Specific and global regulation of mRNA stability during osmotic stress in Saccharomyces cerevisiae

Abstract

Hyperosmotic stress yields reprogramming of gene expression in Saccharomyces cerevisiae cells. Most of this response is orchestrated by Hog1, a stress-activated, mitogen-activated protein kinase (MAPK) homologous to human p38. We investigated, on a genomic scale, the contribution of changes in transcription rates and mRNA stabilities to the modulation of mRNA amounts during the response to osmotic stress in wild-type and hog1 mutant cells. Mild osmotic shock induces a broad mRNA destabilization; however, osmo-mRNAs are up-regulated by increasing both transcription rates and mRNA half-lives. In contrast, mild or severe osmotic stress in hog1 mutants, or severe osmotic stress in wild-type cells, yields global mRNA stabilization and sequestration of mRNAs into P-bodies. After adaptation, the absence of Hog1 affects the kinetics of P-bodies disassembly and the return of mRNAs to translation. Our results indicate that regulation of mRNA turnover contributes to coordinate gene expression upon osmotic stress, and that there are both specific and global controls of mRNA stability depending on the strength of the osmotic stress.

FIGURE 1.

Changes in RNA polymerase II TR and RA during the response to osmotic stress. Wild-type (wt) and hog1Δ cells were grown in YPD until exponential phase and then treated with 0.4 M NaCl. Samples taken at 0, 2, 4, 6, 8, 10, and 15 min of osmotic shock were processed to measure TR and RA of all yeast genes. (A) Changes in global TR and RA after osmotic shock. These two parameters were normalized to an arbitrary value of 1 at t = 0. Error bars represent the standard error of three independent experiments. (B) Clustering of genes according to their TR and RA profiles. Data set series are referred to their respective time 0 in logarithm scale. Relative repression is shown in green and relative induction in red. The most significant gene functional categories for each cluster are shown with their P-values. (C) Changes in global k _D values in response to osmotic stress. The graph represents the difference between the mean k _D value for all genes at each indicated time and the mean k _D value at time 0, divided by mean k _D value at time 0. Standard error of the normalized k _D deviations was between 0.02 and 0.05 for all time points.

FIGURE 2.

Specific stabilization of osmo-mRNAs during mild osmotic stress. (A) Global distribution of genes according to their destabilization (<0) or stabilization (>0) indexes (see Materials and Methods). Arrows in the histogram indicate the position of the genes whose mRNA half-lives have been checked by conventional methods. (B) Determination of mRNAs half-lives (t _1/2) before and after mild osmotic stress. Strains PAY500 (rpb1-1^ts), PAY498 (rpb1-1^ts tetO₂-RTN2), and PAY495 (rpb1-1^ts tetO₂-STL1) were grown in YPD until exponential phase and, before or after treatment for 5 min with NaCl 0.4 M, transcription was stopped by shifting the cells to 37°C and adding doxycycline (10 μg/mL). Aliquots were taken at the indicated times and RA of the indicated genes were determined by Northern analysis (using rRNA18S signal as a loading control). The half-life value of a representative experiment is shown. (C) mRNA half-life change after mild osmotic stress. Mean half-life change (%) for the indicated mRNAs was calculated from at least three independent experiments. Error bars represent the standard deviation.

FIGURE 3.

General mRNA stabilization in hog1Δ mutant after treatment with 0.4 M NaCl. (A) Half-lives of RPS31, URA1, and DSE2 mRNAs in strain PAY532 (rpb1-1^ts hog1Δ) and of RTN2 mRNA in strain PAY591 (rpb1-1^ts hog1Δ rtn2Δ) containing p416TEF-RTN2 were measured as described in Figure 2. The half-life value of a representative experiment is shown. (B) mRNA half-life change (%) after mild osmotic stress in hog1Δ strain. Means were calculated from at least three independent experiments. Error bars represent the standard deviation.

FIGURE 4.

General mRNA stabilization during severe osmotic stress (1 M KCl). (A) Half-lives of RPS31, URA1, and DSE2 mRNAs in strain PAY500 (rpb1-1^ts) and of RTN2 mRNA in strain PAY498 (rpb1-1^ts tetO₂-RTN2) were measured as described in Figure 2. The half-life value of a representative experiment is shown. (B) Half-life change (%) after severe osmotic stress in a wt strain. Means were calculated from at least three independent experiments. Error bars represent the standard deviation.

FIGURE 5.

General mRNA stabilization in hog1Δ mutant during severe osmotic stress (1 M KCl). Half-lives of RTN2, RPS31, URA1, and DSE2 mRNAs in strain PAY591 (rpb1-1^ts hog1Δ rtn2Δ) containing p416TEF-RTN2 were measured as described in Figure 2. The half-life value of a representative experiment, out of three independent experiments, is shown.

FIGURE 6.

Kinetics of P-bodies increase under osmotic shock depends on the stress conditions and the presence of Hog1. Wild-type and hog1Δ (PAY185) strains expressing a GFP-tagged version of the decapping enzyme Dcp2p or BY4741 and BY4741hog1Δ strains expressing the GFP-tagged version of the decapping activator Dhh1p were grown until exponential phase and then treated with (A) NaCl 0.4 M or (B) KCl 1 M. Dcp2-GFP and Dhh1-GFP were used to visualize P-bodies at the indicated times of osmotic stress. (C) Quantification of the percentage of wt or hog1Δ cells with P-bodies after treatment with 0.4 M NaCl or 1 M KCl as indicated above. At least 100 cells were quantified in each condition.