Low level genome mistranslations deregulate the transcriptome and translatome and generate proteotoxic stress in yeast

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Engineering regulated expression of a heterologous tRNA_CAG^Ser in yeast. A) The recombinant mistranslating tRNA_CAG^Ser gene (cloned into plasmid pRS305K-tetO-tRNA) was integrated into the yeast LEU2 locus by homologous recombination using the KanMX4 gene as a selectable marker. The same yeast strain was transformed with the pGalTRI plasmid containing the GAL1 promoter - tetR construct. Selection was carried out in MMgalactose-URA containing geneticin (200 mg/L). B) Growth curves of tetO-tRNA clones growing in liquid MMgalactose+geneticin at 30°C. Expression of the tRNA_CAG^Ser was induced by addition of 40 μg/mL of tetracycline at OD₆₀₀ = 0.4 to 0.5 (T0'). Yeast growth was monitored by measuring OD₆₀₀ of the culture or by counting the number of cells per mL using a Neubauer cell counting chamber. The dilution shown indicates start of second cultures where the tetracycline concentrations tested are indicated in the inset key in μg/mL. C) Left panel shows the amount of β-gal protein expressed in Control and mistranslating yeast cells at T90'. Center panel shows the residual activity of β-gal after its thermal inactivation at 47°C for 10 minutes. The activity of the β-gal fraction that remained functional after thermal inactivation and refolding (4°C) was determined by incubating cell extracts at 37°C for two minutes in the presence of ONPG. The values in the graph represent activity in tetO-tRNA cells as percent relative to Control cells. The right panel shows increased aggregation of mistranslated β-gal relative to wild type enzyme, confirming that mistranslation is an important source of protein aggregation. The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between tetO-tRNA and Control cells in each graph are shown - *P < 0.05; **P < 0.01.

Figure 2

Transcriptional responses of yeast exposed to gene mistranslations and environmental stressors. A) Gene expression profiles of mistranslating cells at T0', T40', T60', T90', T120' and T180'. B) Overlap of genes differentially expressed (DEGs) in the Environmental Stress Response (ESR) and in mistranslations (> 2-fold deregulation). Approximately 70% of the mistranslations DEGs are related to the stress response. The overlap of genes up-regulated by the ESR and mistranslations increased over time reaching 82% at mistranslation T180'. Similarly, the overlap of down-regulated genes increases significantly at mistranslations T120' and T180'. C) Summary of GO terms of ESR and mistranslations DEGs. Each color square represents the average expression level of the genes annotated with the corresponding GO term for each stress condition. Stress conditions have been hierarchically clustered. Mistranslations activate stress responders and repress translational and ribosomal biogenesis processes, although average fold variation is not as strong as for heat shock or nitrogen depletion. The ESR up- and down-regulated gene lists (ESRup, ESRdown) were obtained from Gasch et al. [42].

Figure 3

High overlap of genes deregulated by mistranslations and environmental stressors. Genes whose expression was deregulated (DEGs) by mistranslations were selected and their expression pattern was compared across environmental stress time points. GO terms enrichment analysis showed the main functional categories affected by mistranslations and environmental stressors. Condition time-points are indicated as small numbers below the stress type (time units are minutes unless otherwise stated: h, hours, d, days). Transcriptional data of yeast responses to environmental stress were obtained from Gasch et al. [42].

