Low level genome mistranslations deregulate the transcriptome and translatome and generate proteotoxic stress in yeast
© Paredes et al; licensee BioMed Central Ltd. 2012
Received: 1 June 2012
Accepted: 20 June 2012
Published: 20 June 2012
Organisms use highly accurate molecular processes to transcribe their genes and a variety of mRNA quality control and ribosome proofreading mechanisms to maintain intact the fidelity of genetic information flow. Despite this, low level gene translational errors induced by mutations and environmental factors cause neurodegeneration and premature death in mice and mitochondrial disorders in humans. Paradoxically, such errors can generate advantageous phenotypic diversity in fungi and bacteria through poorly understood molecular processes.
In order to clarify the biological relevance of gene translational errors we have engineered codon misreading in yeast and used profiling of total and polysome-associated mRNAs, molecular and biochemical tools to characterize the recombinant cells. We demonstrate here that gene translational errors, which have negligible impact on yeast growth rate down-regulate protein synthesis, activate the unfolded protein response and environmental stress response pathways, and down-regulate chaperones linked to ribosomes.
We provide the first global view of transcriptional and post-transcriptional responses to global gene translational errors and we postulate that they cause gradual cell degeneration through synergistic effects of overloading protein quality control systems and deregulation of protein synthesis, but generate adaptive phenotypes in unicellular organisms through activation of stress cross-protection. We conclude that these genome wide gene translational infidelities can be degenerative or adaptive depending on cellular context and physiological condition.
KeywordsYeast mistranslation tRNA protein synthesis mRNA profiling stress proteotoxic stress protein misfolding unfolded protein response
Genome decoding fidelity is essential to maintain cell homeostasis and fitness in all organisms. However, errors in DNA transcription, pre-mRNA splicing and editing, and in mRNA translation, generate mutant proteins whose toxicity creates homeostatic imbalances (proteotoxic stress). At the gene translation level, missense, nonsense, frameshifting and ribosome drop-off errors affect protein synthesis . Missense errors arise from incorrect tRNA selection by the ribosome or incorrect tRNA aminoacylation by aminoacyl-tRNA synthetases (aaRSs) and occur with average frequency of 10-3 to 10-5 per codon decoded [2–4]. Such errors are codon-dependent and are sensitive to the nutritional status of the cell [5, 6]. Translational frameshifting errors occur at a frequency of 10-5 and are caused by tRNA slippage during mRNA decoding , while read-through of stop codons (nonsense errors) results from competition between non-sense suppressor tRNAs and release factors (RFs) and occur at a frequency of 10-3 . Ribosome drop-off errors are poorly understood but have a basal frequency of 4 × 10-4 at ribosome pausing sites [8, 9].
Eukaryotic cells mitigate the deleterious effects of those gene expression infidelities through the ubiquitin-proteasome pathway (UPS), autophagy, ER-associated protein degradation pathway (ERAD) and molecular chaperones [10, 11]. Despite this, mutations that affect protein synthesis efficiency and/or accuracy cause neurodegenerative disease in mice and various human diseases, including mitochondrial diseases and cancer [reviewed in ]. For example, a single mutation in the editing domain of the mouse alanyl-tRNA synthetase (AlaRS) leads to serine (Ser) misincorporation at alanine (Ala) codons and causes rapid loss of Purkinje cells , while mischarging of the tRNAMet with homocysteine (Hcy) causes proteome N-homocysteinylation in vascular endothelial cells (HUVEC) and increases the risk of vascular disease in humans . Moreover, reactive oxygen species (ROS) modify phenylalanine (Phe) to m-tyrosine (m-Tyr), o-tyrosine (o-Tyr) and p-tyrosine (Tyr) and promote m-Tyr misincorporation into proteins by both the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases (PheRS) via mischarging of tRNAPhe (m-Tyr-tRNAPhe), but the consequences of proteome m-tyrosylation are not known . Similarly, mutations in mitochondrial tDNA genes encoding tRNAPhe, tRNALeu, tRNASer, tRNAHis and tRNALys, which affect the accuracy and/or efficiency of translation, cause myopathy, encephalopathy, lactic acidosis, stroke-like episodes or myoclonic epilepsy with ragged-red fibers (MELAS/MERRF syndromes) [16–18], indicating that mitochondria are particularly sensitive to gene translation fidelity and efficiency.
Most surprisingly, elevated gene translational errors (mistranslations) can trigger expression of advantageous phenotypes in yeast and bacteria [19–22]. For example, misincorporation of Ser at Leu CUG codons allows yeast to grow in the presence of high concentrations of arsenite, cadmium, cycloheximide, NaCl and H2O2 [20, 21], while natural epigenetic control of both stop codon read-through and antizyme frameshifting by the [PSI + ] prion generates phenotypic diversity and regulates the cellular concentration of polyamines [23–25]. In the fungal pathogen Candida albicans such mistranslations generate extensive phenotypic diversity, induce expression of novel colony and cell morphotypes and are associated with evolution of a genetic code alteration [26, 27].
Mistranslations are also used to synthesise statistical proteins of high potential to generate antigenic variation in Mycoplasma species which encode threonyl-, phenylayl- and leucyl-tRNA synthetases (ThrRS, LeuRS and PheRS, respectively) with defective amino acid editing domains . In E. coli, mistranslations induce a hypermutagenic phenotype known as translational stress mutagenesis (TSM) [29, 30], raising the fascinating hypothesis that phenotypic outcomes of gene translational errors can be rapidly fixed in the genome. We unveil below hidden features of the biology of genome translational infidelities which help us understand some of the phenotypes described above.
Model system to study gene mistranslations in a controlled manner
Since biologically and biomedically relevant gene mistranslations occur at levels that do not compromise cell viability, we have attempted to determine the mistranslations' induction time and intensity thresholds that produced minimal impact on growth rate. Expression of the tRNACAGSer could be induced with 40 μg/ml of tetracycline at an OD600 of 0.4 to 0.5 without significant alteration in growth rate, small differences were visible in stationary phase only (Figure 1B). Earlier induction of the tRNA (OD600 = 0.1) resulted in higher reduction of cell density in stationary phase and slowed growth of cells diluted into fresh medium (Additional file 1, Figure S1). Putting it simply, gene mistranslations remained phenotypically silent during the first three to four yeast generations but their negative effects increased in intensity over time, as one would predict from gradual accumulation of the mistranslating tRNA. When cells expressing the tRNACAGSer were spotted in MMgalactose agar plates, a decrease in viability or ability to re-grow and form colonies was observed. This effect was stronger when a higher concentration of tetracyclin was used (Additional file 2, Figure S2A). A similar result was observed when Control and tetO-tRNA cells pre-cultured in MMgalactose were directly plated in MMgalactose + tetracycline agar plates (Additional file 2, Figure S2B), indicating that mistranslations become degenerative overtime.
