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. 2006 Oct;17(10):4212–4219. doi: 10.1091/mbc.E06-04-0318

Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

Rachid Mazroui *, Rami Sukarieh *,†, Marie-Eve Bordeleau *,†, Randal J Kaufman ‡,§, Peter Northcote ‖, Junichi Tanaka , Imed Gallouzi *,#, Jerry Pelletier *,@,#,✉
Editor: Peter Walter
PMCID: PMC1635342  PMID: 16870703

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)2α 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 eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α 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 eIF2α phosphorylation-dependent and -independent pathways that target translation initiation.

INTRODUCTION

In response to various assaults, mammalian cells activate a protective mechanism to prevent damage of vital cellular processes required for homeostasis, once the stress is relieved (Nover et al., 1989). The rapid formation of stress granules (SGs) in the cytoplasm is one of the main mechanisms by which the cell inhibits translation of mRNAs encoding for “housekeeping” functions to prioritize the synthesis of chaperones and enzymes needed for the stress response (Anderson and Kedersha, 2002). In addition to mRNAs, SGs contain many RNA-binding proteins, including TIA-1, eukaryotic initiation factor (eIF)3, eIF4E, eIF4G, poly(A) binding protein (PABP), AU-rich element binding protein (HuR), tristetraprolin (TTP), fragile X mental retardation protein (FMRP), and Ras-GTPase activating protein (G3BP) (Anderson and Kedersha, 2006; Kedersha et al., 1999, 2002; Mazroui et al., 2003). Translation inhibitors that stabilize polysomes (e.g., cycloheximide or emetine) induce SG disassembly, whereas compounds that cause disassembly of polysomes (e.g., puromycin) promote SG formation (Kedersha et al., 2000). Based on these observations, it was concluded that SGs constitute dynamic entities where mRNAs assemble for quality control before being redirected for reinitiation, degradation, or storage (Kedersha et al., 2005).

Unlike mRNAs directed to sites of decay known as processing bodies (PBs), the messages found in SGs are stabilized (Anderson and Kedersha, 2006). However, SGs and PBs share several RNA-binding proteins, such as TTP and BRF1, which are known as mRNA decay stimulators, suggesting that under stress conditions, SGs communicate with PBs (Anderson and Kedersha, 2006). The observed mRNA protection in SGs under stress could be explained by either inhibition of these decay factors and/or the recruitment of RNA-stabilizing proteins, such as HuR to SGs (Gallouzi et al., 2000). All these observations indicate that during the cell stress response, a close collaboration between different mRNA processing events, such as decay, stabilization, and translation, is required to ensure cellular protection against a lethal outcome and a rapid recovery after stress.

The process that inhibits translation during the stress response, and which also acts as a stimulus for SG assembly, targets specifically the initiation phase of translation (Anderson and Kedersha, 2006). Indeed, it has been shown that arsenite (AS)- and heat shock-mediated SG formation induce the phosphorylation of eIF2α, leading to a reduction in the cellular levels of eIF2·GTP·Met-tRNAMet ternary complexes, and a concomitant decrease in translation initiation rates. As a consequence, 40S ribosomes and some translation initiation factors are recruited to SGs. SG formation by mitochondrial poisons has been documented to occur in the absence of eIF2α phosphorylation (Kedersha et al., 2002). This suggests that inhibition of translation initiation by stimuli that do not induce eIF2α phosphorylation may also be capable of inducing SG formation, a hypothesis that we address in this study.

