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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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ASH1 and OXA1 mRNA granules interact with PBs.A. ASH1-MS2L cells at A600 = 0.5 with pCP-MS2L-GFPx3 and the PB marker, Edc3mCherry, either untreated, treated with 1 mM arsenate or with 8.8 mM H2O2 for 30 minutes or transferred to SC without glucose for 30 minutes. Merge ×5 represents 5 times enlargement of selected granules indicated with white arrows in the whole cells. B. OXA1-MS2L cells treated as in A., and visualized by confocal microscopy. C. Histograms of ASH1-MS2L cells or D. OXA1-MS2L cells, showing percentages of overlapping, docked, or distinct granule types in a population of cells untreated, treated with 1 mM arsenate or with 8.8 mM H2O2 for 30 minutes or stressed in SC without glucose (n = >100 cells).
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pgen-1002527-g006: ASH1 and OXA1 mRNA granules interact with PBs.A. ASH1-MS2L cells at A600 = 0.5 with pCP-MS2L-GFPx3 and the PB marker, Edc3mCherry, either untreated, treated with 1 mM arsenate or with 8.8 mM H2O2 for 30 minutes or transferred to SC without glucose for 30 minutes. Merge ×5 represents 5 times enlargement of selected granules indicated with white arrows in the whole cells. B. OXA1-MS2L cells treated as in A., and visualized by confocal microscopy. C. Histograms of ASH1-MS2L cells or D. OXA1-MS2L cells, showing percentages of overlapping, docked, or distinct granule types in a population of cells untreated, treated with 1 mM arsenate or with 8.8 mM H2O2 for 30 minutes or stressed in SC without glucose (n = >100 cells).

Mentions: To determine whether all mRNA granules correspond to PBs we assayed two further mRNAs that are specifically localized prior to their translation: ASH1-MS2L mRNA that is transported from the mother cell to the bud [42], and OXA1-MS2L mRNA that is localized to the mitochondria [43]. The PB marker Edc3mCherry was produced in cells that expressed ASH1-MS2L or OXA1-MS2L together with pCP-MS2L-GFPx3 for detection of their mRNAs. The cells were treated with the above stresses and imaged after 30 minutes with the confocal microscope. Both ASH1-MS2L and OXA1-MS2L mRNAs showed a similar response to the stress treatments. A small number of untreated cells showed at most a few Edc3mCherry-marked PBs, however, most cells lacked CPGFP granules of either ASH1-MS2L or OXA1-MS2L mRNAs. After 30 minutes with arsenate, H2O2, or glucose deprivation, we observed CPGFP granules corresponding to ASH1-MS2L (Figure 6A) or OXA1-MS2L (Figure 6B) mRNAs. These granules did not fully colocalize with PBs as did UFO1 mRNA, however, they were often docked, partially colocalized (overlapping), or appeared in the same cells with the PB marker protein Edc3mCherry, but at a distinct location in the same cell (Figure 6C).


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

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

ASH1 and OXA1 mRNA granules interact with PBs.A. ASH1-MS2L cells at A600 = 0.5 with pCP-MS2L-GFPx3 and the PB marker, Edc3mCherry, either untreated, treated with 1 mM arsenate or with 8.8 mM H2O2 for 30 minutes or transferred to SC without glucose for 30 minutes. Merge ×5 represents 5 times enlargement of selected granules indicated with white arrows in the whole cells. B. OXA1-MS2L cells treated as in A., and visualized by confocal microscopy. C. Histograms of ASH1-MS2L cells or D. OXA1-MS2L cells, showing percentages of overlapping, docked, or distinct granule types in a population of cells untreated, treated with 1 mM arsenate or with 8.8 mM H2O2 for 30 minutes or stressed in SC without glucose (n = >100 cells).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pgen-1002527-g006: ASH1 and OXA1 mRNA granules interact with PBs.A. ASH1-MS2L cells at A600 = 0.5 with pCP-MS2L-GFPx3 and the PB marker, Edc3mCherry, either untreated, treated with 1 mM arsenate or with 8.8 mM H2O2 for 30 minutes or transferred to SC without glucose for 30 minutes. Merge ×5 represents 5 times enlargement of selected granules indicated with white arrows in the whole cells. B. OXA1-MS2L cells treated as in A., and visualized by confocal microscopy. C. Histograms of ASH1-MS2L cells or D. OXA1-MS2L cells, showing percentages of overlapping, docked, or distinct granule types in a population of cells untreated, treated with 1 mM arsenate or with 8.8 mM H2O2 for 30 minutes or stressed in SC without glucose (n = >100 cells).
Mentions: To determine whether all mRNA granules correspond to PBs we assayed two further mRNAs that are specifically localized prior to their translation: ASH1-MS2L mRNA that is transported from the mother cell to the bud [42], and OXA1-MS2L mRNA that is localized to the mitochondria [43]. The PB marker Edc3mCherry was produced in cells that expressed ASH1-MS2L or OXA1-MS2L together with pCP-MS2L-GFPx3 for detection of their mRNAs. The cells were treated with the above stresses and imaged after 30 minutes with the confocal microscope. Both ASH1-MS2L and OXA1-MS2L mRNAs showed a similar response to the stress treatments. A small number of untreated cells showed at most a few Edc3mCherry-marked PBs, however, most cells lacked CPGFP granules of either ASH1-MS2L or OXA1-MS2L mRNAs. After 30 minutes with arsenate, H2O2, or glucose deprivation, we observed CPGFP granules corresponding to ASH1-MS2L (Figure 6A) or OXA1-MS2L (Figure 6B) mRNAs. These granules did not fully colocalize with PBs as did UFO1 mRNA, however, they were often docked, partially colocalized (overlapping), or appeared in the same cells with the PB marker protein Edc3mCherry, but at a distinct location in the same cell (Figure 6C).

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