In order to confirm the misreading activity of the tRNACAGSer, we have co-expressed the E. coli β-galactosidase (β-gal) and the tRNACAGSer genes in the same recombinant cells. The E. coli LacZ gene contains 54 CUG codons and misincorporation of Ser at these Leu-codons generates a combinatorial array of mutant β-gal molecules (statistical β-gal) whose altered stability can be quantified using thermal denaturation and aggregation assays [40, 41]. The high number of CUG codons present in the LacZ gene combined with the different chemical properties of Ser (polar amino acid) and Leu (hydrophobic amino acid) make β-gal a highly sensitive reporter, allowing for monitoring low level misreading activity of the tRNACAGSer. As expected, Ser misincorporation at CUGs decreased the cellular concentration of β-gal (Figure 1C, left panel) and a thermal denaturation assay  showed decreased β-gal activity after heat denaturation and refolding (T40' - 25.1% and T90' - 35.0%) (Figure 1C, center panel). Mistranslated β-gal also had higher propensity to aggregate (Figure 1C, right panel), confirming previous data on the role of gene mistranslations on protein aggregation . We have attempted to quantify the expression of the tRNACAGSer by Northern blot analysis but we were unable to do so. This was consistent, however, with our previous quantitative mass-spectrometry studies which showed that constitutive expression of the tRNACAGSer in yeast leads to 1.4% misincorporation of Ser at Leu CUG positions, but the tRNA was very difficult to detect by Northern blot analysis .
General features of the transcriptional response to gene mistranslations
General features of the transcriptional response to genome mistranslations
Genes in term
protein targeting to mitochondrion
regulation of protein metabolic process
macromolecule biosynthetic process
vacuolar protein catabolic process
response to heat
response to toxin
regulation of translation
regulation of translational fidelity
maturation of SSU-rRNA
The cross stress comparison of the complete set of DEGs corroborated and highlighted the generalized down-regulation of the protein synthesis machinery, in particular of genes encoding translation factors, RNA binding and processing proteins, regulation of translational fidelity, ribosomal proteins and ribosome biogenesis and assembly genes (Figures 2 and 3; Additional file 3, Figure S3; Additional file 6, Figure S4; Additional file 7, Table S3). It also showed down-regulation of chaperones linked to the ribosome (CLIPS network), which fold newly synthesized proteins emerging from it (Additional file 7, Table S3; Additional file 8, Figure S5). These CLIPS included Hsp70s SSB2, the Hsp70 partners SSZ1 and ZUO1, the chaperonin TriC/CCTs (TCP1 and CCT2 - CCT8) and members of the prefoldin GimC protein family (GIM3, GIM4 and GIM5), suggesting that down-regulation of the protein synthesis machinery exacerbates protein folding problems caused by gene mistranslations.
Gene mistranslations affect protein synthesis
GO enrichment for the different groups of genes identified in the transcriptome and translatome comparison
Genes in GO term
Genes in group
In group and term
Response to oxidative stress
Response to stress
Ribosomal large subunit biogenesis
Ribosomal small subunit biogenesis
Ribosomal large subunit assembly
Ribosomal small subunit assembly
Regulation of translation
Regulation of translational fidelity
Response to drug
Response to toxin
Response to chemical stimulus
Transposition, RNA mediated
A third category of genes (88 genes) had negative transcriptional and positive translational values (Figure 5A, C; Table 2; Additional file 7, Table S3), indicating that they were regulated at the translational level. Most of these genes encode proteins involved in toxin and chemical stimulus responses (AAD6, AAD10, GPX2, GTT2 and SRX1) and drug transport, for example, FLR1, ATR1, PMA2 and AQR1 (Figure 5C; Table 2; Additional file 7, Table S3). A significant number of genes encoding components of yeast transposons, namely YBL005W-A, YFL002W-B, YOR343W-A, YBL101W-A and YOR343W, appeared in this group (Table 2; Additional file 7, Table S3), suggesting that gene mistranslations generate genome diversity through mobilization of transposon activity.
Unidirectional changes between transcription and translation are associated with a gene expression phenomenon called potentiation [53–55], which is characteristic of specific groups of genes under strong stress intensity . Mistranslations potentiated the expression of the plasma membrane chaperone gene HSP30 (16.8-fold), which represses the H(+)-ATPase Pma1, the cell wall protein gene PST1 (3.5-fold), which is activated in response to cell wall damage, the oxidative stress genes GRX2, CTT1, TRX2, ECM4, AHP1, ALD3 (4.7-, 7.4-, 6.8-, 16.4-, 7.9- and 19.1-fold, respectively), the phospholipid binding protein gene SIP18 (11.4-fold), the cell wall secretory glycoprotein gene YGP1 (9.2-fold), and the multi-stress protein genes DDR2 and OYE3 (55.8- and 10.4-fold, respectively). Interestingly, genes that were negatively represented in the total mRNA profile but had positive representation in the translatome profile (T90') (Figure 5A, C; Table 2; Additional file 7, Table S3) were also involved in the stress response. For example, the plasma membrane multidrug transporter gene FLR1 (6.2-fold), the phospholipid hydroperoxide glutathione peroxidase gene GPX2 (2.9-fold), the bZIP transcription regulator of the UPR (HAC1) (1.6-fold), the putative aryl alcohol dehydrogenase genes AAD6 and AAD10 (14.7- and 2.9-fold, respectively), the sulfiredoxin gene SRX1 (2.3-fold) whose protein reduces cysteine-sulfinic acid groups in the peroxiredoxins Tsa1 and Ahp1 and contributes to oxidative stress resistance which was further enhanced by overexpression of the glutathione S-transferase gene GTT2 (10.3-fold).
Gene mistranslations activate the unfolded protein response
Expression of the transcription factor Hac1p, which regulates UPR genes through the UPR enhancer (UPRE) [61–63], was slightly up-regulated at the translatome level at T90' and was down-regulated 6.7-fold at the same time point in the total mRNA profile (Figure 5C; Additional file 7, Table S3). This post-transcriptional regulation of HAC1 expression was consistent with processing and activation of the HAC1 mRNA since its pre-mRNA contains a 252 bp intron whose retention in the HAC1 pre-mRNA renders its mRNA untranslatable (HAC1 u ). Splicing of this intron allows for translation of the HAC1 mRNA (translatable HAC1 i ) and subsequent activation of the UPR via transcription of ER genes [64–68]. The spliced (HAC i ) and unspliced (HAC1 u ) forms of HAC1 mRNA were detected at T0' by RT-PCR and increased HAC i levels were observed between T90' and T180' (Figure 6C, D), confirming that the UPR was activated, explaining the increased transcription of UPR genes containing UPREs from T90' to T180' (Figure 6A). This delay in the activation of the UPR (T90') contrasted with the early detection of mistranslations (T40') (Figure 1C, center panel) and with the early up-regulation of stress-induced chaperones (Additional file 6, Figure S4; Additional file 8, Figure S5). Therefore, steady state activity of proteome quality control systems, in particular of stress-induced molecular chaperones and the ubiquitin-proteasome pathway, likely mitigated the early proteome disruption caused by mistranslations, but above a certain threshold those quality control systems probably became overloaded and proteome quality maintenance required the UPR.
