scholarly journals Temperature-Sensitive Mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 Genes Result in Defects in mRNA Turnover

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 35-47 ◽  
Author(s):  
Dorit Zuk ◽  
Jonathan P Belk ◽  
Allan Jacobson
Keyword(s):  
Mrna Decay ◽  
Trna Synthetase ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Significant Drop ◽  
Nonsense Mediated Decay ◽  
Mrna Stabilization ◽  
Yeast Strains ◽  
Two Hybrid

Abstract In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37°. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p.

scholarly journals Dynamic profiling of mRNA turnover reveals gene-specific and system-wide regulation of mRNA decay

2011 ◽  
Vol 22 (15) ◽  
pp. 2787-2795 ◽  
Author(s):  
Sarah E. Munchel ◽  
Ryan K. Shultzaberger ◽  
Naoki Takizawa ◽  
Karsten Weis
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Mrna Decay ◽  
Global Analysis ◽  
Decay Rates ◽  
Mrna Turnover ◽  
Control Of Gene Expression ◽  
Mrna Stabilization ◽  
Highly Sensitive ◽  
Wide Range

RNA levels are determined by the rates of both transcription and decay, and a mechanistic understanding of the complex networks regulating gene expression requires methods that allow dynamic measurements of transcription and decay in living cells with minimal perturbation. Here, we describe a metabolic pulse-chase labeling protocol using 4-thiouracil combined with large-scale RNA sequencing to determine decay rates of all mRNAs in Saccharomyces cerevisiae. Profiling in various growth and stress conditions reveals that mRNA turnover is highly regulated both for specific groups of transcripts and at the system-wide level. For example, acute glucose starvation induces global mRNA stabilization but increases the degradation of all 132 detected ribosomal protein mRNAs. This effect is transient and can be mimicked by inhibiting the target-of-rapamycin kinase. Half-lives of mRNAs critical for galactose (GAL) metabolism are also highly sensitive to changes in carbon source. The fast reduction of GAL transcripts in glucose requires their dramatically enhanced turnover, highlighting the importance of mRNA decay in the control of gene expression. The approach described here provides a general platform for the global analysis of mRNA turnover and transcription and can be applied to dissect gene expression programs in a wide range of organisms and conditions.

Identification of Edc3p as an Enhancer of mRNA Decapping in Saccharomyces cerevisiae

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 729-739
Author(s):  
Meenakshi Kshirsagar ◽  
Roy Parker
Keyword(s):  
Mrna Decay ◽  
Temperature Sensitive ◽  
Early Step ◽  
Major Pathway ◽  
Mrna Decapping ◽  
P Bodies ◽  
Exonuclease Digestion ◽  
Decapping Enzyme ◽  
Two Hybrid ◽  
New Protein

Abstract The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5′-3′ exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5′-3′ degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5′-3′ exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3Δ had no effect when combined with the lsm1Δ, dhh1Δ, or pat1Δ mutations, which appear to affect an early step in the decapping pathway. This suggests that Edc3p specifically affects the function of the decapping enzyme per se. Consistent with a functional role in decapping, GFP-tagged Edc3p localizes to cytoplasmic foci involved in mRNA decapping referred to as P-bodies. These results identify Edc3p as a new protein involved in the decapping reaction.

High-level expression of Bacillus subtilis tryptophanyl-tRNA synthetase in Escherichia coli

1990 ◽  
Vol 68 (2) ◽  
pp. 492-495 ◽  
Author(s):  
Wen Shi ◽  
King-Chuen Chow ◽  
J. Tze-Fei Wong
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Large Scale ◽  
Trna Synthetase ◽  
Start Codon ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Scale Preparation ◽  
High Level

The trpS gene encoding Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was prepared from the pUC8-derived pTSQ2 plasmid, mutagenized to introduce an EcoRI site immediately in front of the ATG start codon, and inserted into the pKK223-3 vector downstream to the tac promoter to yield the pKSW1 plasmid. Upon induction with isopropyl-β-D-thiogalactopyranoside, Escherichia coli JM109[pKSW1] cells synthesized TrpRS to a level corresponding to 45% of total cell proteins. This high level of gene expression facilitates large scale preparation of TrpRS for physical studies, detection of in vivo degradation of mutant forms of TrpRS, and comparative assays of TrpRS by [3H]Trp-tRNA formation and by Trp-hydroxamate formation for the purpose of mutant characterization. Finally, since pKSW1 could complement the temperature-sensitive TrpRS mutation on E. coli trpS 10343 cells, defective mutations of the trpS gene on pKSW1 would be detectible on the basis of complementation testing.Key words: tryptophan-tRNA, aminoacyl-tRNA synthetase, Bacillus subtilis.

scholarly journals Mutations in trans-acting factors affecting mRNA decapping in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (10) ◽  
pp. 5830-5838 ◽  
Author(s):  
L Hatfield ◽  
C A Beelman ◽  
A Stevens ◽  
R Parker
Keyword(s):  
Mrna Decay ◽  
Decay Rates ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Subsequent Work ◽  
Factors Affecting ◽  
Mrna Decapping ◽  
Specific Effects ◽  
Cell Extracts ◽  
Decapping Enzyme