Recently, two novel small molecule inhibitors of translation initiation, pateamine and hippuristanol, have been identified and characterized (Bordeleau et al., 2005, 2006; Low et al., 2005). Both compounds target eIF4A, an RNA helicase required for recruitment of ribosomes to cellular, and most viral, mRNAs (Rogers et al., 2002). Pateamine stimulates eIF4A RNA-dependent ATPase, RNA binding, and helicase activity, whereas hippuristanol is an inhibitor of eIF4A RNA binding. eIF4A is the most abundant translation initiation factor, present at three copies per ribosome (Duncan et al., 1987). There are two highly related eIF4A gene products, eIF4AI and eIF4AII (90–95% identical), both implicated in translation and functionally interchangeable in vitro (Conroy et al., 1990; Yoder-Hill et al., 1993). eIF4A exists as a free form (eIF4Af) and as a subunit of eIF4F (eIF4Ac), a heterotrimeric complex that also contains eIF4E (the m7GpppN cap binding protein) and the scaffolding protein eIF4G (Edery et al., 1983; Grifo et al., 1983). The helicase activity of eIF4Ac is ∼20-fold more efficient than eIF4Af (Pause and Sonenberg, 1992; Rogers et al., 1999), suggesting that eIF4Ac is the functional helicase required to unwind local secondary structure in the mRNA 5′ untranslated region during ribosome recruitment. A recent report indicates that exposure of cells to pateamine induces the formation of cytoplasmic granules containing TIA-1, eIF4A, and eIF4B (Low et al., 2005). Whether the formation of these granules is an indirect consequence of eIF2α phosphorylation has not been investigated. To further characterize a potential relationship between the ribosome recruitment step of translation initiation and SG formation, we made use of several strategies to interdict this phase of translation. Our data indicate that SG formation can occur as a consequence of impaired translation initiation independently of eIF2α phosphorylation.

MATERIALS AND METHODS

General Methods and Cell Line Maintenance

The compounds pateamine and hippuristanol were stored at −70°C in dimethyl sulfoxide as stocks of 10 mM. The characterization of these compounds has been documented previously (Bordeleau et al., 2005, 2006). HeLa cells (obtained from American Type Culture Collection, Manassas, VA), wild-type (wt) mouse embryonic fibroblasts (MEFs), and MEFs harboring the mutation eIF2αS51A/S51A were maintained in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin.

Antibodies

The monoclonal anti-eIF4A antibody has been described previously (Edery et al., 1983). Phospho-specific anti-eIF2α and the pan anti-eIF2 were obtained from Cell Signaling Technology (Beverly, MA). Anti-HuR and anti-G3BP antibodies were described previously (Gallouzi et al., 1998, 2000). The use of anti-Dcp1α anti-FMRP and antibodies has been documented previously (Sheth and Parker, 2003).

Small-interfering RNA (siRNA) Transfections

siRNA transfections were performed in HeLa cells essentially as described previously (Ferraiuolo et al., 2004). Briefly, siRNA transfections were performed in HeLa cells by using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). Twenty-four hours before transfection, cells were trypsinized to obtain 50–60% confluence on the day of transfection. For a six-well plate, 15 μl of siRNA duplex (20 μM annealed duplex; Dharmacon RNA Technologies, Lafayette, CO) was mixed with 100 μl of OPTI-MEM and 3.5 μl of Plus reagent and incubated for 15 min at room temperature. A mixture of 4 μl of Lipofectamine (Invitrogen) and 100 μl of OPTI-MEM was then added to the precomplexed RNA mix and incubated for an additional 15 min before adding to cells. Cells were harvested 48 h after transfection and processed for immunofluorescence, or proteins were extracted in 3× SDS-PAGE sample buffer and used for Western blots. The efficiency of knockdown was determined by quantitation of the signal on films using ImageQuant (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). The sense sequences of the siRNAs used in this study are eIF4AI-1, 5′GCCCAAUCUGGGACUGGGAdTdT3′) (nucleotides [nt] 226–244), and eIF4AI-2, 5′UGAUAUGCUUAACCGGAGAdTdT3′ (nt 488–506).

Fluorescence Microscopy

Cells were processed for immunofluorescence as described previously (Mazroui et al., 2003). Essentially, cells were fixed in 3% paraformaldehyde and permeabilized with 0.1% Triton X-100/phosphate-buffered saline. Slides were incubated with primary antibodies diluted in 0.1% normal goat serum for 1 h at room temperature. After washing, slides were incubated with goat anti-mouse/rabbit IgG (H+L) secondary antibodies coupled to goat Alexa Fluor 488/594. Fluorescence microscopy was performed using a Zeiss AxioVision 3.1 microscope equipped with AxioCam HR (Carl Zeiss, Jena, Germany) digital camera. Images were compiled using Adobe Photoshop software (Adobe Systems, Mountain View, CA).

