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, 15 (10), 1814-21

Metazoan Stress Granule Assembly Is Mediated by P-eIF2alpha-dependent and -Independent Mechanisms

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Metazoan Stress Granule Assembly Is Mediated by P-eIF2alpha-dependent and -Independent Mechanisms

Natalie G Farny et al. RNA.

Abstract

Stress granules (SGs) are cytoplasmic bodies wherein translationally silenced mRNAs are recruited for triage in response to environmental stress. We report that Drosophila cells form SGs in response to arsenite and heat shock. Drosophila SGs, like mammalian SGs, are distinct from but adjacent to processing bodies (PBs, sites of mRNA silencing and decay), require polysome disassembly, and are in dynamic equilibrium with polysomes. We further examine the role of the two Drosophila eIF2alpha kinases, PEK and GCN2, in regulating SG formation in response to heat and arsenite stress. While arsenite-induced SGs are dependent upon eIF2alpha phosphorylation, primarily via PEK, heat-induced SGs are phospho-eIF2alpha-independent. In contrast, heat-induced SGs require eIF2alpha phosphorylation in mammalian cells, as non-phosphorylatable eIF2alpha Ser51Ala mutant murine embryonic fibroblasts do not form SGs even after severe heat shock. These results suggest that mammals evolved alternative mechanisms for dealing with thermal stress.

Figures

FIGURE 1.
FIGURE 1.
Arsenite or heat shock causes the formation of reversible, cytoplasmic, poly(A)+ RNA granules in Drosophila cells. (A) 500 μM arsenite was applied to S2R+ cells for times indicated, or applied for 2 h then removed and recovered for times indicated, then cells were fixed and hybridized with Cy3-Oligo-dT(30) to visualize poly(A)+ RNA. (B) S2R+ cells were incubated at the indicated temperatures for 1 h, or incubated for 1 h followed by 2 h of recovery, then processed for RNA visualization as in A. Bars, 5 μm. (C,D) Western blots of arsenite (C) and heat shock (D) time course samples, parallel to those shown in A and B, respectively. P-2α/total is the ratio of the density of the P-eIF2α bands divided by total eIF2α bands, normalized to the control lane. (E) S2R+ cells were heat-shocked as indicated, and then processed for Western blot. (F) S2R+ cells were pre-incubated with MG132 (MG) at 100 μM for 3 h, or 75 nm of okadaic acid (OA) for 3 h, then heat-shocked for 1 h and processed for Western blot. (G) A replicate of the −/+ MG132, 25°C samples from panel F (lanes 1,2) was processed for Western blot and probed with anti-ubiquitin antibody.
FIGURE 2.
FIGURE 2.
Stress-induced, poly(A)+ RNA granules co-localize with markers of SGs. (A) S2R+ cells were stressed with 500 μm arsenite for 2 h, or heat shock at 40°C for 1 h, or left untreated, then fixed and hybridized with Cy3-Oligo-dT(30) (red, center panels). Then, cells were co-stained with antibodies to FMR1 (green, left panels). (B) After stress treatment as in A, cells were fixed and co-stained with antibodies to FMR1 (green, left panels) and Dcp1 (red, center panels). White boxes indicate regions enlarged at right. (C,E) Control or stressed cells were stained with antibodies to RPL P0 (C, red) or eIF4E (E, red) and co-stained with anti-FMR1 (green). (D) Control or stressed cells were hybridized with a Cy3-labeled probe specific for 18S rRNA and co-stained with anti-FMR1. A second, nonoverlapping probe yielded identical results (data not shown). White boxes in arsenite and heat shock “merge” panels of C and D indicate areas enlarged in the lower right hand corners of these panels. (F–H) S2R+ cells were transfected with V5-eIF3 S8 (F), GFP-PABP (G), or GFP-Rox8 (H), and co-stained with anti-FMR1 (red). Bars, 5 μm.
FIGURE 3.
FIGURE 3.
Polysome disassembly is required for the formation of Drosophila SGs. (A,B,E,F) Sucrose gradient analysis of polysomes. S2R+ cells were untreated (A,E, black traces) or treated with 500 μM arsenite (A, gray trace) or heat shock at 40°C for 1 h (E, gray trace). Where indicated, cells were treated with CHX (50 μg/mL) for 3 h (B,F, black traces) or pretreated with CHX for 2 h followed by 1 h of arsenite (B, gray trace) or heat shock at 40°C (F, gray trace). (C,G) Parallel samples to those in A–B and E–F were hybridized with Cy3-Oligo-dT(30) to visualize SGs. Bars, 5 μm. (D,H) Western blots of parallel samples to those in C and G. Bands shown are from the same film and exposure for each antibody; Intermediate lanes containing irrelevant samples were removed for simplicity.
FIGURE 4.
FIGURE 4.
P-eIF2α is required for heat shock SGs in mammals, but not in flies. (A) S2R+ cells were treated with indicated dsRNAs for 96 h, followed by 500 μm arsenite for 2 h or 40°C heat shock for 1 h, then hybridized with Cy3-Oligo-dT(30). Samples were blinded and photographed, and cells in each image were scored for the presence of SGs. (B) Western blot of parallel samples to the data shown in A. (C) Quantitative real-time PCR analysis of PEK and GCN2 mRNA levels in knockdown cells. Error bars in A and C represent one standard deviation from the average of three independent experiments. (D) eIF2α Ser51Ala MEFs (denoted “M”) were co-plated with human-derived U2OS cells (denoted “H”), then either exposed to heat shock at 48°C for 30 min (top panels), pre-treated with CHX at 50 μg/mL for 30 min, and then heat-shocked (center panels), or treated with 50 μM pateamine A (patA) for 1 h (bottom panels). Samples were fixed and stained with anti-eIF3 (green), anti-eIF4E (red), and Hoechst (DNA, blue). Mouse cells can be distinguished from human cells by the distinct nuclear speckling pattern in DNA panels. Bar, 25 μm. (E) A model of eIF2α-dependent and -independent translational arrest and SG formation in Drosophila and mammals. In Drosophila, arsenite results in the phosphorylation of eIF2α via the kinase PEK, whereas in mammals the critical arsenite responsive kinase is HRI. In both cases, P-eIF2α inhibits translation initiation, and the initiation complex, associated mRNAs and RNA binding proteins are recruited to SGs. However, the formation of Drosophila heat shock SGs does not depend on P-eIF2α, whereas P-eIF2α is required for mammalian heat shock SGs. The mammalian eIF2α kinase(s) activated by heat shock is unknown.

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