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Stress granule-defective mutants deregulate stress responsive transcripts.

Yang X, Shen Y, Garre E, Hao X, Krumlinde D, Cvijović M, Arens C, Nyström T, Liu B, Sunnerhagen P - PLoS Genet. (2014)

Bottom Line: We found several mutations affecting the Ran GTPase, regulating nucleocytoplasmic transport of RNA and proteins, to confer SG defects.Unexpectedly, we found stress-regulated transcripts to reach more extreme levels in mutants unable to form SGs: stress-induced mRNAs accumulate to higher levels than in the wild-type, whereas stress-repressed mRNAs are reduced further in such mutants.The absence of SGs thus leads the cell to excessive, and potentially deleterious, reactions to stress.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Engineering, Harbin Institute of Technology, Harbin, China.

ABSTRACT
To reduce expression of gene products not required under stress conditions, eukaryotic cells form large and complex cytoplasmic aggregates of RNA and proteins (stress granules; SGs), where transcripts are kept translationally inert. The overall composition of SGs, as well as their assembly requirements and regulation through stress-activated signaling pathways remain largely unknown. We have performed a genome-wide screen of S. cerevisiae gene deletion mutants for defects in SG formation upon glucose starvation stress. The screen revealed numerous genes not previously implicated in SG formation. Most mutants with strong phenotypes are equally SG defective when challenged with other stresses, but a considerable fraction is stress-specific. Proteins associated with SG defects are enriched in low-complexity regions, indicating that multiple weak macromolecule interactions are responsible for the structural integrity of SGs. Certain SG-defective mutants, but not all, display an enhanced heat-induced mutation rate. We found several mutations affecting the Ran GTPase, regulating nucleocytoplasmic transport of RNA and proteins, to confer SG defects. Unexpectedly, we found stress-regulated transcripts to reach more extreme levels in mutants unable to form SGs: stress-induced mRNAs accumulate to higher levels than in the wild-type, whereas stress-repressed mRNAs are reduced further in such mutants. Our findings are consistent with the view that, not only are SGs being regulated by stress signaling pathways, but SGs also modulate the extent of stress responses. We speculate that nucleocytoplasmic shuttling of RNA-binding proteins is required for gene expression regulation during stress, and that SGs modulate this traffic. The absence of SGs thus leads the cell to excessive, and potentially deleterious, reactions to stress.

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Stress-induced mutation rate in strains with SG defects.The indicated mutant strains, selected for having strongly reduced ability to accumulate SGs (Fig. 2, Fig. S4) were shocked by either heat or NaCl. The frequency of CanR cells was recorded thereafter as detailed in Materials and Methods, and compared to the mutation frequency of untreated cells of the same strain. A) Mutation rate after heat shock (44°C for 1 h). Statistically significant increases in heat-induced mutation rate in mutants compared to wt are marked with the respective numbers of stars. P values are listed in Table S3. B) Mutation rate after NaCl shock (1.5 M for 1 h). Data are mean ± standard error of measurement of three independent determinations.
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pgen-1004763-g004: Stress-induced mutation rate in strains with SG defects.The indicated mutant strains, selected for having strongly reduced ability to accumulate SGs (Fig. 2, Fig. S4) were shocked by either heat or NaCl. The frequency of CanR cells was recorded thereafter as detailed in Materials and Methods, and compared to the mutation frequency of untreated cells of the same strain. A) Mutation rate after heat shock (44°C for 1 h). Statistically significant increases in heat-induced mutation rate in mutants compared to wt are marked with the respective numbers of stars. P values are listed in Table S3. B) Mutation rate after NaCl shock (1.5 M for 1 h). Data are mean ± standard error of measurement of three independent determinations.

Mentions: Heat stress increases mutation frequency in budding yeast, which has been suggested to result from increased ROS production in mitochondria [36]. This mutation peak can be suppressed by overexpression of Pbp1 or Dhh1, both of which cause increased SG formation [15]. The authors concluded that SGs formed under stress serve as a dampening factor of some aspects of the heat-induced stress response, which attenuate the mutagenic effects of the heat shock. We were interested to see if the converse was true, that the absence of SGs under stress per se would enhance the mutation rate. We also wanted to investigate the generality of this phenomenon, and so recorded the frequency of inactivating mutations in the CAN1 gene, after heat shock (60 min at 44°C, as applied by Takahara, 2012 [15]) for 12 mutants with strongly negative SG phenotypes upon heat shock (Fig. 2, Fig. S4) but otherwise without any obvious functional bias. As seen in Fig. 4 A, indeed a marked increase in canavanine resistant (canR) clones (3–15 fold) was observed for four of the mutants; top3Δ, set3Δ, ski3Δ and gtr1Δ. However, for the remaining 8 mutants, no significant increased mutation frequency was found; thus, an increased heat-induced mutation rate does not occur in all SG-defective mutants. We wanted to see if other stress types normally causing SG formation would similarly lead to an elevated mutation frequency in SG defective mutants. Thus, we examined top3Δ and vta1Δ mutants for the frequency of canR clones after hyperosmotic shock (1.5 M NaCl). Both mutants are unable to form SGs under these conditions (Fig. 2). However, no increased mutation frequency was observed neither in the wt nor in either mutant (Fig. 4 B), indicating that the stress-induced mutability may be specific to heat shock.