Regulation of the stress response triggered by mistranslation
The similarities between the transcriptional and translational responses elicited by environmental stressors and the gene mistranslations allow one to get the first insight into the gene regulatory networks involved in the cellular response to genome translational infidelities. Enrichment of transcription factor (TF) binding motifs present in the DEGs promoters (Additional file 12, Figure S9) identified the general stress response element (STRE; AGGGGA/T), the heat-shock responsive element (HSE; nGAAn), the proteasome associated control element (PACE; GGTGGCAAA; targeted by Rpn4p) and the pleiotropic drug resistance element (PDRE; TCCGCGGA targeted by Pdr1p/Pdr3p), as the main cis regulatory elements of the transcriptional responses to gene mistranslations (Additional file 12, Figure S9).
The enrichment in STREs (Additional file 12, Figure S9A) indicates that the transcriptional response to gene mistranslations is partly regulated by the cyclic AMP (cAMP) protein kinase A (PKA) (cAMP-PKA) and the TORC1 pathways, which control the transcription factors Msn2p and Msn4p [69, 70]. Since ATP and cAMP regulate PKA signalling through the RAS activators (Ras1/2) of the adenylate cyclase Cyr1 , gene mistranslations likely decrease cAMP production because Hsp70 Ssa1 regulates positively the guanine nucleotide exchange factor for RAS (Cdc25). Indeed, mistranslated proteins are folding substrates of Hsp70 chaperones and can deviate Ssa1 from its interaction with Cdc25p , lowering its activity and decreasing Ras1/2 - Cyr1 activity, cAMP production and PKA activity . The enrichment in STRE elements also provides strong evidence for a role of the TORC1 signalling pathway as it regulates Msn2p/4p by promoting their phosphorylation (see below) . On the other hand, the enrichment in HSE (Additional file 12, Figure S9A) indicates that the observed up-regulation of molecular chaperones is mediated through the heat-shock factor (Hsf1p) . Regulation of Hsf1p involves phosphorylation, conformational alterations and chromatin structure remodelling and it is difficult to understand how gene mistranslations activate it on the sole basis of the comparative transcriptomic studies that we have carried out. Nevertheless, the known down-regulation of Hsf1p via direct interaction with Ssa1-4 members of the Hsp70 family  is of particular relevance here as mistranslated proteins likely reduce the pool of free Hsp70 allowing for release and activation of Hsf1p and transcriptional up-regulation of HSE-containing genes.
The enrichment in PACE-containing genes (Additional file 12, Figure S9B) indicates that gene mistranslations up-regulate the UPS through the Rpn4p transcription factor, which is one of the main regulators of proteasome biosynthesis [76, 77]. Interestingly, the promoter of the RPN4 gene contains HSE (Hsf1p), YRE (Yap1p) and PDRE (Pdr1p/3p) elements and it is likely that mistranslated proteins activate transcription of PACE genes through synergistic interactions between Hsf1p, Yap1p and Pdr1/3p transcription factors. This is consistent with delayed UPS activation under gene mistranslations (Additional file 3, Figure S3; Additional file 7, Table S3; Additional file 13, Figure S10) and suggests that while Hsf1p, Yap1p and Pdr1/3p are directly activated by mistranslated proteins, the UPS is activated by a second wave of transcriptional regulation. An alternative hypothesis is that Rpn4p is stabilized by mistranslated proteins. Rpn4p has a very short half-life under non-stress conditions (approximately two minutes) but is stable under stress ; therefore, UPS overloading with mistranslated proteins may stabilize it, providing additional signals for up-regulation of genes encoding proteasome subunits and other PACE genes.
Regarding the up-regulation of stress genes regulated by PDREs (Additional file 12, Figure S9B), there is again an interesting connection with Hsp70 family members as Pdr3p is negatively regulated by Hsp70-Ssa1, while Pdr1p is positively regulated by the CLIP Hsp70 Ssz1 [78, 79]. Hence, mistranslated proteins likely activate Pdr3p by freeing it from the repressive interaction with Hsp70-Ssa1/2, suggesting that activation of multidrug response genes is mediated through Pdr3p rather than Pdr1p as the latter is likely down-regulated under mistranslations due to decreased expression of the ribosome-associated activator Ssz1p (Additional file 3, Figure S3; Additional file 8, Figure S5; Additional file 7, Table S3). Mistranslated proteins translocated into mitochondria should also compete for Ssa1/Ssa2 and may activate the retrograde mechanism, which is known to increase multidrug resistance . This is consistent with increased ROS production and deregulation of mitochondrial genes, including the mitochondrial chaperones Hsp78, Hsp60 and Hsp10 by the gene mistranslations.
The down-regulation of CLIPS, RP and RiBi regulons
Co-down-regulation of CLIPS and the translational machinery is expected to exacerbate the consequences of the gene mistranslations due to the critical role of these chaperones in folding newly synthesized proteins. Indeed, deletion of SSB1/2 results in accumulation of misfolded polyubiquitinated proteins and activation of stress HSE regulated genes [81, 82], as is also the case in strains harboring deletions in GimC/GIM or TriC/CCT . The down-regulation of these CLIPS may also explain the high expression of HSPs as the latter are essential for survival in ΔSSB1/2 or ΔGIMc deleted cells and mildly beneficial in cells lacking the RAC complex . Interestingly, accumulation of misfolded proteins is not a major problem in strains lacking GIM2, ZUO1, SSZ1 and CCT. Ssb1/2p are the main players in folding newly synthesized proteins while the other CLIPS play alternative roles. Hence, down-regulation of SSB1/2 in the mistranslating cells likely increases accumulation of misfolded proteins, which may explain why cells integrated mistranslations as a strong stressor.
Strains construction and growth
The Saccharomyces cerevisiae BMA64 strain (EUROSCARF acc. no. 20000D; genotype: MATa/MATα; ura3-52/ura3-52; trp1Δ2/trp1Δ2; leu2-3_112/leu2-3_112; his3-11/his3-11; ade2-1/ade2-1; can1-100/can1-100) was used for the genetic manipulations described below. BMA64 cells were transformed with pGalTR1 (kind gift of T. Winckler and T. Dingermann), which encodes the prokaryotic tet-repressor protein (tetR) whose expression is activated in the presence of galactose . Yeast transformations were carried out using the lithium acetate method . Clones were grown in MMgalactose-URA (Minimal Medium without uracil: 0.67% yeast nitrogen base, 2% galactose, 0.2% Drop-out mix). For construction of the inducible system, the misreading tRNACAGSer gene was amplified by PCR and SalI/BamHI restriction sites were inserted at the 5'- and 3'-ends during the amplification. The tet-operator sequence (tetO) was inserted three nucleotides upstream of the mature tRNA 5'-end. These amplified fragments were cloned into the pRS305K plasmid  yielding the plasmid pRS305K-tetOtRNA. These recombinant tRNA genes were integrated into the genome of the yeast strain BMA64A (previously transformed with the plasmid pGalTR1) by homologous recombination using linear DNA fragments containing long tails with homology to the leu2 integration locus and the geneticin-resistance KanMX4 gene. Transformed clones were selected in MMgalactose-URA containing 200 mg/L of geneticin. The integration into the yeast leu2 locus were checked by colony PCR followed by Sanger DNA sequencing. For monitoring Ser misincorporation at Leu CUG codons using the β-gal thermal stability assay the above clones were transformed with the pGL-C1 plasmid , which encodes a GST-β-gal chimeric gene fusion.
