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. 2006 Oct;17(10):4212-9.
doi: 10.1091/mbc.e06-04-0318. Epub 2006 Jul 26.

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation

Rachid Mazroui  1 , Rami Sukarieh, Marie-Eve Bordeleau, Randal J Kaufman, Peter Northcote, Junichi Tanaka, Imed Gallouzi, Jerry Pelletier
Affiliations

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation

Rachid Mazroui et al. Mol Biol Cell. 2006 Oct.

Abstract

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

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Figures

Figure 1.
Figure 1.
Perturbing eIF4A activity and levels induces SG formation. (A) eIF4A localizes to cytoplasmic granules upon inhibition of translation initiation. HeLa cells were treated with 1 μM hippuristanol (5–8), 0.1 μM pateamine (9–12), or 0.5 mM arsenite (13–16) for 30 min; permeabilized; and fixed. The primary antibodies used were a monoclonal anti-eIF4A antibody (5D3) and a polyclonal anti-G3BP antibody. The percentage of cells harboring SGs (>5 granules/cell), from three different fields and three different experiments containing a total of 450 cells, is indicated to the right bottom of 4, 8, 12, and 16. (B) Reduction of eIF4AI levels by siRNA induces SG formation. Cells were transfected with siRNA against eIF4AI (eIF4AI-1) or a control siRNA and fixed 2 d later. The distribution of HuR and G3BP was visualized by immunofluorescence. (C) Knockdown of eIF4A has a modest effect on cellular translation. Cells were treated with eIF4AI-1 or a control siRNA (Ctr) and 48 h later they were labeled for 30 min with 50 μCi/ml [35S]methionine. (D) Western blot analysis of protein extracts prepared from cells treated with eIF4AI-1 or control siRNA (Ctr). The blot was first probed with a monoclonal anti-eIF4A antibody (5D3), stripped, and reprobed with an anti-G3BP antibody.
Figure 2.
Figure 2.
Granules induced by perturbation of eIF4A activity are similar to SGs and distinct from processing bodies. (A) Granules induced by perturbation of eIF4A activity contain TIA-1 and FMRP. HeLa cells were treated with 1 μM hippuristanol or 0.1 μM pateamine and then processed for immunofluorescence. The distribution of TIA-1 and FMRP was monitored with anti-TIA-1 and anti-FMRP (1C3) antibodies, respectively. The percentage of cells harboring SGs is indicated to the bottom right of 3 and 6. (B) Cellular distribution of HuR and DCP1α was visualized with anti-HuR (3A2) and anti-DCP1α antibodies, respectively. Yellow arrows indicate the location of granules induced by perturbing eIF4A activity, whereas the white arrows indicate the position of PBs.
Figure 3.
Figure 3.
Assembly of eIF4A-inhibition induced granules is independent of eIF2α phosphorylation status. (A) Hippuristanol and pateamine do not induce phosphorylation of eIF2α. Cells were treated with 1 μM hippuristanol (lane 2), 0.1 μM pateamine (lane 3), or 0.5 mM of arsenite (lane 4) for 1 h. Protein extracts were prepared and analyzed by Western blotting using an anti-phospho eIF2α (top) or pan anti-eIF2α (bottom) antibody. (B) MEFs derived from wt and eIF2αS51A/S51A knockin mice were treated with 1 μM hippuristanol (3, 4, 9, and 10) or 0.5 mM arsenite (5, 6, 11, and 12) for 30 min. The localization of HuR (1, 3, 5, 7, 9, and 11) and G3BP (2, 4, 6, 8, 10, and 12) was assessed by immunofluorescence. The percentage of cells harboring SGs is indicated to the bottom right. (C) Exposure of cells to pateamine or hippuristanol does not block arsenite-mediated phosphorylation of eIF2α. Cells were exposed to hippuristanol (lanes 2 and 5) or pateamine (lanes 3 and 6) for 1.5 h. In some instances, arsenite was added to the cells 30 min after the addition of hippuristanol or pateamine, and the incubation was continued for 1 h. (lanes 4–6). Cell extracts were prepared and probed for eIF2α phosphorylation (top, p-eIF2α) as well as for total eIF2α levels (bottom, eIF2α).
Figure 4.
Figure 4.
Inhibition of cap-dependent translation initiation by poliovirus is sufficient to induce cytoplasmic granule formation. (A) Cellular localization of HuR and G3BP was determined in control, uninfected cells (1 and 2) and in cells 1.5 h PI (3 and 4) and 3 h PI (5–8). Infection with poliovirus was performed in the absence (1–6) or presence (7 and 8) of guanidine hydrochloride (GuHCl). The percentage of cells harboring SGs is indicated to the bottom right. (B) Western blot analysis of eIF4GII integrity during poliovirus infection. Cell extracts were prepared from uninfected cells (lanes 1 and 5) or cells 1.5 h PI (lanes 2 and 6) 3 h PI (lanes 3 and 7), or 6 h PI (lanes 4 and 8). Poliovirus infections were performed in the absence (lanes 2–4) or presence (lanes 6–8) of GuHCl. The antibodies used to probe the blot are indicated to the right. Note that full-length eIF4GII was not detected well in this experiment, and only the cleavage product is clearly apparent (indicated by an asterisk). (C) Western blot analysis of eIF2α phosphorylation status during poliovirus infection. The same blot as in B was probed for eIF2α phosphorylation.
Figure 5.
Figure 5.
Schematic representation of the relationship between the ribosome recruitment phase of translation, eIF2α phosphorylation, and SG formation. The class of mRNA whose translation is increased as a consequence of eIF2α phosphorylation is depicted with 2 uORFs (small white boxes) and a larger coding region (gray box).
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