Poliovirus Infection

HeLa cells were incubated with the Mahoney strain of poliovirus type 1 (10 plaque-forming units/cell) in serum-free DMEM at room temperature for 30 min, after which time the medium was replaced with DMEM containing 10% fetal bovine serum. The infection was then allowed to proceed to the indicated times at 37°C. When present, guanidine HCl was used at a final concentration of 1.5 mM. Cells were then fixed and processed for immunofluorescence, or total protein was harvested for extraction and analyzed by Western blots.

RESULTS

In the course of characterizing the cellular effects of pateamine and hippuristanol, we noted that cells exposed to these compounds showed granules that were reminiscent of SGs induced by arsenite (Figure 1A, 1–16). SGs are thought to be sites of initiation factor and mRNA storage during translation inhibition, and the presence of eIF3, eIF4E, eIF4G, and PABP in these granules has been well documented (Kedersha et al., 2002; McEwen et al., 2005; McInerney et al., 2005). AS, hippuristanol, and pateamine exposure induced the recruitment of eIF4A to these granules, as revealed by immunostaining using an anti-eIF4A monoclonal antibody (Figure 1A, compare 5, 9, and 13 with 1), confirming a previous report suggesting the presence of eIF4A in SGs (Low et al., 2005). These granules harbor the classical SG marker G3BP (Tourriere et al., 2003) (Figure 1A, compare 6, 10, and 14 with 2). In these experiments, translation was reduced by >95% when cells were treated with pateamine or hippuristanol, as judged by [35S]methionine metabolic labeling (our unpublished data). These observations are consistent with previous reports linking SG formation and translation inhibition (Kedersha et al., 2002; McEwen et al., 2005; McInerney et al., 2005).

Figure 1.

Figure 1.

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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.

Because both pateamine and hippuristanol target eIF4A, we addressed whether reducing eIF4A activity by an independent method would also trigger SG formation. To this end, HeLa cells were treated with an siRNA duplex directed to eIF4AI (eIF4AI-1) or a control siRNA (Figure 1B, 1–8). Treatment of cells with eIF4AI-1, but not the control siRNA, was sufficient to induce SG formation in ∼5% of the cells visualized, as judged by the presence of HuR and G3BP in these granules (Figure 1B, compare 1 and 2 with 5 and 6). Metabolic labeling indicated that cellular protein synthesis was reduced by 40% (Figure 1C and as judged by quantitation of trichloroacetic acid-precipitable counts; our unpublished data). Western blot analysis of total cellular extracts revealed that the efficiency of eIF4AI knockdown in this experiment was 85% (Figure 1D, compare lane 1 with lane 2), consistent with what has been reported previously (Ferraiuolo et al., 2004). These results were reproduced with a second siRNA targeting a different region of eIF4AI (eIF4AI-2), arguing that they are unlikely a consequence of off-target effects (Supplemental Figure 1). These results suggest that perturbing eIF4A activity with small molecules or by siRNA knockdown is sufficient to induce SG formation.

The granules formed by cellular exposure to hippuristanol and pateamine also were found to contain TIA-1 (Figure 2A, compare 2 and 3 with 1), FMRP (Figure 2A, compare panels 5 and 6 with 4), and HuR (Figures 2B and S2, compare panels 5 and 9 with 1). In HeLa cells, these proteins are present in SGs (Kedersha et al., 1999; Gallouzi et al., 2000; Mazroui et al., 2003). In contrast, neither hippuristanol nor pateamine significantly affected the appearance of PBs, as shown by immunofluorescence of DCP1α, a known PB marker (Figures 2B and S2, compare panels 2, 6, and 10) (Sheth and Parker, 2003), although we cannot rule out subtle effects not detectable in our assay. Together, our data suggest that the cytoplasmic granules induced by hippuristanol and pateamine are similar in composition to SGs.

Figure 2.

Figure 2.

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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.