Pre-cultures of yeast cells containing the tetO-tRNA cassette (tetO-tRNA cells) or the empty cassette (Control cells) were prepared in MMgalactose-URA+geneticin (200 mg/L) media for approximately 16 to 20 hours, at 30°C. Such pre-cultures were used to inoculate fresh cultures of MMgalactose+geneticin (200 mg/L) at OD600 of approximately 0.05, which were allowed to grow at 30°C. Tetracycline (40 μg/mL) was then added at OD600 0.4 to 0.5. Control cells and tetO-tRNA cells were harvested (50 mL) at T0', T40', T60', T90', T120' and T180' of tRNACAGSer expression induction with tetracycline. Cell pellets were immediately frozen in liquid nitrogen and were stored at -80°C for later use.
β-galactosidase activity assays
A total of 500 μl of exponentially growing (OD approximately 0.5) Control cells and cells expressing the Ser tRNACAGSer were harvested at time points T0', T40', T90', T180' after mistranslations induction with tetracycline. Cells were washed and resuspended in 800 μl of Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4·2H2O, 10 mM KCl, 1 mM MgSO4·7H2O, 50 mM 2-mercaptoethanol, pH 7.0), 20 μl of 0.1% SDS and 50 μl of chloroform. Cell suspensions were mixed (vortex) for 30 seconds and incubated in triplicate at 47°C in a water bath for 10 minutes. This β-gal unfolding step was followed by a refolding step, which was carried out by incubating samples on ice for 30 minutes. Residual β-gal activity was then quantified at 37°C. For this, the assay tubes (200 μl) were incubated for five minutes at 37°C and then 4 mg/mL of the o-nitrophenyl-β-D-galactopyranoside (ONPG) substrate were added to each tube and reactions were allowed to proceed for two minutes and were stopped by the addition of 400 μl of 1M Na2CO3. β-gal activity was determined by monitoring o-nitrophenol synthesis at 420 nm.
β-galactosidase aggregation assay
Protein aggregation assays were adapted from . Briefly, 10 A600 units of exponentially growing cells were harvested by centrifugation, washed and resuspended in 300 μl of lysis buffer (50 mM potassium phosphate buffer pH 7, 1 mM ethylenediaminetetraacetic acid (EDTA), 5% v/v glycerol, 1 mM phenylmethylsulfonyl fluoride, and complete mini protease inhibitor cocktail from Roche Diagnostics (Mannheim, Germany). Cells were disrupted by vortexing with glass beads (0.5 mm diameter) for 3 × 1 minute, with 1-minute incubation on ice between each disruption cycle. Intact cells were removed by centrifugation of the crude extract at 5,000 rpm for 15 minutes. Aggregated proteins were isolated by centrifugation at 13,000 rpm for 20 minutes and membrane proteins were removed by washing the pellet with a lysis buffer containing 2% Triton X-100. The final pellet was resuspended in 100 μl of lysis buffer.
Western blot analysis
Total and aggregated protein fractions were analyzed under reducing conditions using 12% SDS-PAGE and blotted onto nitrocellulose membranes according to standard procedures. β-Gal was detected using a rabbit anti-β-Gal primary antibody (Molecular Probes, Leiden, The Netherlands) at 1:5,000 dilution. Bound antibody was visualized by incubating membranes with a IRDye680 goat anti-rabbit secondary antibody (Li-cor Biosciences, Lincoln, NE, USA) at 1:10,000 dilution. Detection was carried out using an Odyssey Infrared Imaging system (Li-cor Biosciences). The amount of aggregated β-Gal was normalized to the amount of β-Gal present in the total protein fraction.
RNA isolation and labeling
RNA isolation and labeling were carried out as described by van de Peppel , with minor modifications. Briefly, total yeast RNA extracts were prepared using hot phenol (T0' to T180'). cDNA synthesis was carried out using 40 μg of total RNA extracted from T0' to T180' samples and SuperscriptII Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). A pool of mRNAs extracted from Control cells at several time points was used as reference RNA sample. For labeling, all cDNAs were synthesized in presence of aminoallyl-dUTP (Sigma-Aldrich, Munich, Germany), purified using Microcon-30 (Millipore, Billerica, MA, USA) columns and were coupled to Cy3 or Cy5 fluorophores (Amersham Biosciences, Piscataway, NJ, USA). Before hybridization, free dyes were removed using Chromaspin-30 (Clontech, Palo Alto, CA, USA) columns and the efficiency of cDNA synthesis and dye incorporation was measured using a Nanodrop spectrophotometer by determining the full spectrum of absorption in the 190 to 750 nm range and registering the OD values at 260 nm, 550 nm and 649 nm points for each sample. For each hybridization 300 ng of Cy3- and Cy5-labelled cDNAs were mixed with in house printed yeast arrays (YAUAv 1.0, DNA Microarray Facility, Department of Biology, University of Aveiro, Aveiro, Portugal) and hybridized for 20 hours at 42°C using an Agilent (Santa Clara, CA, USA) hybridization oven. Slides were scanned using an Agilent G2565AA scanner and raw data were extracted using the QuantArray v3.0 software (PerkinElmer, Waltham, MA, USA).
Preparation of yeast polysomal RNA
Polysomes were isolated as previously described by Arava , with minor modifications. For each sample, yeast cultures (80 mL) were harvested by centrifugation at 4,000 rpm, for four minutes, at 4°C, in the presence of 100 μg/mL cycloheximide to freeze protein synthesis elongation. Cells were then washed twice using 2 mL of lysis buffer (20 mM Tris-HCl at pH 8.0, 140 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 100 μg/mL cycloheximide, 1 mg/mL heparin, 1% Triton X-100), and were resuspended in 700 μl of the same buffer supplemented with 0.6 volumes of chilled glass beads. Cell lysis was carried out using eight cycles of 30 seconds vortexing and 1 minute cooling on ice. Lysates were transferred to clean microfuge tubes and centrifuged for five minutes at 8,000 rpm at 4°C. Supernatants were transferred to clean microfuge tubes and 40 units A280 nm of sample were loaded onto 11 mL 15% to 50% sucrose gradients containing 20 mM Tris-HCl at pH 8.0, 140 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 100 μg/mL cyclohexamide, 500 μg/ml heparin. Gradients were centrifuged at 35,000 rpm for 2 hours and 45 minutes, using a SW41 rotor and an Optima series ultracentrifuge (Beckman Coulter, Brea, CA, USA). Polysomal profiles were visualized by monitoring RNA absorbance at 254 nm using a Bio-Rad (Hercules, CA, USA) Biologic LP system adapted for this assay. The polysomal fraction of the gradient was recovered and RNA was precipitated as previously described by Arava . mRNA was isolated from polysomal RNA using Oligotex (Qiagen, Hilden, Germany) beads and cDNA synthesis was carried out using 3 μg of purified mRNA. Labeling and hybridization were carried out as described above.
