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. 2015 Apr 15;26(8):1476-90.
doi: 10.1091/mbc.E14-11-1523. Epub 2015 Feb 18.

Cytoplasmic hGle1A Regulates Stress Granules by Modulation of Translation

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Free PMC article

Cytoplasmic hGle1A Regulates Stress Granules by Modulation of Translation

Aditi et al. Mol Biol Cell. .
Free PMC article

Abstract

When eukaryotic cells respond to stress, gene expression pathways change to selectively export and translate subsets of mRNAs. Translationally repressed mRNAs accumulate in cytoplasmic foci known as stress granules (SGs). SGs are in dynamic equilibrium with the translational machinery, but mechanisms controlling this are unclear. Gle1 is required for DEAD-box protein function during mRNA export and translation. We document that human Gle1 (hGle1) is a critical regulator of translation during stress. hGle1 is recruited to SGs, and hGLE1 small interfering RNA-mediated knockdown perturbs SG assembly, resulting in increased numbers of smaller SGs. The rate of SG disassembly is also delayed. Furthermore, SG hGle1-depletion defects correlate with translation perturbations, and the hGle1 role in SGs is independent of mRNA export. Interestingly, we observe isoform-specific roles for hGle1 in which SG function requires hGle1A, whereas mRNA export requires hGle1B. We find that the SG defects in hGle1-depleted cells are rescued by puromycin or DDX3 expression. Together with recent links of hGLE1 mutations in amyotrophic lateral sclerosis patients, these results uncover a paradigm for hGle1A modulating the balance between translation and SGs during stress and disease.

