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Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.

Lavut A, Raveh D - PLoS Genet. (2012)

Bottom Line: However, not all the induced genes undergo translation, and mutants of many induced genes do not show elevated sensitivity to the particular stress.These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses.Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beersheba, Israel.

ABSTRACT
Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress.

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Induction of Hsp12 protein and mRNA, and mRNA decay after stress.A. WB of protein produced from genomic HSP12-GFP in response to stress. B. UFO1 and HSP12 mRNA levels in untreated cells analyzed by qRT-PCR. mRNA levels were normalized to ACT1. C. Induction of HSP12 mRNA by stress. Wild type cells at A600 = 0.5, treated with 1 mM arsenate, 8.8 mM H2O2, irradiated with 40 mJ/cm2 UV or shifted from 30°C to 37°C for 40 minutes. Aliquots were collected at the times indicated and analyzed by qRT-PCR. D. HSP12 mRNA decay. pGAL-HSP12 was expressed in hsp12Δ mutants by overnight induction with 2% galactose. Next morning cells at A600 = 0.5 were untreated, or stressed with 1 mM arsenate or 8.8 mM H2O2 for 30 minutes, or irradiated with 40 mJ/cm2 UV. The cells were washed and transferred to SC medium with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to ACT1 and to time 0 (untreated log cells).
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pgen-1002527-g007: Induction of Hsp12 protein and mRNA, and mRNA decay after stress.A. WB of protein produced from genomic HSP12-GFP in response to stress. B. UFO1 and HSP12 mRNA levels in untreated cells analyzed by qRT-PCR. mRNA levels were normalized to ACT1. C. Induction of HSP12 mRNA by stress. Wild type cells at A600 = 0.5, treated with 1 mM arsenate, 8.8 mM H2O2, irradiated with 40 mJ/cm2 UV or shifted from 30°C to 37°C for 40 minutes. Aliquots were collected at the times indicated and analyzed by qRT-PCR. D. HSP12 mRNA decay. pGAL-HSP12 was expressed in hsp12Δ mutants by overnight induction with 2% galactose. Next morning cells at A600 = 0.5 were untreated, or stressed with 1 mM arsenate or 8.8 mM H2O2 for 30 minutes, or irradiated with 40 mJ/cm2 UV. The cells were washed and transferred to SC medium with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to ACT1 and to time 0 (untreated log cells).

Mentions: Both whole-genome microarray experiments [7], [8] and our qRT-PCR data (Figure 1) indicate that UFO1 steady state mRNA levels are elevated in response to stress. However, it is only by using 10-fold the amount of cells compared with our standard protocols that we are able to detect Ufo1GFP protein after any of the stresses applied. We therefore examined the fate of the mRNA of HSP12, the protein of which is highly expressed in response to stress [40]. We treated cells with arsenate, H2O2, UV, glucose deprivation, 37°C or NaCl and analyzed induction of the protein by Western blotting. Hsp12GFP protein was induced after all the treatments, particularly after 37°C (Figure 7A and Figure S2). To examine mRNA localization we fused the MS2L tag to the HSP12 ORF and treated the cells with the same stresses. In contrast to UFO1-MS2L, we did not detect granules in HSP12-MS2L cells and the CPGFP signal remained diffuse throughout the cytoplasm (Figure S3 and [50]). The basal level of HSP12 mRNA in untreated cells was higher than that of UFO1 (Figure 7B). however, the relative increase of HSP12 steady state mRNA level after stress was lower than the high induced levels of UFO1 mRNA in these samples (cf. Figure 7C and Figure 1A). The elevation of HSP12 mRNA level results from increased transcription as HSP12 mRNA is slightly destablized after arsenate, whereas after H2O2, and UV treatments the stability is similar to the untreated cells with a half-life of ca. 10 minutes in both untreated and stressed cells (Figure 7D). There was no stabilization of the HSP12 mRNA after UV irradiation as observed for UFO1 mRNA (Figure 1B).


Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.

