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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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Mutants defective in control of the Ran GTPase homolog have a deregulated SG stress response.A) Schematic representation of the relations between the proteins concerned. B–I: SG phenotype was recorded after 90 min in 400 mM 2-DG. Numbers above bars in diagrams indicate P-values for a change relative to the wt phenotype. B,C) Mutations in the Ran homolog (gsp1-P162I) and its GTPase activating protein Rna1 (rna1-1, rna1-S116P) confer strong SG defects. D,E) Different mutant alleles of the Gsp1 GDP/GTP exchange factor Srm1 (srm1-ts, srm1-G282S) have opposing effects on SG formation. F,G) Increased SRM1 dosage enhances SG accumulation after challenge with 400 mM 2-DG. H,I) SG phenotypes of other mutants (slm4Δ, ltv1Δ) functionally linked to the EGO/GSE complex and to gtr1/gtr2 but not to RanGDP/GTP function. In A, C, and F, data are mean ± standard error of measurement of three independent determinations; in E, five independent determinations.
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pgen-1004763-g005: Mutants defective in control of the Ran GTPase homolog have a deregulated SG stress response.A) Schematic representation of the relations between the proteins concerned. B–I: SG phenotype was recorded after 90 min in 400 mM 2-DG. Numbers above bars in diagrams indicate P-values for a change relative to the wt phenotype. B,C) Mutations in the Ran homolog (gsp1-P162I) and its GTPase activating protein Rna1 (rna1-1, rna1-S116P) confer strong SG defects. D,E) Different mutant alleles of the Gsp1 GDP/GTP exchange factor Srm1 (srm1-ts, srm1-G282S) have opposing effects on SG formation. F,G) Increased SRM1 dosage enhances SG accumulation after challenge with 400 mM 2-DG. H,I) SG phenotypes of other mutants (slm4Δ, ltv1Δ) functionally linked to the EGO/GSE complex and to gtr1/gtr2 but not to RanGDP/GTP function. In A, C, and F, data are mean ± standard error of measurement of three independent determinations; in E, five independent determinations.

Mentions: Gtr1 and Gtr2 have been implicated both in activation of TORC1 as members of the GSE complex [37], and as regulators of the budding yeast Ran homolog, Gsp1, required for a large fraction of all nucleocytoplasmic transport of protein and RNA in the cell. We wanted to examine if the SG defects of gtr1Δ and gtr2Δ mutants were due to interactions with the TOR pathway, with Ran functions, or both. As many of the Ran function genes are essential (GSP1, RNA1, SRM1), we resorted to temperature-sensitive (ts) alleles of those [38]. Cells carrying the gsp1-P162I allele are highly defective in SG formation in glucose starvation at the restrictive temperature (36°C) (Fig. 5 B). RNA1 encodes the GTPase-activating protein for Gsp1 (RanGAP). The rna1-1 allele likewise confers decreased SG numbers; the rna1-S116P only moderately so (Fig. 5 B). Srm1 (Prp20) encodes the guanine nucleotide exchange factor (GEF) of Gsp1 (RCC1 in human cells). In glucose starvation, the srm1-G282S allele instead causes a marked increase of the SG-positive fraction, to 90% of all cells, whereas the srm1-ts allele gives lower SG numbers (Fig. 5 D). These results show that those mutants with a functional connection both with Gtr1 and Gtr2 and with the Ran nucleocytoplasmic transport system, are also affected in their SG forming ability. The other EGO/GSE mutants, slm4Δ and ltv1Δ, also did display reduced SG numbers, though with weaker phenotypes (Fig. 5 H,I).


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)

Mutants defective in control of the Ran GTPase homolog have a deregulated SG stress response.A) Schematic representation of the relations between the proteins concerned. B–I: SG phenotype was recorded after 90 min in 400 mM 2-DG. Numbers above bars in diagrams indicate P-values for a change relative to the wt phenotype. B,C) Mutations in the Ran homolog (gsp1-P162I) and its GTPase activating protein Rna1 (rna1-1, rna1-S116P) confer strong SG defects. D,E) Different mutant alleles of the Gsp1 GDP/GTP exchange factor Srm1 (srm1-ts, srm1-G282S) have opposing effects on SG formation. F,G) Increased SRM1 dosage enhances SG accumulation after challenge with 400 mM 2-DG. H,I) SG phenotypes of other mutants (slm4Δ, ltv1Δ) functionally linked to the EGO/GSE complex and to gtr1/gtr2 but not to RanGDP/GTP function. In A, C, and F, data are mean ± standard error of measurement of three independent determinations; in E, five independent determinations.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004763-g005: Mutants defective in control of the Ran GTPase homolog have a deregulated SG stress response.A) Schematic representation of the relations between the proteins concerned. B–I: SG phenotype was recorded after 90 min in 400 mM 2-DG. Numbers above bars in diagrams indicate P-values for a change relative to the wt phenotype. B,C) Mutations in the Ran homolog (gsp1-P162I) and its GTPase activating protein Rna1 (rna1-1, rna1-S116P) confer strong SG defects. D,E) Different mutant alleles of the Gsp1 GDP/GTP exchange factor Srm1 (srm1-ts, srm1-G282S) have opposing effects on SG formation. F,G) Increased SRM1 dosage enhances SG accumulation after challenge with 400 mM 2-DG. H,I) SG phenotypes of other mutants (slm4Δ, ltv1Δ) functionally linked to the EGO/GSE complex and to gtr1/gtr2 but not to RanGDP/GTP function. In A, C, and F, data are mean ± standard error of measurement of three independent determinations; in E, five independent determinations.
Mentions: Gtr1 and Gtr2 have been implicated both in activation of TORC1 as members of the GSE complex [37], and as regulators of the budding yeast Ran homolog, Gsp1, required for a large fraction of all nucleocytoplasmic transport of protein and RNA in the cell. We wanted to examine if the SG defects of gtr1Δ and gtr2Δ mutants were due to interactions with the TOR pathway, with Ran functions, or both. As many of the Ran function genes are essential (GSP1, RNA1, SRM1), we resorted to temperature-sensitive (ts) alleles of those [38]. Cells carrying the gsp1-P162I allele are highly defective in SG formation in glucose starvation at the restrictive temperature (36°C) (Fig. 5 B). RNA1 encodes the GTPase-activating protein for Gsp1 (RanGAP). The rna1-1 allele likewise confers decreased SG numbers; the rna1-S116P only moderately so (Fig. 5 B). Srm1 (Prp20) encodes the guanine nucleotide exchange factor (GEF) of Gsp1 (RCC1 in human cells). In glucose starvation, the srm1-G282S allele instead causes a marked increase of the SG-positive fraction, to 90% of all cells, whereas the srm1-ts allele gives lower SG numbers (Fig. 5 D). These results show that those mutants with a functional connection both with Gtr1 and Gtr2 and with the Ran nucleocytoplasmic transport system, are also affected in their SG forming ability. The other EGO/GSE mutants, slm4Δ and ltv1Δ, also did display reduced SG numbers, though with weaker phenotypes (Fig. 5 H,I).

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