The decay of several yeast mRNAs occurs by a mechanism in which deadenylation precedes decapping and subsequent 5'-to-3' exonucleolytic decay. In order to identify gene products required for this process of mRNA turnover, we screened a library of temperature-sensitive strains for mutants with altered mRNA degradation. We identified seven mutations in four genes that inhibited mRNA turnover. Two mutations were alleles of the XRN1 5'-to-3' exoribonuclease known to degrade mRNAs following decapping. One mutation defined a new gene, termed DCP1, which in subsequent work was demonstrated to encode a decapping enzyme or a necessary component of a decapping complex. The other mutations defined two additional genes, termed MRT1 and MRT3 (for mRNA turnover). Mutations in the MRT1 and MRT3 genes slow the rate of deadenylation-dependent decapping, show transcript-specific effects on mRNA decay rates, and do not affect the rapid turnover of an mRNA containing an early nonsense codon, which is degraded by a deadenylation-independent decapping mechanism. Importantly, cell extracts from mrt1 and mrt3 strains contain normal levels of the decapping activity required for mRNA decay. These observations suggest that the products of the MRT1 and MRT3 genes function to modulate the rates of decapping that occur following deadenylation.

Glutamyl and Glutaminyl-tRNA Synthetases Are a Promising Target for the Design of Threonine-Producing Strain

2019 ◽  
Vol 35 (6) ◽  
pp. 39-50
Author(s):  
T.V. Yuzbashev ◽  
A.S. Fedorov ◽  
F.V. Bondarenko ◽  
A.S. Savchenko ◽  
T.V. Vybornaya ◽  
...  
Keyword(s):  
Biomass Accumulation ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Original Strain ◽  
Trna Synthetases ◽  
Resource Center ◽  
Promising Target ◽  
The Russian Federation ◽  
Increase In Productivity ◽  
Culture Growth

The present work describes an approach that improves the properties of the strain producing L-threonine via the reduction in the biomass accumulation during fermentation. Glutamyl- and glutaminyl-tRNA synthetases were chosen as targets. Mutants carrying temperature-sensitive alleles were obtained. It was shown that the used system caused the suppression of the function of tRNA synthetases which led to a rapid arrest of the culture growth, and an increase in productivity and yield of the L-threonine synthesis. One of the temperature-sensitive strains was used to obtain under non-permissive conditions of mutants with the suppressed above phenotype. Some of these mutants accumulate less biomass and produce by 10-12% more threonine than the original strain. Escherichia coli, producing strain, threonine, aminoacyl-tRNA synthetase, ts-mutation This work was supported by the Ministry of Science and Higher Education of the Russian Federation (project code RFMEFI61017X0011), and it was carried out using the equipment of the National Bio-Resource Center All-Russian Collection of Industrial Microorganisms, NRC «Kurchatov Institute» - GosNIIgenetika.

scholarly journals A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

1998 ◽  
Vol 9 (8) ◽  
pp. 2201-2216 ◽  
Author(s):  
Thu Nguyen ◽  
Dani B.N. Vinh ◽  
Douglas K. Crawford ◽  
Trisha N. Davis
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Amino Terminus ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Synthetic Lethal ◽  
Lethal Phenotype ◽  
Amino Terminal ◽  
N Terminus ◽  
Two Hybrid

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations inSPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, thespc97 alleles are synthetic lethal withspc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals Suppressors of a Saccharomyces cerevisiae pkc1 mutation identify alleles of the phosphatase gene PTC1 and of a novel gene encoding a putative basic leucine zipper protein.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1275-1285 ◽  
Author(s):  
K N Huang ◽  
L S Symington
Keyword(s):  
Protein Kinase ◽  
Leucine Zipper ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Basic Leucine Zipper ◽  
Mapk Cascades ◽  
Synthetically Lethal

Abstract The PKC1 gene product, protein kinase C, regulates a mitogen-activated protein kinase (MAPK) cascade, which is implicated in cell wall metabolism. Previously, we identified the pkc1-4 allele in a screen for mutants with increased rates of recombination, indicating that PKC1 may also regulate DNA metabolism. The pkc1-4 allele also conferred a temperature-sensitive (ts) growth defect. Extragenic suppressors were isolated that suppress both the ts and hyperrecombination phenotypes conferred by the pkc1-4 mutation. Eight of these suppressors for into two complementation groups, designated KCS1 and KCS2. KCS1 was cloned and found to encode a novel protein with homology to the basic leucine zipper family of transcription factors. KCS2 is allelic with PTC1, a previously identified type 2C serine/threonine protein phosphatase. Although mutation of either KCS1 or PTC1 causes little apparent phenotype, the kcs1 delta ptc1 delta double mutant fails to grow at 30 degrees. Furthermore, the ptc1 deletion mutation is synthetically lethal in combination with a mutation in MPK1, which encodes a MAPK homologue proposed to act in the PKC1 pathway. Because PTC1 was initially isolated as a component of the Hog1p MAPK pathway, it appears that these two MAPK cascades share a common regulatory feature.

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