◽

Ribosomal Subunit ◽

Single Copy ◽

5S Rrna ◽

Gene Encoding ◽

Ribosomal Subunits ◽

Ribosomal Protein L1 ◽

The Stability ◽

And Function

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

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