Figures

FIGURE 1:
FIGURE 1:
hGle1A is not required for mRNA export. (A) Endogenous hGle1 protein levels are reduced upon hGLE1 depletion in HeLa cells. hGLE1 or CTRL siRNA-treated cells were transfected with indicated EGFP-tagged plasmids, and cell lysates were analyzed by immunoblotting using anti-hGle1, GFP, and actin antibodies. (B) Expression of EGFP-hGle1B but not EGFP-hGle1A rescues mRNA export defects in hGle1-depleted HeLa cells. Nuclear poly(A)+ mRNA accumulation was detected by Cy3-labeled oligo-dT in situ hybridization in the CTRL or hGLE1 siRNA-treated samples expressing either EGFP, EGFP-hGLE1AR, or EGFP-hGLE1BR plasmids. Scale bar: 10 μm. (C) Quantification of N/C ratio of poly(A)+ RNA in CTRL and hGLE1 siRNA-treated samples expressing indicated plasmids. Error bars represent mean ± 95% confidence interval from at least three independent experiments. (D) Both hGle1A and hGle1B localize to the cytoplasmic face of NPC in hGle1-depleted cells. hGLE1 or CTRL siRNA-treated HeLa cells were transfected with indicated EGFP-tagged plasmids, and cells were imaged live using superresolution structural microscopy with Pom121-mCherry marking the nuclear envelope. Scale bar: 1 μm.
FIGURE 2:
FIGURE 2:
hGle1 is recruited specifically to SGs upon heat shock. (A) Endogenous hGle1 is localized to SGs. HeLa cells were either left untreated or exposed to heat shock at 45°C for 60 min. Cells were processed for immunofluorescence using anti-hGle1 and G3BP antibodies. Scale bar: 10 μm. (B) hGle1 is not a component of P bodies. Immunofluorescence of hGle1 and P-body marker Dcp1a in HeLa cells treated with either heat shock or left untreated. The white line in A and B indicates the position of the line scan to assess colocalization in merged images using ImageJ. Arrow points toward the position of white line showing colocalization. Scale bar: 10 μm.
FIGURE 3:
FIGURE 3:
hGle1 is required for SG assembly and SG disassembly. (A) hGle1-depleted cells show SG-assembly defects. HeLa cells transfected with CTRL or hGLE1 siRNAs were subjected to heat shock at 45°C for 60 min and processed for immunofluorescence using anti-hGle1 and G3BP antibodies. hGle1-depleted cells show either increased numbers of SGs or fail to assemble SGs. Scale bar: 10 μm. (B) Expression of EGFP-hGle1A but not EGFP-hGle1B rescues SG-assembly defects in hGle1-depleted cells. CTRL or hGLE1 siRNA-treated HeLa cells were transfected with EGFP, EGFP-hGLE1AR, or EGFP-hGLE1BR plasmids, heat shocked, and processed for immunofluorescence detection of G3BP and hGle1. Scale bar: 10 μm. (C) Quantification of SG numbers in CTRL and hGLE1 siRNA cells expressing indicated plasmids. (D, E) Analysis of SG formation in CTRL and hGLE1 siRNA-treated samples. hGLE1 siRNA or CTRL siRNA-treated HeLa cells were heat shocked at 45°C. Samples were fixed across a time course of 0–60 min and processed for immunofluorescence detection of G3BP and hGle1. p < 0.000001 for each data pair. (F) SG disassembly is delayed in hGle1-depleted cells. Following heat shock at 45°C for 60 min, cells were incubated at 37°C for the indicated times and processed for immunofluorescence using anti-G3BP and hGle1 antibodies. Error bar represents mean ± SE from three independent experiments. **, p < 0.001.
FIGURE 4:
FIGURE 4:
hGle1-dependent SG defects are not linked with microtubules. (A–C) HeLa cells transfected with CTRL and hGLE1 siRNAs were treated with (A) vehicle alone, (B) 5 μM nocodazole, or (C) 100 nM Taxol for 120 min at 37°C followed by heat shock at 45°C for 60 min. Cells were processed for immunofluorescence with anti-G3BP, α-tubulin, and hGle1 antibodies. Scale bar: 10 μm. (D) Quantification of SG numbers in hGLE1 and CTRL siRNA cells treated with indicated drugs after heat shock.
FIGURE 5:
FIGURE 5:
hGle1 modulates SG assembly by regulating translation. (A, B) Nascent protein synthesis is deregulated in hGle1-depleted cells. HeLa cells treated with CTRL and hGLE1 siRNAs were subjected to heat shock at 45°C or left untreated. After 15 min, AHA was added to the incubations, and heat shock treatment was continued for an additional 30 min. Samples were processed by Alexa Fluor-488 alkyne staining followed by immunofluorescence with anti-G3BP and hGle1 antibodies. Scale bar: 10 μm. (C) Quantification of AHA-488 staining. Mean fluorescence intensity of AHA-488 staining in individual cells was calculated in CTRL and hGLE1 siRNA cells using ImageJ. (D) CTRL or hGLE1 siRNA-treated HeLa cells were either heat shocked at 45°C or left untreated followed by metabolic labeling with 100 μCi/ml [35S]methionine/cysteine for 30 min at either 37°C or 45°C. Cells were lysed, and 35S incorporation was measured by liquid scintillation counter. Counts per minutes (cpm) are shown for hGLE1 and CTRL siRNA-treated cells. (E) hGle1-depleted cells have polysome profile defects under normal conditions. CTRL or hGLE1 siRNA cells were lysed, and polysome profiles were generated by subjecting cells to a 7–47% sucrose gradient centrifugation. The 40S, 60S, 80s, and polysome peaks are labeled. (F) Expression of hGle1A but not hGle1B rescues translation defect in hGle1-depleted cells. CTRL or hGLE1 siRNA-treated HeLa cells were transfected with mCherry, mCherry-hGLE1AR, or mCherry-hGLE1BR plasmids, heat shocked, and processed for metabolic labeling using AHA. AHA incorporation was detected with Alexa Fluor-488 alkyne using click chemistry. Scale bar: 10 μm. (G) Quantification of AHA-488 staining in CTRL and hGLE1 siRNA cells expressing the indicated plasmids.
FIGURE 6:
FIGURE 6:
Phosphorylation of eIF2α is reduced in hGle1-depleted cells. (A, B) HeLa cells transfected with either CTRL or hGLE1 siRNA were left untreated or heat shocked at 45°C for 30 min or 60 min. Lysates were analyzed by immunoblotting using anti-eIF2α and anti–phospho-eIF2α (Ser-51) antibodies. Phospho-eIF2α levels were quantified by densitometry and normalized to total eIF2α protein levels. Error bar represents SD from mean from four independent experiments.
FIGURE 7:
FIGURE 7:
hGle1 regulates balance between active and stalled translation upon stress. (A, B) Puromycin rescues SG-assembly defects in hGle1-depleted cells. CTRL or hGLE1 siRNA HeLa cells were treated with vehicle alone, 0.1 mg/ml, or 0.5 mg/ml puromycin for 60 min at 45°C. Cells were processed for immunofluorescence using anti-hGle1 and G3BP antibodies. Scale bar: 10 μm. (C, D) EGFP-hGle1A but not EGFP-hGle1B overexpression results in bigger SGs: HeLa cells were transfected with either EGFP, EGFP-hGLE1AR, or EGFP-hGLE1BR, and either (C) left untreated or (D) heat shocked for 60 min at 45°C. After heat shock, cells were processed for immunofluorescence using anti-hGle1 and G3BP antibodies. Data represent mean ± SE from three independent experiments. Scale bar: 10 μm.
FIGURE 8:
FIGURE 8:
DDX3 rescues hGle1-dependent SG and translation defects. (A) DDX3 coimmunoprecipitates with hGle1. HeLa cells were either left untreated or heat shocked for 30 min or 60 min, and cell lysates were immunoprecipitated using anti-hGle1 or IgG control antibodies and immunoblotted with anti-hGle1 or DDX3 antibodies. (B) DDX3 partially rescues SG defects in hGle1-depleted cells. CTRL or hGLE1 siRNA-treated cells were transfected with EGFP, EGFP-DDX19B, or DDX3-EGFP plasmids and heat shocked. Cells were processed for immunofluorescence detection of G3BP and hGle1. Scale bar: 10 μm. (C) SG numbers in CTRL and hGLE1 siRNA cells expressing indicated plasmids were quantified. (D) DDX3 partially rescues translation defects in hGle1-depleted cells. CTRL or hGLE1 siRNA-treated HeLa cells transfected with mCherry or mCherry-DDX3 plasmids were heat shocked at 45°C. After 15 min of heat shock, AHA was added to the incubations, and heat shock treatment was continued for an additional 30 min. Samples were processed for detection of AHA-labeled proteins with Alexa Fluor-488 alkyne using click chemistry. Scale bar: 10 μm. (E) Quantification of AHA-488 staining. Mean fluorescence intensity of AHA-488 staining in individual cells was calculated in CTRL and hGLE1 siRNA cells expressing the indicated plasmids.

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