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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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SG phenotypes of individual mutants are mostly general for different stress types.The indicated mutant cells were exposed to the following stress conditions: 400 mM 2-DG (90 min); 44°C (45 min); 1.5 M KCl (90 min); 1.5 M NaCl (90 min). SG phenotype is shown as the deviation from wt level (his3Δ) scaled as in Fig. 1 C. Error bars indicate standard error for 25 independent determinations (about 80 cells per image) for each data point.
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pgen-1004763-g002: SG phenotypes of individual mutants are mostly general for different stress types.The indicated mutant cells were exposed to the following stress conditions: 400 mM 2-DG (90 min); 44°C (45 min); 1.5 M KCl (90 min); 1.5 M NaCl (90 min). SG phenotype is shown as the deviation from wt level (his3Δ) scaled as in Fig. 1 C. Error bars indicate standard error for 25 independent determinations (about 80 cells per image) for each data point.

Mentions: It is known that defects in a particular stress signaling pathway can affect SG formation ability specifically for that stress condition [24] whereas other mutations cause a general SG deficiency. We investigated the SG phenotype of the 194 mutants using other stress conditions: heat shock (44°C for 45 min) or hyperosmosis (1.5 M KCl or 1.5 M NaCl for 90 min). As seen in Fig. 2 and Fig. S4, some of the mutants display a strong phenotype uniformly across all the investigated stress conditions (e.g. tif4632Δ, arc1Δ, set3Δ, top3Δ, mft1Δ, ypr172wΔ, and ski3Δ). For tif4632Δ (lacking the gene encoding eIF4G2, one of two isoforms of the translation initiation factor eIF4G in yeast), a defect has previously been observed in glucose starvation [11]. However, a substantial fraction were specific to either 2-DG or to various combinations of the stress conditions (e.g. gtr2Δ, ncs6Δ, and hgh1Δ) (Fig. 2, Fig. S4). Among the set of 73 mutants with verified phenotypes in 2-DG, 10 showed defects under all four stress conditions, and a further 10 under at least three of the four conditions. Mutants with a strong phenotype in 2-DG were more likely to display phenotypes also in other stress conditions (Table S1). Over half of the mutants, 40, were unique to 2-DG. The greatest degree of overlap with the 2-DG SG phenotype was found with NaCl stress (24 mutants); all the mutants that displayed a defect in KCl were also defective in NaCl (Fig. S4).


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)

SG phenotypes of individual mutants are mostly general for different stress types.The indicated mutant cells were exposed to the following stress conditions: 400 mM 2-DG (90 min); 44°C (45 min); 1.5 M KCl (90 min); 1.5 M NaCl (90 min). SG phenotype is shown as the deviation from wt level (his3Δ) scaled as in Fig. 1 C. Error bars indicate standard error for 25 independent determinations (about 80 cells per image) for each data point.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004763-g002: SG phenotypes of individual mutants are mostly general for different stress types.The indicated mutant cells were exposed to the following stress conditions: 400 mM 2-DG (90 min); 44°C (45 min); 1.5 M KCl (90 min); 1.5 M NaCl (90 min). SG phenotype is shown as the deviation from wt level (his3Δ) scaled as in Fig. 1 C. Error bars indicate standard error for 25 independent determinations (about 80 cells per image) for each data point.
Mentions: It is known that defects in a particular stress signaling pathway can affect SG formation ability specifically for that stress condition [24] whereas other mutations cause a general SG deficiency. We investigated the SG phenotype of the 194 mutants using other stress conditions: heat shock (44°C for 45 min) or hyperosmosis (1.5 M KCl or 1.5 M NaCl for 90 min). As seen in Fig. 2 and Fig. S4, some of the mutants display a strong phenotype uniformly across all the investigated stress conditions (e.g. tif4632Δ, arc1Δ, set3Δ, top3Δ, mft1Δ, ypr172wΔ, and ski3Δ). For tif4632Δ (lacking the gene encoding eIF4G2, one of two isoforms of the translation initiation factor eIF4G in yeast), a defect has previously been observed in glucose starvation [11]. However, a substantial fraction were specific to either 2-DG or to various combinations of the stress conditions (e.g. gtr2Δ, ncs6Δ, and hgh1Δ) (Fig. 2, Fig. S4). Among the set of 73 mutants with verified phenotypes in 2-DG, 10 showed defects under all four stress conditions, and a further 10 under at least three of the four conditions. Mutants with a strong phenotype in 2-DG were more likely to display phenotypes also in other stress conditions (Table S1). Over half of the mutants, 40, were unique to 2-DG. The greatest degree of overlap with the 2-DG SG phenotype was found with NaCl stress (24 mutants); all the mutants that displayed a defect in KCl were also defective in NaCl (Fig. S4).

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