Transient transcriptional responses to stress are generated by opposing effects of mRNA production and degradation

Ophir Shalem^1,2, Orna Dahan¹, Michal Levo¹, Maria Rodriguez Martinez^1,3, Itay Furman¹, Eran Segal² & Yitzhak Pilpel¹

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Synopsis

The ability to follow genome-wide changes in mRNA abundance over time, using microarray technology, has revolutionized the study of gene expression regulation. In a typical microarray experiment, cell samples are taken over a defined time course following a treatment, RNA is extracted and then hybridized to the microarrays to quantify the relative amount of each gene's mRNA at each sample. Such studies have revealed that in response to environmental stimuli the mRNA abundance of a large fraction of the genome changes either by increasing or decreasing its levels (Gasch et al, 2000, 2001; Jelinsky et al, 2000; Gasch and Werner-Washburne, 2002). In the study of transcriptional regulation, this data are often assumed to reflect transcriptional changes. Although this approach has greatly improved our understanding of transcriptional regulation, it does not address the fact that changes in mRNA abundance are influenced by both changes in transcription rates and mRNA stability. Not only that an increase or decrease in mRNA abundance can be achieved by both changes in production or degradation, different strategies of changing production and degradation rates will result in different kinetic behaviors of mRNA abundance over time (Perez-Ortin et al, 2007). Thus, to understand the dynamics of the transcriptome under varying conditions, the role of both production and degradation of mRNAs must be examined.

Here, we investigate changes in mRNA stability and study their effect on key kinetic parameters of the mRNA response to stress in the yeast Saccharomyces cerevisiae. By using a strain bearing a temperature-sensitive mutation in RNA polymerase II, we were able to stop transcription of mRNA and follow mRNA decay kinetics by microarray hybridization. For the first time, genome-wide mRNA stability is measured directly, in more than one external condition, revealing mRNA stability as a dynamic feature that not only varies between genes, as was shown earlier (Bernstein et al, 2002; Wang et al, 2002; Yang et al, 2003; Grigull et al, 2004; Narsai et al, 2007), but is also modulated between different conditions sometimes even in opposite directions in each condition.

To examine the effect of changes in mRNA stability on the temporal kinetics of mRNA abundance, we chose two intensively investigated conditions, an oxidative and a DNA-damaging stress. The two stresses were selected such that they will differ in the kinetics of the mRNA response they induce. In oxidative stress, the majority of the responding genes show fast response followed by relaxation resulting in a quick and transient response, whereas in the DNA damage experiment, the response is slow and long enduring (Figure 1A). In each of the two conditions, mRNA decay experiments were performed in parallel to conventional mRNA abundance measurements. In addition, as a control, we performed experiments in which no stress was applied prior to stopping transcription to obtain a reference decay rate for all genes. These experiments yield three data sets for each stressful condition: genome-wide mRNA abundance profiles, genome-wide mRNA decay profiles in the stressful condition and genome-wide mRNA decay profiles in a reference condition. The majority of the genes showed exponential decay in all three decay experiments, which enabled us to calculate the half-life of each gene in each of the three conditions. For each gene, we used the ratio between the mRNA half-life in each stressful condition to the half-life measured in the reference condition as a measure of the modulation of mRNA stability in response to stress. We assessed the correlation between this measure to the maximal change in mRNA abundance for each gene in each of the two conditions and found two opposing relationships (Figure 2). By examining the response to DNA damage, we observed a somewhat more intuitive trend. In this condition, the mRNA of induced genes shows a tendency to be stabilized, whereas that of repressed genes show a tendency to be destabilized (Figure 2). In the oxidative stress on the other hand, we found a surprising negative correlation—genes whose mRNAs are induced in response to the stress are typically destabilized, whereas repressed genes show a weaker, though still significant, tendency toward stabilization.

Figure 1

Distinct transcriptome responses at the two conditions. (A) Mean expression profile of all induced and repressed genes (fold change >2) in oxidative and MMS stress (blue and red curves, respectively). (B) The proteasomal genes as an example for a group of genes showing coherent change in mRNA stability in response to each stress. The mean of the fitted decay profiles is shown; black, blue and red represent the reference, oxidative stress and DNA damage conditions, respectively. (C) The mRNA abundance profiles of the proteasomal genes (after mean and variance normalization) are shown for the oxidative stress and DNA damage stress (left and right panels, respectively).

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Figure 2

Distinct relationships between the change in mRNA abundance and the change in stability between the two conditions. For each stress, the change in mRNA stability relative to the reference state (log₂(t_{1/2 stress}/t_{1/2 reference})) is plotted against the maximal fold change (defined as described in Materials and methods). Two different trends are observed, a negative trend (R=-0.38, p-value<3 10^-150) for the oxidative stress and a positive trend for the DNA damage stress (R=0.27, p-value<4.2 10^-70).

Full figure and legend (279K)Figures & Tables index

In our paper, we show that the distinct global transcriptome responses between the two conditions are a direct outcome of this opposite relationships between changes in mRNA abundance to changes in mRNA stability. We divide the genes in both conditions according to the time at which the maximal change in mRNA abundance is reached and show that when the changes in mRNA abundance are counteracted by the changes in mRNA stability a transient change in mRNA abundance is observed, e.g. induced genes that are also destabilized show a transient induction. When the change in mRNA abundance is accompanied by a change in mRNA stability in the same direction, a sustained response is observed. These results suggest that mRNA decay is a key feature regulating the response duration of mRNA abundance.

Our findings suggest that there exists coordination between regulation of mRNA production to mRNA degradation in the cell, and that this coordination is important for shaping the overall mRNA kinetics in response to a given stimulus. We propose two alternative models to explain such coordination. The first model assumes that mRNA decay rates are directly affected during the stage of production. Indications for a potential mechanism that could generate such coupling have been reported in previous studies based on a few genes (Lotan et al, 2005, 2007). According to the second indirect model, sensing of the stress results in the activation of the transcriptional response and, independently of that, it also induces a change in stability of the transcripts. Future work will be needed to determine the relative validity of these models. Still, it is clear that mRNA stability plays a central role in shaping the temporal profile of mRNA abundance changes in response to stress and its contribution should be taken into consideration in studies aiming to reveal the mechanisms of gene expression regulation.

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