Normalization and analysis of DNA microarray data
Raw data were normalized using limmaGUI software (R/Bioconductor, Boston, MA, USA)  and print-tip lowess normalization within arrays. Heatmaps and clustering of genes were carried out using MeV software . Functional analysis of expression data obtained was carried out using the EXPANDER software (Algorithms in Computational Genomics group, Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel)  and the YEASTRACT online tool (Biological Sciences Research Group, IBB and Knowledge Discovery and Bioinformatics group, INESC-ID, Lisbon, Portugal)  as well as the R/Bioconductor limma package (R/Bioconductor, Boston, MA, USA) . The microarray raw data were submitted to the ArrayExpress database (EMBL-EBI, Hinxton, UK) and are available under the accession codes E-MTAB-153 and E-MTAB-166.
Gene expression deregulation analysis
Differentially expressed genes (DEGs) for each of the mistranslations' time-points were extracted using a linear model analysis (R/Bioconductor package limma , considering as differentially expressed a variation equal or higher than 2X or 1X between each time-point and the initial time-point. Only genes with a significance level below a Benjamini-Hochberg corrected P-value of 10-3 were considered as differentially expressed. The relaxed 1X DEGs were used in order to avoid a low number of genes in the GO analysis which could raise spurious enriched GO terms and distort the data analysis. The more strict 2X DEGs were used for other analysis, namely for ESR comparisons. GO term enrichment for DEGs listed at each time-point was carried out using the hypergeometric test developed by Falcon and Gentleman in GOstats , applied over each GO biological process, and then selecting GO terms enriched with a P-value lower than 10-3. The GO terms considered for mistranslation are either enriched in four or more time points or have a significance level below a P-value of 10-9 for a single time point. This method provided approximately 40 GO terms that, after manually removing redundant and generic terms, resulted in a dozen terms (Table 1). The hierarchical clustering was carried out by constructing an expression matrix containing the stress profiles of genes annotated in the enriched GO terms. The values of this matrix were also averaged by time point and GO term and stress conditions were clustered again (Figures 2C, 5B, 6B). For the comparison of ESR vs. mistranslation, the ESR up- and down-regulated gene lists (ESRup, ESRdown) from Gasch et al.  were compared with the mistranslation time-point specific DEGs and with the combination of these DEGs lists into a mistranslations DEGs list.
Real time quantitative PCR
Total RNA was extracted from yeast cells and genomic DNA contamination was removed using DNase I (Invitrogen), followed by phenol extraction. Total RNA quantity and quality were accessed using the Nanodrop 1000 and Agilent 2100 Bioanalyzer systems, respectively. Total RNA (40 μg) was reverse-transcribed to cDNA using Superscript II RT enzyme (Invitrogen) and oligo dT (12 to 18) primers, following the manufacturer's recommendations. First-strand cDNA templates were then used for PCR amplification of short (100 to 150 bp) gene fragments using appropriate primers. PCRs were carried out in triplicate using a Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) and analyzed using a 7500 real-time PCR system (Applied Biosystems), following the manufacturer's recommendations. A dissociation curve was generated at the end of each PCR cycle to check for primer dimerization. Standard dilution curves were determined for each primer set and their amplification efficiencies calculated. cDNA concentration in each sample was normalized to ACT1. Relative quantification of target cDNA was determined by calculating the difference in cross-threshold (Ct) values after normalization to the ACT1 signal, according to the Pfaffl's method  and the Excel-based program REST (Technical University of Munich, Munich, Germany) .
For RT-PCR, total RNA extracts were prepared as above from T0', T40', T90' and T180'. RNA samples were prepared for HAC1 mRNA for reverse transcription (see above) and RT-PCR using the PCR primers 5'-ATGACTGATTTTGAACTAACTAG and 5'-CAATTCAAATGAATTCAAACCTG.
Protein pulse labeling with [14C]-Leucine
Amino acid incorporation was performed at time points T0', T40', T90', T180' and T240' after inducing the gene mistranslations with tetracycline. Briefly, 2 × 107cells were collected and resuspended into 2 ml of pre-warmed minimal medium, 20 μl of cold [14C(U)]-L-Amino Acid Mixture were added, (Perkin Elmer, 0.1 mCi/ml) and the mixture was incubated 10 minutes at 30°C with agitation. Amino acid incorporation was stopped by the addition of 60 μl of cicloheximide (20 mg/ml) and ice incubation. Cells were washed once with cold water and frozen at -80°C. Protein was then extracted by resuspending cell pellets in 200 μl Lysis buffer (50 mM potassium phosphate buffer pH 7, 1 mM EDTA, 5% (vol/vol) glycerol, 1 mM phenylmethylsulfonyl fluoride, and complete mini protease inhibitor cocktail (Roche) and 120 μl of glass beads. Cells were disrupted using a Precellys (Bertin Technologies, Montigny-le-Bretonneux, France) disrupter (5 cycles of 10 sec at 5,000 rpm and 1 minute on ice between cycles) and centrifuged at 3,000 × g for 10 minutes. A total of 30 μl of supernatant was applied on 1 cm2 square paper microfiber filter (GF/C, Whatman, Maidstone, UK). Amino acid incorporation was measured using a scintillation counter (Beckman) and protein extracts were quantified using the BCA protein quantification Kit (Pierce. Rockford, IL, USA). [14C(U)]-L-Amino acid incorporation was normalized against the total protein for each sample and compared to Control amino acid incorporation at each time point.