We considered the possibility that hippuristanol- or pateamine-induced SG formation was an indirect consequence of eIF2α phosphorylation. To assess this possibility, total extracts were prepared from HeLa cells treated for 1 h with arsenite, pateamine, or hippuristanol, and the extracts were used for Western blot analysis. We observed that eIF2α phosphorylation was observed only upon arsenite treatment and not when cells were exposed to pateamine or hippuristanol (Figure 3A). These observations indicate that pateamine- and hippuristanol-induced SG formation does not correlate with eIF2α phosphorylation. This conclusion was further supported by the fact that hippuristanol induced SG formation in MEFs expressing the nonphosphorylatable eIF2α mutant (eIF2αS51A/S51A), obtained from knockin mice (Scheuner et al., 2001) (Figure 3B, compare 9 and 10 with 7 and 8). In contrast, arsenite exposure did not induce SG assembly in eIF2αS51A/S51A-derived MEFs (Figure 3B, compare 11 and 12 with 7 and 8), as documented previously (McEwen et al., 2005). Both compounds induced SG formation in wt MEFs (Figure 3B, 1–6). (Note that in these experiments, staining of G3BP was not restricted to the cytoplasm, unlike what was observed for HeLa cells [e.g., Figure 1].) Nuclear localization of G3BP has previously been documented in quiescent fibroblasts (Tourriere et al., 2001). We therefore conclude that formation of SGs can occur via eIF2α phosphorylation-independent mechanisms. To assess whether the effect of our two compounds was dominant over stimuli that induced eIF2α phosphorylation, we pretreated cells with hippuristanol or pateamine, followed by exposure to arsenite. Probing for the phosphorylation status of eIF2α showed that arsenite is still capable of inducing eIF2α phosphorylation in hippuristanol- and pateamine-treated cells (Figure 3C, compare lanes 5 and 6 with 2 and 3), indicating that eIF4A-mediated SG formation occurs before eIF2α phosphorylation or resides in an independent pathway. These results demonstrate that perturbing eIF4A activity, in the absence of eIF2α phosphorylation, is sufficient to induce SG formation.

Figure 3.

Figure 3.

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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).

SGs could represent cytoplasmic entities where the cell assembles all components of the translation initiation machinery before commencing protein synthesis. Therefore, that these entities are highly dynamic could be one reason why they are not detected under normal cellular homeostasis and only become visible when translation initiation is blocked (Anderson and Kedersha, 2006). We have analyzed a number of additional translation inhibitors for their ability to induce SG formation. We tested compounds expected to stabilized polysomes (cepaeline, bouvardin, didemnin B, verrucarin A, and borrelidin) as well as two compounds that allow ribosomes to run off mRNA templates but block subsequent cycles of elongation by impairing the activity of newly initiated ribosomes (bruceantin and homoharringtonine) (Pelletier and Peltz, 2006). Although exposure of HeLa cells to concentrations of these elongation inhibitors was sufficient to inhibit protein synthesis (our unpublished data), they failed to induce SG formation (Supplemental Table 1).

We next determined whether other stimuli that inhibit translation initiation could also induce SG formation. For this purpose, we used poliovirus, because infected cells show inhibition of cap-dependent protein synthesis as a consequence of eIF4GI and eIF4GII subunit cleavage (Gradi et al., 1998). At 3 h postinfection (PI), we noted the appearance of SGs that were restricted to poliovirus-infected cells (Figure 4A, compare 5 and 6 with 1 and 2). At this time point, cleavage of eIF4GII was apparent (Figure 4B, compare lane 3 with lanes 1 and 2). SGs were not observed in poliovirus-infected cells that had been incubated with guanidine HCl, an inhibitor of poliovirus replication (Figure 4A, compare 7 and 8 with 5 and 6). Guanidine-HCl inhibits the function of poliovirus 2C protein, a protease that plays an essential role in viral replication (Pincus and Wimmer, 1986). As a consequence, synthesis of the poliovirus protease 2Apro is diminished, and eIF4GII cleavage is delayed (Gradi et al., 1998) (Figure 4B, compare lanes 5–8 with lanes 1–4). Although phosphorylation of eIF2α has been reported after infection with poliovirus (Black et al., 1989; O’Neill and Racaniello, 1989), this is a late event that occurs after eIF4G cleavage. Indeed, Western blot analysis of extracts from infected cells indicated that phospho-eIF2α is detectable only at 6 h PI (Figure 4C, compare lane 4 with lanes 1–3). We note that large cytoplasmic aggregates after poliovirus infection have been reported previously in poliovirus-infected Hep-2 cells and shown to contain both positive- and negative-strand viral RNA (Bolten et al., 1998). We have not further investigated whether the composition of these granules is similar to the ones we described herein. These results support our conclusion that impairment of the cap-dependent ribosome recruitment phase of translation initiation, independent of eIF2α phosphorylation, is sufficient to induce SG formation.