Figure 4

The effect of gene mistranslations on protein synthesis. A) Validation of expression of ribosomal protein genes by real time quantitative PCR (n = 3) confirmed the down regulation of ribosome biogenesis (*P < 0.05; **P < 0.01 for T90' vs. T0' two-tailed unpaired Student's t-test comparison). B) Mistranslations decreased protein synthesis. Control and mistranslating cells (12 OD₆₀₀ units) were pulse labeled with 62.5 μCi of [¹⁴C]-Leu for 30' at 30°C. Labeled cells were then disrupted and cleared protein extracts (30 μl) were applied onto paper filters for counting incorporated radioactivity using a scintillation counter. The values in the graph represent incorporated radioactivity in tetO-tRNA cells as percent relative to Control cells. The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between tetO-tRNA and Control cells are shown - **P < 0.01. C) Polysomal profiles of yeast cells mistranslating at T0', T40' and T90', showing reduction in the number of polysomes engaged in mRNA translation. The relative area of the polysomal (P) vs. total area (P+M) of Control and tetO-tRNA cells at each time point and the ratios between total polysomes+monosome material (P+M) in tetO-tRNA cells and in Control cells before (T0') and after mistranslation induction (T40' and T90'), are shown.

Figure 5

The translatome of mistranslating cells. A) Graphical representation of the correlation between the profiles of total mRNA (transcriptome) and polysome-associated (translatome) mRNA. The y-axis represents M values of the ratio of total mRNA transcript fractions at time-points T90' and T0'. The x-axis represents M values of the ratio of polysome-associated transcripts at time-points T90' and T0'. B) Cross stress comparison of translatome DEGs using GO terms enrichment analysis [99]. Mistranslation translatome DEGs (T90') were used for this comparative analysis. The translatome data of cells exposed to environmental stress were obtained from Halbeisen et al. [46]. C) Schematic representation of genes shown in the panel-A quadrants, which have higher variation of fold values between the transcriptome and translatome. The data show that gene expression deregulation was independent of the CUG content and that ribosomal proteins are not directly affected by Ser misincorporation at CUGs.

Figure 6

The UPR is activated by gene mistranslations. A) Gene expression profile highlighting genes involved in the Unfolded Protein Response (UPR) elicited by mistranslations. B) Cross stress comparison of DEGs using GO terms enrichment analysis. Mistranslations up-regulate ERAD, ER translocation, protein folding and oxidative stress genes. C) Activation of the UPR regulator HAC1 (HAC1i) through splicing. The panel shows RT-PCR fragments of the HAC1 mRNA amplified using specific primers and the presence (HAC1^u) and absence (HAC1ⁱ) of a 252-bp intron in the HAC1 primary transcript. A microfluidics gel image showing increased quantity of the HACⁱ isoform at induction times T120' and T180'. D) The graph shows the quantification of both forms of the HAC1 mRNA displayed in B. The data were normalized to the ACT1 internal control (*P < 0.05 for HAC1ⁱ vs. HAC1^u two-tailed unpaired Student's t-test comparison).

Figure 7

Working model of the yeast response to gene mistranslations. Mistranslated proteins are folding substrates of molecular chaperones that compete with wild type client substrates for folding/refolding. Gradual accumulation of mistranslated proteins shifts the HSP-client binding equilibrium to the subpopulation of misfolded proteins, releasing client substrates from HSPs. This activates or inactivates natural HSP-client substrates depending on whether the HSP-client interaction is positive or negative. HSP substrate release deregulates cellular processes mediated by the HSP client proteins and remodels chaperone-chaperone networks, which are critical for cellular homeostasis. Increased protein folding/refolding and degradation increase ATP consumption leading to up-regulation of mitochondrial metabolism and ROS accumulation. Accumulation of mistranslated proteins in mitochondria also increases mitochondrial stress and ROS production. Mistranslated proteins that enter the secretory pathway accumulate in the ER and up-regulate ER resident chaperones, activating the UPR, further increasing ROS production. This leads to a deficit in protein secretion with consequences for cell membranes and cell wall structure and function. Aggregation of mistranslated proteins exacerbates proteotoxic stress, increases ATP consumption and activates the general stress response (ESR) through the transcription factors Msn2/4p and Yaps. Mistranslations also down-regulate ribosome biosynthesis, translational factors and CLIPS, exacerbating the negative consequences of mistranslations due to the critical role of CLIPS in folding newly synthesized proteins.