leucine labelled with carbon 14
chaperones linked to protein synthesis
environmental stress response
heat shock proteins
unfolded protein response
The authors are most grateful to the Portuguese Foundation for Science and Technology (FCT) for funding our work through project REF: FCT/FEDER/PTDC/BIA-BCM/64745/2006, PTDC/SAU-GMG/098850/2008 and also to the EU FP7 Sybaris consortium through project SYBARIS/HEALTH-F3-2009-242220. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Farabaugh PJ, Bjork GR: How translational accuracy influences reading frame maintenance. EMBO J. 1999, 18: 1427-1434. 10.1093/emboj/18.6.1427.PubMedPubMed Central
- Jakubowski H, Goldman E: Editing of errors in selection of amino acids for protein synthesis. Microbiol Rev. 1992, 56: 412-429.PubMedPubMed Central
- Loftfield RB, Vanderjagt D: The frequency of errors in protein biosynthesis. Biochem J. 1972, 128: 1353-1356.PubMedPubMed Central
- Stansfield I, Jones KM, Herbert P, Lewendon A, Shaw WV, Tuite MF: Missense translation errors in Saccharomyces cerevisiae. J Mol Biol. 1998, 282: 13-24. 10.1006/jmbi.1998.1976.PubMed
- Elf J, Nilsson D, Tenson T, Ehrenberg M: Selective charging of tRNA isoacceptors explains patterns of codon usage. Science. 2003, 300: 1718-1722. 10.1126/science.1083811.PubMed
- Kramer EB, Farabaugh PJ: The frequency of translational misreading errors in E. coli is largely determined by tRNA competition. RNA. 2007, 13: 87-96.PubMedPubMed Central
- Valente L, Kinzy TG: Yeast as a sensor of factors affecting the accuracy of protein synthesis. Cell Mol Life Sci. 2003, 60: 2115-2130. 10.1007/s00018-003-2334-2.PubMed
- Menninger JR: Peptidyl transfer RNA dissociates during protein synthesis from ribosomes of Escherichia coli. J Biol Chem. 1976, 251: 3392-3398.PubMed
- Buchan JR, Stansfield I: Halting a cellular production line: responses to ribosomal pausing during translation. Biol Cell. 2007, 99: 475-487. 10.1042/BC20070037.PubMed
- Gregersen N, Bross P, Vang S, Christensen JH: Protein misfolding and human disease. Annu Rev Genomics Hum Genet. 2006, 7: 103-124. 10.1146/annurev.genom.7.080505.115737.PubMed
- Bukau B, Weissman J, Horwich A: Molecular chaperones and protein quality control. Cell. 2006, 125: 443-451. 10.1016/j.cell.2006.04.014.PubMed
- Scheper GC, van der Knaap MS, Proud CG: Translation matters: protein synthesis defects in inherited disease. Nat Rev Genet. 2007, 8: 711-723. 10.1038/nrg2142.PubMed
- Lee JW, Beebe K, Nangle LA, Jang J, Longo-Guess CM, Cook SA, Davisson MT, Sundberg JP, Schimmel P, Ackerman SL: Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature. 2006, 443: 50-55. 10.1038/nature05096.PubMed
- Jakubowski H: The pathophysiological hypothesis of homocysteine thiolactone-mediated vascular disease. J Physiol Pharmacol. 2008, 59 (Suppl 9): 155-167.PubMed
- Klipcan L, Moor N, Kessler N, Safro MG: Eukaryotic cytosolic and mitochondrial phenylalanyl-tRNA synthetases catalyze the charging of tRNA with the meta-tyrosine. Proc Natl Acad Sci USA. 2009, 106: 11045-11048. 10.1073/pnas.0905212106.PubMedPubMed Central
- Shoffner JM, Lott MT, Lezza AM, Seibel P, Ballinger SW, Wallace DC: Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation. Cell. 1990, 61: 931-937. 10.1016/0092-8674(90)90059-N.PubMed
- Borner GV, Zeviani M, Tiranti V, Carrara F, Hoffmann S, Gerbitz KD, Lochmuller H, Pongratz D, Klopstock T, Melberg A, Holme E, Pääbo S: Decreased aminoacylation of mutant tRNAs in MELAS but not in MERRF patients. Hum Mol Genet. 2000, 9: 467-475. 10.1093/hmg/9.4.467.PubMed
- Enriquez JA, Chomyn A, Attardi G: MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA(Lys) and premature translation termination. Nat Genet. 1995, 10: 47-55. 10.1038/ng0595-47.PubMed
- Pezo V, Metzgar D, Hendrickson TL, Waas WF, Hazebrouck S, Doring V, Marliere P, Schimmel P, De Crecy-Lagard V: Artificially ambiguous genetic code confers growth yield advantage. Proc Natl Acad Sci USA. 2004, 101: 8593-8597. 10.1073/pnas.0402893101.PubMedPubMed Central
- Silva RM, Paredes JA, Moura GR, Manadas B, Lima-Costa T, Rocha R, Miranda I, Gomes AC, Koerkamp MJ, Perrot M, Holstege FC, Boucherie H, Santos MA: Critical roles for a genetic code alteration in the evolution of the genus Candida. EMBO J. 2007, 26: 4555-4565. 10.1038/sj.emboj.7601876.PubMedPubMed Central
- Santos MA, Cheesman C, Costa V, Moradas-Ferreira P, Tuite MF: Selective advantages created by codon ambiguity allowed for the evolution of an alternative genetic code in Candida spp. Mol Microbiol. 1999, 31: 937-947. 10.1046/j.1365-2958.1999.01233.x.PubMed
- Bacher JM, Waas WF, Metzgar D, de Crecy-Lagard V, Schimmel P: Genetic code ambiguity confers a selective advantage on Acinetobacter baylyi. J Bacteriol. 2007, 189: 6494-6496. 10.1128/JB.00622-07.PubMedPubMed Central
- True HL, Berlin I, Lindquist SL: Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits. Nature. 2004, 431: 184-187. 10.1038/nature02885.PubMed
- True HL, Lindquist SL: A yeast prion provides a mechanism for genetic variation and phenotypic diversity. Nature. 2000, 407: 477-483. 10.1038/35035005.PubMed
- Namy O, Galopier A, Martini C, Matsufuji S, Fabret C, Rousset JP: Epigenetic control of polyamines by the prion [PSI(+)]. Nat Cell Biol. 2008, 10: 1069-1075. 10.1038/ncb1766.PubMed
- Gomes AC, Miranda I, Silva RM, Moura GR, Thomas B, Akoulitchev A, Santos MA: A genetic code alteration generates a proteome of high diversity in the human pathogen Candida albicans. Genome Biol. 2007, 8: R206-10.1186/gb-2007-8-10-r206.PubMedPubMed Central
- Miranda I, Rocha R, Santos MC, Mateus DD, Moura GR, Carreto L, Santos MA: A genetic code alteration is a phenotype diversity generator in the human pathogen Candida albicans. PLoS One. 2007, 2: e996-10.1371/journal.pone.0000996.PubMedPubMed Central
- Li L, Boniecki MT, Jaffe JD, Imai BS, Yau PM, Luthey-Schulten ZA, Martinis SA: Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites. Proc Natl Acad Sci USA. 2011, 108: 9378-9383. 10.1073/pnas.1016460108.PubMedPubMed Central
- Al Mamun AA, Marians KJ, Humayun MZ: DNA polymerase III from Escherichia coli cells expressing mutA mistranslator tRNA is error-prone. J Biol Chem. 2002, 277: 46319-46327. 10.1074/jbc.M206856200.PubMed
- Balashov S, Humayun MZ: Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells. J Mol Biol. 2002, 315: 513-527. 10.1006/jmbi.2001.5273.PubMed
- Grant CM, Firoozan M, Tuite MF: Mistranslation induces the heat-shock response in the yeast Saccharomyces cerevisiae. Mol Microbiol. 1989, 3: 215-220. 10.1111/j.1365-2958.1989.tb01810.x.PubMed
- Al Mamun AA, Gautam S, Humayun MZ: Hypermutagenesis in mutA cells is mediated by mistranslational corruption of polymerase, and is accompanied by replication fork collapse. Mol Microbiol. 2006, 62: 1752-1763. 10.1111/j.1365-2958.2006.05490.x.PubMed
- Grant CM, Tuite MF: Mistranslation of human phosphoglycerate kinase in yeast in the presence of paromomycin. Curr Genet. 1994, 26: 95-99. 10.1007/BF00313794.PubMed
- Kohanski MA, Dwyer DJ, Wierzbowski J, Cottarel G, Collins JJ: Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death. Cell. 2008, 135: 679-690. 10.1016/j.cell.2008.09.038.PubMedPubMed Central
- Nagel R, Chan A: Mistranslation and genetic variability: the effect of streptomycin. Mutat Res. 2006, 601: 162-170. 10.1016/j.mrfmmm.2006.06.012.PubMed
- Santos MA, Perreau VM, Tuite MF: Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans. EMBO J. 1996, 15: 5060-5068.PubMedPubMed Central
- Santos MA, Tuite MF: The CUG codon is decoded in vivo as serine and not leucine in Candida albicans. Nucleic Acids Res. 1995, 23: 1481-1486. 10.1093/nar/23.9.1481.PubMedPubMed Central
- Suzuki T, Ueda T, Watanabe K: The 'polysemous' codon--a codon with multiple amino acid assignment caused by dual specificity of tRNA identity. EMBO J. 1997, 16: 1122-1134. 10.1093/emboj/16.5.1122.PubMedPubMed Central
- Dingermann T, Frank-Stoll U, Werner H, Wissmann A, Hillen W, Jacquet M, Marschalek R: RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system. EMBO J. 1992, 11: 1487-1492.PubMedPubMed Central
- Branscomb EW, Galas DJ: Progressive decrease in protein synthesis accuracy induced by streptomycin in Escherichia coli. Nature. 1975, 254: 161-163. 10.1038/254161a0.PubMed
- Finkelstein DB, Strausberg S: Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae. Mol Cell Biol. 1983, 3: 1625-1633.PubMedPubMed Central
- Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO: Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell. 2000, 11: 4241-4257.PubMedPubMed Central
- Liberek K, Lewandowska A, Zietkiewicz S: Chaperones in control of protein disaggregation. EMBO J. 2008, 27: 328-335. 10.1038/sj.emboj.7601970.PubMedPubMed Central
- Tadi D, Hasan RN, Bussereau F, Boy-Marcotte E, Jacquet M: Selection of genes repressed by cAMP that are induced by nutritional limitation in Saccharomyces cerevisiae. Yeast. 1999, 15: 1733-1745. 10.1002/(SICI)1097-0061(199912)15:16<1733::AID-YEA490>3.0.CO;2-L.PubMed
- Malys N, Carroll K, Miyan J, Tollervey D, McCarthy JE: The 'scavenger' m7GpppX pyrophosphatase activity of Dcs1 modulates nutrient-induced responses in yeast. Nucleic Acids Res. 2004, 32: 3590-3600. 10.1093/nar/gkh687.PubMedPubMed Central
- Halbeisen RE, Gerber AP: Stress-dependent coordination of transcriptome and translatome in yeast. PLoS Biol. 2009, 7: e105-10.1371/journal.pbio.1000105.
