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.

scholarly journals Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 719-730
Author(s):  
A G Paulovich ◽  
J R Thompson ◽  
J C Larkin ◽  
Z Li ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Gene Fusion ◽  
Null Allele ◽  
Ribosomal Subunit ◽  
Null Alleles ◽  
Protein Synthesis Inhibitor ◽  
Lacz Gene ◽  
Ribosomal Subunits ◽  
Cry1 Gene

Abstract The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype. (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.

scholarly journals Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

2014 ◽  
Vol 42 (15) ◽  
pp. 9838-9853 ◽  
Author(s):  
Saeed Kaboli ◽  
Takuya Yamakawa ◽  
Keisuke Sunada ◽  
Tao Takagaki ◽  
Yu Sasano ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Interaction Network ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

scholarly journals Pol5 is an essential ribosome biogenesis factor required for 60S ribosomal subunit maturation in Saccharomyces cerevisiae

RNA ◽  
2019 ◽  
Vol 25 (11) ◽  
pp. 1561-1575 ◽  
Author(s):  
Ana Ramos-Sáenz ◽  
Daniel González-Álvarez ◽  
Olga Rodríguez-Galán ◽  
Alfonso Rodríguez-Gil ◽  
Sonia G. Gaspar ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit

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 Bni1p Regulates Microtubule-Dependent Nuclear Migration through the Actin Cytoskeleton in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (12) ◽  
pp. 8016-8027 ◽  
Author(s):  
Takeshi Fujiwara ◽  
Kazuma Tanaka ◽  
Eiji Inoue ◽  
Mitsuhiro Kikyo ◽  
Yoshimi Takai
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Nuclear Migration ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Synthetic Lethal ◽  
Downstream Target ◽  
Synthetically Lethal ◽  
Mother Cells

ABSTRACT The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identifiedPAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150Glued, a component of the dynein-activated dynactin complex. Disruption ofBNI1 in either the pac1 or nip100mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.

scholarly journals The DEAH-box RNA helicase Dhr1 contains a remarkable carboxyl terminal domain essential for small ribosomal subunit biogenesis

2019 ◽  
Vol 47 (14) ◽  
pp. 7548-7563 ◽  
Author(s):  
Amlan Roychowdhury ◽  
Clément Joret ◽  
Gabrielle Bourgeois ◽  
Valérie Heurgué-Hamard ◽  
Denis L J Lafontaine ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Rna Helicases ◽  
Range Interaction ◽  
Small Ribosomal Subunit ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Assembly Factors ◽  
Carboxyl Terminal

Abstract Ribosome biogenesis is an essential process in all living cells, which entails countless highly sequential and dynamic structural reorganization events. These include formation of dozens RNA helices through Watson-Crick base-pairing within ribosomal RNAs (rRNAs) and between rRNAs and small nucleolar RNAs (snoRNAs), transient association of hundreds of proteinaceous assembly factors to nascent precursor (pre-)ribosomes, and stable assembly of ribosomal proteins. Unsurprisingly, the largest group of ribosome assembly factors are energy-consuming proteins (NTPases) including 25 RNA helicases in budding yeast. Among these, the DEAH-box Dhr1 is essential to displace the box C/D snoRNA U3 from the pre-rRNAs where it is bound in order to prevent premature formation of the central pseudoknot, a dramatic irreversible long-range interaction essential to the overall folding of the small ribosomal subunit. Here, we report the crystal structure of the Dhr1 helicase module, revealing the presence of a remarkable carboxyl-terminal domain essential for Dhr1 function in ribosome biogenesis in vivo and important for its interaction with its coactivator Utp14 in vitro. Furthermore, we report the functional consequences on ribosome biogenesis of DHX37 (human Dhr1) mutations found in patients suffering from microcephaly and other neurological diseases.

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 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.

scholarly journals RelA Functionally Suppresses the Growth Defect Caused by a Mutation in the G Domain of the Essential Der Protein

2008 ◽  
Vol 190 (9) ◽  
pp. 3236-3243 ◽  
Author(s):  
Jihwan Hwang ◽  
Masayori Inouye
Keyword(s):  
Cell Growth ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Synthetic Activity ◽  
Growth Defect ◽  
Multicopy Plasmid ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Binding Domains ◽  
Gtp Binding

ABSTRACT A unique bacterial GTPase, Der, containing two tandem GTP-binding domains, is essential for cell growth and plays a crucial role in a large ribosomal subunit in Escherichia coli. The depletion of Der resulted in accumulation of both large and small ribosomal subunits and also affected the stability of large ribosomal subunits. However, its exact cellular function still remains elusive. Previously, we have shown that two G domain mutants, DerN118D and DerN321D, cannot support cell growth at low temperatures, suggesting that both GTP-binding domains are indispensable. In this study, we show that both Der variants are defective in ribosome biogenesis. Genetic screening of an E. coli genomic library was performed to identify the genes which, when expressed from a multicopy plasmid, can restore the growth defect of the DerN321D mutant at restrictive temperatures. Among seven suppressors isolated, four were located at 62.7 min on the E. coli genomic map, and the gene responsible for the suppression of DerN321D was identified as the relA gene which encodes a ribosome-associated (p)ppGpp synthetase. The synthetic activity of RelA was found to be essential for its DerN321D suppressor activity. Overexpression of RelA in a suppressor strain did not affect the expression of DerN321D but suppressed the polysome defects caused by the DerN321D mutant. This is the first demonstration of suppression of impaired function of Der by a functional enzyme. A possible mechanism of the suppression of DerN321D by RelA overproduction is discussed.

scholarly journals Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.

1997 ◽  
Vol 8 (4) ◽  
pp. 647-662 ◽  
Author(s):  
F P Finger ◽  
P Novick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Synthetic Lethal ◽  
Growth Defects ◽  
Actin Structure ◽  
Synthetically Lethal ◽  
Bud Site ◽  
Slow Growing ◽  
Late Stages ◽  
Synthetic Growth

Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.

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