Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress - PubMed (original) (raw)
Release of extraction-resistant mRNA in stationary phase Saccharomyces cerevisiae produces a massive increase in transcript abundance in response to stress
Anthony D Aragon et al. Genome Biol. 2006.
Erratum in
- Genome Biol. 2006; 7(8):403
Abstract
Background: As carbon sources are exhausted, Saccharomyces cerevisiae cells exhibit reduced metabolic activity and cultures enter the stationary phase. We asked whether cells in stationary phase cultures respond to additional stress at the level of transcript abundance.
Results: Microarrays were used to quantify changes in transcript abundance in cells from stationary phase cultures in response to stress. More than 800 mRNAs increased in abundance by one minute after oxidative stress. A significant number of these mRNAs encode proteins involved in stress responses. We tested whether mRNA increases were due to new transcription, rapid poly-adenylation of message (which would not be detected by microarrays), or potential release of mature mRNA present in the cell but resistant to extraction during RNA isolation. Examination of the response to oxidative stress in an RNA polymerase II mutant, rpb1-1, suggested that new transcription was not required. Quantitative RT-PCR analysis of a subset of these transcripts further suggested that the transcripts present in isolated total RNA from stationary phase cultures were polyadenylated. In contrast, over 2,000 transcripts increased after protease treatment of cell-free lysates from stationary phase but not exponentially growing cultures. Different subsets of transcripts were released by oxidative stress and temperature upshift, suggesting that mRNA release is stress-specific.
Conclusions: Cells in stationary phase cultures contain a large number of extraction-resistant mRNAs in a protease-labile, rapidly releasable form. The transcript release appears to be stress-specific. We hypothesize that these transcripts are associated with P-bodies.
Figures
Figure 1
Time course at 30 minute intervals in cells from stationary phase cultures exposed to 50 μM menadione. (a) Heat map of results from unsupervised, hierarchical clustering (Pearson's centered, average-linkage) of approximately 2,800 transcripts. Microarrays were of samples taken at 30 minute intervals over 8 hours. The color scale at the bottom indicates the log2 values of changes in mRNA abundance. (b) Median values for the four major temporal patterns of gene expression identified on the right side of the heat map in (a). RNA was isolated using the modified Gentra method described in Materials and methods.
Figure 2
Time course at 1 minute intervals in cells from stationary phase cultures exposed to 50 μM menadione. (a) Heat map of results from unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 2,000 transcripts from samples harvested at 1 minute intervals for 35 minutes with an additional sample taken at one hour. (b) Median values for the two major temporal patterns of changes in mRNA abundance plotted from the median values of mRNAs clustered in (a). RNA was isolated using the modified Gentra method described in Materials and methods.
Figure 3
Time course of gene expression in wild type (S), parental (P), and rpb1-1 mutant (M) stationary phase cultures exposed to 50 μM menadione for 0, 2, and 30 minutes. Heat map of results from unsupervised hierarchical clustering (Pearson's centered, average-linkage). Approximately 1,000 transcripts were included in this analysis. Samples were taken at T0, T2, and T30 minutes after exposure to 50 μM menadione. The color scale at the bottom indicates the log2 values for changes in mRNA abundance.
Figure 4
Quantitative RT-PCR analysis to detect presence of non-adenylated transcripts in T0 samples and samples 2 minutes after oxidative stress (T2). cDNA was synthesized using oligo-dT (to identify polyadenylated transcripts) or random hexamer primers. To determine if 5' or 3' ends of transcripts were more abundant, primer pairs were made to amplify 3' or 5' ends of each of four transcripts. Fold change represents the difference in abundance of 5' or 3' ends of transcripts in cDNAs synthesized using random hexamers versus oligo-dT primers. Measurements were obtained by quantitative RT-PCR and error bars represent the standard deviation of three measurements. The red horizontal bar at Fold Change = 1 indicates no difference in transcript abundance between oligo-dT-primed cDNA and random hexamer-primed cDNA. If non-adenylated transcripts were present, Fold Change > 1 would be expected.
Figure 5
mRNA abundance in samples treated with or without protease. Unsupervised hierarchical clustering (Pearson's centered, average-linkage) of approximately 3,800 transcripts. Samples were incubated with buffer alone (-) or protease (+): trypsin (T), proteinase K (K), Qiagen protease (P). Results were normalized to untreated samples (lanes 1, 5, 7, 9, 11, or 13). Lanes 1 to 8: samples from stationary phase cultures. Lanes 3 and 4: stationary phase samples 2 minutes after treatment with menadione (+). Lanes 9 to 14: exponential samples treated with or without protease. The color scale at the bottom represents the log2 values for changes in mRNA abundance.
Figure 6
mRNA abundance in samples isolated using two different RNA isolation methods or treated with proteinase K. Unsupervised hierarchical clustering (Person's centered, average-linkage) of approximately 4,000 transcripts. RNA was isolated from unstressed cells from stationary phase cultures using the modified Gentra isolation method, hot phenol, or treated with proteinase K. Results were normalized to samples isolated using our RNA isolation method. Biological replicates for each RNA isolation method are shown. The color scale at the bottom represents the log2 values for changes in mRNA abundance.
Figure 7
Venn diagram of transcripts that increased after oxidative stress or proteinase K treatment of T0 cell lysates. Transcripts were evaluated that had a ≥2-fold increase in abundance by 1 and 30 minutes after oxidative stress or after proteinase K treatment. Transcripts were also required to have good spots in 80% of the time points.
Figure 8
Venn diagram of transcripts that increased by 1 minute after oxidative stress, 30 minutes after temperature upshift, or after proteinase K treatment of T0 cell lysates. Transcripts used for this analysis were filtered as described in Figure 7.
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