- Helgstrand M, Mandava CS, Mulder FA, Liljas A, Sanyal S, Akke M: The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain. J Mol Biol. 2007, 365: 468-479. 10.1016/j.jmb.2006.10.025.PubMed
- Krokowski D, Boguszewska A, Abramczyk D, Liljas A, Tchorzewski M, Grankowski N: Yeast ribosomal P0 protein has two separate binding sites for P1/P2 proteins. Mol Microbiol. 2006, 60: 386-400. 10.1111/j.1365-2958.2006.05117.x.PubMed
- Stevens SW, Abelson J: Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins. Proc Natl Acad Sci USA. 1999, 96: 7226-7231. 10.1073/pnas.96.13.7226.PubMedPubMed Central
- Dobbyn HC, O'Keefe RT: Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA. RNA. 2004, 10: 308-320. 10.1261/rna.5970404.PubMedPubMed Central
- Sickmann A, Reinders J, Wagner Y, Joppich C, Zahedi R, Meyer HE, Schonfisch B, Perschil I, Chacinska A, Guiard B, Rehling P, Pfanner N, Meisinger C: The proteome of Saccharomyces cerevisiae mitochondria. Proc Natl Acad Sci USA. 2003, 100: 13207-13212. 10.1073/pnas.2135385100.PubMedPubMed Central
- Reinders J, Zahedi RP, Pfanner N, Meisinger C, Sickmann A: Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics. J Proteome Res. 2006, 5: 1543-1554. 10.1021/pr050477f.PubMed
- Preiss T, Baron-Benhamou J, Ansorge W, Hentze MW: Homodirectional changes in transcriptome composition and mRNA translation induced by rapamycin and heat shock. Nat Struct Biol. 2003, 10: 1039-1047. 10.1038/nsb1015.PubMed
- Smirnova JB, Selley JN, Sanchez-Cabo F, Carroll K, Eddy AA, McCarthy JE, Hubbard SJ, Pavitt GD, Grant CM, Ashe MP: Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways. Mol Cell Biol. 2005, 25: 9340-9349. 10.1128/MCB.25.21.9340-9349.2005.PubMedPubMed Central
- Melamed D, Pnueli L, Arava Y: Yeast translational response to high salinity: global analysis reveals regulation at multiple levels. RNA. 2008, 14: 1337-1351. 10.1261/rna.864908.PubMedPubMed Central
- Patil C, Walter P: Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. Curr Opin Cell Biol. 2001, 13: 349-355. 10.1016/S0955-0674(00)00219-2.PubMed
- Spear E, Ng DT: The unfolded protein response: no longer just a special teams player. Traffic. 2001, 2: 515-523. 10.1034/j.1600-0854.2001.20801.x.PubMed
- Rose MD, Misra LM, Vogel JP: KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell. 1989, 57: 1211-1221. 10.1016/0092-8674(89)90058-5.PubMed
- Okamura K, Kimata Y, Higashio H, Tsuru A, Kohno K: Dissociation of Kar2p/BiP from an ER sensory molecule, Ire1p, triggers the unfolded protein response in yeast. Biochem Biophys Res Commun. 2000, 279: 445-450. 10.1006/bbrc.2000.3987.PubMed
- Kimata Y, Kimata YI, Shimizu Y, Abe H, Farcasanu IC, Takeuchi M, Rose MD, Kohno K: Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins. Mol Biol Cell. 2003, 14: 2559-2569. 10.1091/mbc.E02-11-0708.PubMedPubMed Central
- Kohno K, Normington K, Sambrook J, Gething MJ, Mori K: The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. Mol Cell Biol. 1993, 13: 877-890.PubMedPubMed Central
- Mori K, Ogawa N, Kawahara T, Yanagi H, Yura T: Palindrome with spacer of one nucleotide is characteristic of the cis-acting unfolded protein response element in Saccharomyces cerevisiae. J Biol Chem. 1998, 273: 9912-9920. 10.1074/jbc.273.16.9912.PubMed
- Mori K, Sant A, Kohno K, Normington K, Gething MJ, Sambrook JF: A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. EMBO J. 1992, 11: 2583-2593.PubMedPubMed Central
- Cox JS, Walter P: A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell. 1996, 87: 391-404. 10.1016/S0092-8674(00)81360-4.PubMed
- Sidrauski C, Walter P: The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell. 1997, 90: 1031-1039. 10.1016/S0092-8674(00)80369-4.PubMed
- Chapman RE, Walter P: Translational attenuation mediated by an mRNA intron. Curr Biol. 1997, 7: 850-859. 10.1016/S0960-9822(06)00373-3.PubMed
- Kawahara T, Yanagi H, Yura T, Mori K: Endoplasmic reticulum stress-induced mRNA splicing permits synthesis of transcription factor Hac1p/Ern4p that activates the unfolded protein response. Mol Biol Cell. 1997, 8: 1845-1862.PubMedPubMed Central
- Ruegsegger U, Leber JH, Walter P: Block of HAC1 mRNA translation by long-range base pairing is released by cytoplasmic splicing upon induction of the unfolded protein response. Cell. 2001, 107: 103-114. 10.1016/S0092-8674(01)00505-0.PubMed
- Gorner W, Durchschlag E, Martinez-Pastor MT, Estruch F, Ammerer G, Hamilton B, Ruis H, Schuller C: Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev. 1998, 12: 586-597. 10.1101/gad.12.4.586.PubMedPubMed Central
- Beck T, Hall MN: The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature. 1999, 402: 689-692. 10.1038/45287.PubMed
- Thevelein JM: Fermentable sugars and intracellular acidification as specific activators of the RAS-adenylate cyclase signalling pathway in yeast: the relationship to nutrient-induced cell cycle control. Mol Microbiol. 1991, 5: 1301-1307. 10.1111/j.1365-2958.1991.tb00776.x.PubMed
- Geymonat M, Wang L, Garreau H, Jacquet M: Ssa1p chaperone interacts with the guanine nucleotide exchange factor of ras Cdc25p and controls the cAMP pathway in Saccharomyces cerevisiae. Mol Microbiol. 1998, 30: 855-864. 10.1046/j.1365-2958.1998.01118.x.PubMed