Lavut A, Raveh D - PLoS Genet. (2012)

Induction of Hsp12 protein and mRNA, and mRNA decay after stress.A. WB of protein produced from genomic HSP12-GFP in response to stress. B. UFO1 and HSP12 mRNA levels in untreated cells analyzed by qRT-PCR. mRNA levels were normalized to ACT1. C. Induction of HSP12 mRNA by stress. Wild type cells at A600 = 0.5, treated with 1 mM arsenate, 8.8 mM H2O2, irradiated with 40 mJ/cm2 UV or shifted from 30°C to 37°C for 40 minutes. Aliquots were collected at the times indicated and analyzed by qRT-PCR. D. HSP12 mRNA decay. pGAL-HSP12 was expressed in hsp12Δ mutants by overnight induction with 2% galactose. Next morning cells at A600 = 0.5 were untreated, or stressed with 1 mM arsenate or 8.8 mM H2O2 for 30 minutes, or irradiated with 40 mJ/cm2 UV. The cells were washed and transferred to SC medium with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to ACT1 and to time 0 (untreated log cells).
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pgen-1002527-g007: Induction of Hsp12 protein and mRNA, and mRNA decay after stress.A. WB of protein produced from genomic HSP12-GFP in response to stress. B. UFO1 and HSP12 mRNA levels in untreated cells analyzed by qRT-PCR. mRNA levels were normalized to ACT1. C. Induction of HSP12 mRNA by stress. Wild type cells at A600 = 0.5, treated with 1 mM arsenate, 8.8 mM H2O2, irradiated with 40 mJ/cm2 UV or shifted from 30°C to 37°C for 40 minutes. Aliquots were collected at the times indicated and analyzed by qRT-PCR. D. HSP12 mRNA decay. pGAL-HSP12 was expressed in hsp12Δ mutants by overnight induction with 2% galactose. Next morning cells at A600 = 0.5 were untreated, or stressed with 1 mM arsenate or 8.8 mM H2O2 for 30 minutes, or irradiated with 40 mJ/cm2 UV. The cells were washed and transferred to SC medium with 4% glucose. Samples were collected immediately after addition of glucose and at the times indicated and analyzed by qRT-PCR. mRNA levels were normalized to ACT1 and to time 0 (untreated log cells).
Mentions: Both whole-genome microarray experiments [7], [8] and our qRT-PCR data (Figure 1) indicate that UFO1 steady state mRNA levels are elevated in response to stress. However, it is only by using 10-fold the amount of cells compared with our standard protocols that we are able to detect Ufo1GFP protein after any of the stresses applied. We therefore examined the fate of the mRNA of HSP12, the protein of which is highly expressed in response to stress [40]. We treated cells with arsenate, H2O2, UV, glucose deprivation, 37°C or NaCl and analyzed induction of the protein by Western blotting. Hsp12GFP protein was induced after all the treatments, particularly after 37°C (Figure 7A and Figure S2). To examine mRNA localization we fused the MS2L tag to the HSP12 ORF and treated the cells with the same stresses. In contrast to UFO1-MS2L, we did not detect granules in HSP12-MS2L cells and the CPGFP signal remained diffuse throughout the cytoplasm (Figure S3 and [50]). The basal level of HSP12 mRNA in untreated cells was higher than that of UFO1 (Figure 7B). however, the relative increase of HSP12 steady state mRNA level after stress was lower than the high induced levels of UFO1 mRNA in these samples (cf. Figure 7C and Figure 1A). The elevation of HSP12 mRNA level results from increased transcription as HSP12 mRNA is slightly destablized after arsenate, whereas after H2O2, and UV treatments the stability is similar to the untreated cells with a half-life of ca. 10 minutes in both untreated and stressed cells (Figure 7D). There was no stabilization of the HSP12 mRNA after UV irradiation as observed for UFO1 mRNA (Figure 1B).

Bottom Line: However, not all the induced genes undergo translation, and mutants of many induced genes do not show elevated sensitivity to the particular stress.These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses.Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beersheba, Israel.

ABSTRACT
Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress.

Show MeSH
Related in: MedlinePlus