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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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Overview of screening results.A) Network analysis of SG-defective mutants. Mutants showing SG formation defect (nodes linked with brown arrows) were grouped into modules based on their known physical interactions (blue arrows) and published cellular components information. Functional groups and cellular components are shown in different colored nodes. Blue circles indicate subunits or complexes within groups. B) Functional enrichment analysis of SG-defective mutants. Confirmed SG-defective mutants were analyzed for enrichment of the GO biological process, function and component categories. Enriched groups were scored by comparing to a background list of SGA-V2+slower array (4691 genes) using a cut-off of P<0.05. C) Distribution of SG phenotypes among cytoplasmic ribosomal protein mutants. D,E) Formation of PBs is undisturbed in the SG-defective mutants studied after addition of 400 mM 2-DG in wt and mutant cells. Data represent mean ± standard error of measurement of three independent determinations. D) Micrographs of mutants expressing both Dcp2-GFP (PB marker) and Pab1-RFP (SG marker) E) Time course of appearance of SGs and PBs.
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pgen-1004763-g003: Overview of screening results.A) Network analysis of SG-defective mutants. Mutants showing SG formation defect (nodes linked with brown arrows) were grouped into modules based on their known physical interactions (blue arrows) and published cellular components information. Functional groups and cellular components are shown in different colored nodes. Blue circles indicate subunits or complexes within groups. B) Functional enrichment analysis of SG-defective mutants. Confirmed SG-defective mutants were analyzed for enrichment of the GO biological process, function and component categories. Enriched groups were scored by comparing to a background list of SGA-V2+slower array (4691 genes) using a cut-off of P<0.05. C) Distribution of SG phenotypes among cytoplasmic ribosomal protein mutants. D,E) Formation of PBs is undisturbed in the SG-defective mutants studied after addition of 400 mM 2-DG in wt and mutant cells. Data represent mean ± standard error of measurement of three independent determinations. D) Micrographs of mutants expressing both Dcp2-GFP (PB marker) and Pab1-RFP (SG marker) E) Time course of appearance of SGs and PBs.

Mentions: A survey of this group of mutants with a verified decrease in SG number reveals a heterogeneous composition of biochemical and cellular functions, a reflection of the complexity of the functions required to assemble SGs, and to regulate their formation under different stress conditions. The network overview of functional clusters in this group (Fig. 3 A) displays large groups related to protein translation and endosomal vesicles, and the interrelated groups of the EGO/GSE complex and the Ran GTPase (Gsp1) with regulators.


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)

Overview of screening results.A) Network analysis of SG-defective mutants. Mutants showing SG formation defect (nodes linked with brown arrows) were grouped into modules based on their known physical interactions (blue arrows) and published cellular components information. Functional groups and cellular components are shown in different colored nodes. Blue circles indicate subunits or complexes within groups. B) Functional enrichment analysis of SG-defective mutants. Confirmed SG-defective mutants were analyzed for enrichment of the GO biological process, function and component categories. Enriched groups were scored by comparing to a background list of SGA-V2+slower array (4691 genes) using a cut-off of P<0.05. C) Distribution of SG phenotypes among cytoplasmic ribosomal protein mutants. D,E) Formation of PBs is undisturbed in the SG-defective mutants studied after addition of 400 mM 2-DG in wt and mutant cells. Data represent mean ± standard error of measurement of three independent determinations. D) Micrographs of mutants expressing both Dcp2-GFP (PB marker) and Pab1-RFP (SG marker) E) Time course of appearance of SGs and PBs.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004763-g003: Overview of screening results.A) Network analysis of SG-defective mutants. Mutants showing SG formation defect (nodes linked with brown arrows) were grouped into modules based on their known physical interactions (blue arrows) and published cellular components information. Functional groups and cellular components are shown in different colored nodes. Blue circles indicate subunits or complexes within groups. B) Functional enrichment analysis of SG-defective mutants. Confirmed SG-defective mutants were analyzed for enrichment of the GO biological process, function and component categories. Enriched groups were scored by comparing to a background list of SGA-V2+slower array (4691 genes) using a cut-off of P<0.05. C) Distribution of SG phenotypes among cytoplasmic ribosomal protein mutants. D,E) Formation of PBs is undisturbed in the SG-defective mutants studied after addition of 400 mM 2-DG in wt and mutant cells. Data represent mean ± standard error of measurement of three independent determinations. D) Micrographs of mutants expressing both Dcp2-GFP (PB marker) and Pab1-RFP (SG marker) E) Time course of appearance of SGs and PBs.
Mentions: A survey of this group of mutants with a verified decrease in SG number reveals a heterogeneous composition of biochemical and cellular functions, a reflection of the complexity of the functions required to assemble SGs, and to regulate their formation under different stress conditions. The network overview of functional clusters in this group (Fig. 3 A) displays large groups related to protein translation and endosomal vesicles, and the interrelated groups of the EGO/GSE complex and the Ran GTPase (Gsp1) with regulators.

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