- Estruch F: Stress-controlled transcription factors, stress-induced genes and stress tolerance in budding yeast. FEMS Microbiol Rev. 2000, 24: 469-486. 10.1111/j.1574-6976.2000.tb00551.x.PubMed
- Sorger PK: Heat shock factor and the heat shock response. Cell. 1991, 65: 363-366. 10.1016/0092-8674(91)90452-5.PubMed
- Craig EA, Weissman JS, Horwich AL: Heat shock proteins and molecular chaperones: mediators of protein conformation and turnover in the cell. Cell. 1994, 78: 365-372. 10.1016/0092-8674(94)90416-2.PubMed
- Mannhaupt G, Schnall R, Karpov V, Vetter I, Feldmann H: Rpn4p acts as a transcription factor by binding to PACE, a nonamer box found upstream of 26S proteasomal and other genes in yeast. FEBS Lett. 1999, 450: 27-34. 10.1016/S0014-5793(99)00467-6.PubMed
- Xie Y, Varshavsky A: RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit. Proc Natl Acad Sci USA. 2001, 98: 3056-3061. 10.1073/pnas.071022298.PubMedPubMed Central
- Shahi P, Gulshan K, Moye-Rowley WS: Negative transcriptional regulation of multidrug resistance gene expression by an Hsp70 protein. J Biol Chem. 2007, 282: 26822-26831. 10.1074/jbc.M704772200.PubMed
- Hallstrom TC, Moye-Rowley WS: Hyperactive forms of the Pdr1p transcription factor fail to respond to positive regulation by the hsp70 protein Pdr13p. Mol Microbiol. 2000, 36: 402-413. 10.1046/j.1365-2958.2000.01858.x.PubMed
- Liu Z, Butow RA: Mitochondrial retrograde signaling. Annu Rev Genet. 2006, 40: 159-185. 10.1146/annurev.genet.40.110405.090613.PubMed
- Albanese V, Reissmann S, Frydman J: A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis. J Cell Biol. 2010, 189: 69-81. 10.1083/jcb.201001054.PubMedPubMed Central
- Koplin A, Preissler S, Ilina Y, Koch M, Scior A, Erhardt M, Deuerling E: A dual function for chaperones SSB-RAC and the NAC nascent polypeptide-associated complex on ribosomes. J Cell Biol. 2010, 189: 57-68. 10.1083/jcb.200910074.PubMedPubMed Central
- Albanese V, Yam AY, Baughman J, Parnot C, Frydman J: Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells. Cell. 2006, 124: 75-88. 10.1016/j.cell.2005.11.039.PubMed
- Temple MD, Perrone GG, Dawes IW: Complex cellular responses to reactive oxygen species. Trends Cell Biol. 2005, 15: 319-326. 10.1016/j.tcb.2005.04.003.PubMed
- Rutherford SL: Between genotype and phenotype: protein chaperones and evolvability. Nat Rev Genet. 2003, 4: 263-274. 10.1038/nrg1041.PubMed
- Cowen LE, Lindquist S: Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science. 2005, 309: 2185-2189. 10.1126/science.1118370.PubMed
- Queitsch C, Sangster TA, Lindquist S: Hsp90 as a capacitor of phenotypic variation. Nature. 2002, 417: 618-624. 10.1038/nature749.PubMed
- Rutherford SL, Lindquist S: Hsp90 as a capacitor for morphological evolution. Nature. 1998, 396: 336-342. 10.1038/24550.PubMed
- Sangster TA, Bahrami A, Wilczek A, Watanabe E, Schellenberg K, McLellan C, Kelley A, Kong SW, Queitsch C, Lindquist S: Phenotypic diversity and altered environmental plasticity in Arabidopsis thaliana with reduced Hsp90 levels. PLoS One. 2007, 2: e648-10.1371/journal.pone.0000648.PubMedPubMed Central
- Gietz RD, Woods RA: Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol. 2006, 313: 107-120.PubMed
- Taxis C, Knop M: System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. Biotechniques. 2006, 40: 73-78. 10.2144/000112040.PubMed
- Rand JD, Grant CM: The thioredoxin system protects ribosomes against stress-induced aggregation. Mol Biol Cell. 2006, 17: 387-401.PubMedPubMed Central
- van de Peppel J, Kemmeren P, van Bakel H, Radonjic M, van Leenen D, Holstege FC: Monitoring global messenger RNA changes in externally controlled microarray experiments. EMBO Rep. 2003, 4: 387-393. 10.1038/sj.embor.embor798.PubMedPubMed Central
- Arava Y, Wang Y, Storey JD, Liu CL, Brown PO, Herschlag D: Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 2003, 100: 3889-3894. 10.1073/pnas.0635171100.PubMedPubMed Central
- Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004, 5: R80-10.1186/gb-2004-5-10-r80.PubMedPubMed Central
- Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, Li J, Thiagarajan M, White JA, Quackenbush J: TM4 microarray software suite. Methods Enzymol. 2006, 411: 134-193.PubMed
- Shamir R, Maron-Katz A, Tanay A, Linhart C, Steinfeld I, Sharan R, Shiloh Y, Elkon R: EXPANDER--an integrative program suite for microarray data analysis. BMC Bioinformatics. 2005, 6: 232-10.1186/1471-2105-6-232.PubMedPubMed Central
- Teixeira MC, Monteiro P, Jain P, Tenreiro S, Fernandes AR, Mira NP, Alenquer M, Freitas AT, Oliveira AL, Sa-Correia I: The YEASTRACT database: a tool for the analysis of transcription regulatory associations in Saccharomyces cerevisiae. Nucleic Acids Res. 2006, 34: (Database issue):D446-451.PubMed
- Falcon S, Gentleman R: Using GOstats to test gene lists for GO term association. Bioinformatics. 2007, 23: 257-258. 10.1093/bioinformatics/btl567.PubMed
- Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29: e45-10.1093/nar/29.9.e45.PubMedPubMed Central
- Pfaffl MW, Horgan GW, Dempfle L: Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002, 30: e36-10.1093/nar/30.9.e36.PubMedPubMed Central
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.