Stress granule-defective mutants deregulate stress responsive transcripts.

Yang X, Shen Y, Garre E, Hao X, Krumlinde D, Cvijović M, Arens C, Nyström T, Liu B, Sunnerhagen P - PLoS Genet. (2014)

Stress-induced mutation rate in strains with SG defects.The indicated mutant strains, selected for having strongly reduced ability to accumulate SGs (Fig. 2, Fig. S4) were shocked by either heat or NaCl. The frequency of CanR cells was recorded thereafter as detailed in Materials and Methods, and compared to the mutation frequency of untreated cells of the same strain. A) Mutation rate after heat shock (44°C for 1 h). Statistically significant increases in heat-induced mutation rate in mutants compared to wt are marked with the respective numbers of stars. P values are listed in Table S3. B) Mutation rate after NaCl shock (1.5 M for 1 h). Data are mean ± standard error of measurement of three independent determinations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222700&req=5

pgen-1004763-g004: Stress-induced mutation rate in strains with SG defects.The indicated mutant strains, selected for having strongly reduced ability to accumulate SGs (Fig. 2, Fig. S4) were shocked by either heat or NaCl. The frequency of CanR cells was recorded thereafter as detailed in Materials and Methods, and compared to the mutation frequency of untreated cells of the same strain. A) Mutation rate after heat shock (44°C for 1 h). Statistically significant increases in heat-induced mutation rate in mutants compared to wt are marked with the respective numbers of stars. P values are listed in Table S3. B) Mutation rate after NaCl shock (1.5 M for 1 h). Data are mean ± standard error of measurement of three independent determinations.
Mentions: Heat stress increases mutation frequency in budding yeast, which has been suggested to result from increased ROS production in mitochondria [36]. This mutation peak can be suppressed by overexpression of Pbp1 or Dhh1, both of which cause increased SG formation [15]. The authors concluded that SGs formed under stress serve as a dampening factor of some aspects of the heat-induced stress response, which attenuate the mutagenic effects of the heat shock. We were interested to see if the converse was true, that the absence of SGs under stress per se would enhance the mutation rate. We also wanted to investigate the generality of this phenomenon, and so recorded the frequency of inactivating mutations in the CAN1 gene, after heat shock (60 min at 44°C, as applied by Takahara, 2012 [15]) for 12 mutants with strongly negative SG phenotypes upon heat shock (Fig. 2, Fig. S4) but otherwise without any obvious functional bias. As seen in Fig. 4 A, indeed a marked increase in canavanine resistant (canR) clones (3–15 fold) was observed for four of the mutants; top3Δ, set3Δ, ski3Δ and gtr1Δ. However, for the remaining 8 mutants, no significant increased mutation frequency was found; thus, an increased heat-induced mutation rate does not occur in all SG-defective mutants. We wanted to see if other stress types normally causing SG formation would similarly lead to an elevated mutation frequency in SG defective mutants. Thus, we examined top3Δ and vta1Δ mutants for the frequency of canR clones after hyperosmotic shock (1.5 M NaCl). Both mutants are unable to form SGs under these conditions (Fig. 2). However, no increased mutation frequency was observed neither in the wt nor in either mutant (Fig. 4 B), indicating that the stress-induced mutability may be specific to heat shock.

Bottom Line: We found several mutations affecting the Ran GTPase, regulating nucleocytoplasmic transport of RNA and proteins, to confer SG defects.Unexpectedly, we found stress-regulated transcripts to reach more extreme levels in mutants unable to form SGs: stress-induced mRNAs accumulate to higher levels than in the wild-type, whereas stress-repressed mRNAs are reduced further in such mutants.The absence of SGs thus leads the cell to excessive, and potentially deleterious, reactions to stress.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Engineering, Harbin Institute of Technology, Harbin, China.

ABSTRACT
To reduce expression of gene products not required under stress conditions, eukaryotic cells form large and complex cytoplasmic aggregates of RNA and proteins (stress granules; SGs), where transcripts are kept translationally inert. The overall composition of SGs, as well as their assembly requirements and regulation through stress-activated signaling pathways remain largely unknown. We have performed a genome-wide screen of S. cerevisiae gene deletion mutants for defects in SG formation upon glucose starvation stress. The screen revealed numerous genes not previously implicated in SG formation. Most mutants with strong phenotypes are equally SG defective when challenged with other stresses, but a considerable fraction is stress-specific. Proteins associated with SG defects are enriched in low-complexity regions, indicating that multiple weak macromolecule interactions are responsible for the structural integrity of SGs. Certain SG-defective mutants, but not all, display an enhanced heat-induced mutation rate. We found several mutations affecting the Ran GTPase, regulating nucleocytoplasmic transport of RNA and proteins, to confer SG defects. Unexpectedly, we found stress-regulated transcripts to reach more extreme levels in mutants unable to form SGs: stress-induced mRNAs accumulate to higher levels than in the wild-type, whereas stress-repressed mRNAs are reduced further in such mutants. Our findings are consistent with the view that, not only are SGs being regulated by stress signaling pathways, but SGs also modulate the extent of stress responses. We speculate that nucleocytoplasmic shuttling of RNA-binding proteins is required for gene expression regulation during stress, and that SGs modulate this traffic. The absence of SGs thus leads the cell to excessive, and potentially deleterious, reactions to stress.

Show MeSH
Related in: MedlinePlus