Figure 4.

Figure 4.

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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.

To establish a link between exposure to hippuristanol and pateamine and the cell stress response, we monitored the expression of the heat shock protein heat-shock protein (HSP)70 in wt and eIF2αS51A/S51A MEF cells. It is well established that the expression of HSP70 protein is induced during cell recovery from stress (Pelham, 1984). Therefore, we treated wt and eIF2αS51A/S51A MEF cells with heat shock, arsenite, pateamine, or hippuristanol for 30 min and allowed them to recover 6 h at 37°C (Supplemental Figure 3). Western blot analysis in which cellular extracts were probed with an anti-HSP70 antibody revealed that HSP70 expression is induced upon heat-shock and arsenite treatment in both cell lines, but not in cells exposed to pateamine or hippuristanol (Supplemental Figure 3, compare lanes 4 and 5 with lanes 2 and 3). Furthermore, we noted that eIF2α phosphorylation is not required for HSP70 induction by arsenite (compare lane 8 with lane 3) but may be involved in the heat-shock response (compare lane 7 with lane 2). These data suggest that pateamine and hippuristanol may induce SG assembly by activating a cellular pathway that is independent of the well-known stress response process.

DISCUSSION

Herein, we demonstrate that perturbing eIF4A and/or inhibiting eIF4F activity induces SG assembly without the need to phosphorylate eIF2α. All of the stimuli we have used are expected to affect ribosome recruitment to mRNAs during translation initiation. Hippuristanol inhibits the RNA binding activity of both eIF4Af and eIF4Ac (Bordeleau et al., 2006), whereas pateamine increases the RNA binding activity of eIF4Af removing it from the pool that is available for recycling through the eIF4F complex (Bordeleau et al., 2005; Low et al., 2005). In contrast, infection of cells by poliovirus cleaves the eIF4GI and eIF4GII subunits of eIF4F, preventing the recruitment of eIF4A to mRNA 5′ cap structures (Gradi et al., 1998). These results are consistent with the fact that SGs lack 60S ribosomes and agree with previous suggestions that this component of the translation apparatus may be antagonistic to SG formation or is the feature that removes the mRNA (and associated factors) out of SGs (Kedersha et al., 2000). To better define the molecular mechanisms by which SGs are formed, an extended approach to the one described here could be applied in which each step of the initiation phase leading to 80S complex formation at the AUG codon could be targeted for inhibition, defining the critical boundary required for SG formation.

Our study defines a new mechanism by which SGs can form (Figure 5). Previously published data demonstrated that SG assembly upon inhibition of translation is triggered by stimuli that induce eIF2α phosphorylation (Anderson and Kedersha, 2006). Phosphorylation of eIF2α converts eIF2 from a substrate to a competitive inhibitor of eIF2B, preventing guanine nucleotide exchange and reducing ternary complex availability (for review, see Dever, 2002). Although this results in a severe block of general translation, the translation of a special class of mRNAs (e.g., ATF4), containing upstream ORFs is up-regulated (Figure 5) (Dever, 2002). This is a consequence of reduced ternary complex levels enabling newly initiated ribosomes to bypass some of the inhibitory upstream ORFs (uORFs) and commence protein synthesis more frequently at the appropriate downstream initiation codon (Dever, 2002). Our findings allow us to eliminate a potential role of these uORF-containing mRNAs in SG formation, because inhibiting the ribosome recruitment phase of translation initiation will also decrease translation of these mRNAs and yet is sufficient to induce SG formation (Figure 5). In addition, it is likely that it is reduced ternary complex availability (and its subsequent effects on global protein synthesis), and not phosphorylation of eIF2α per se, that is responsible for SG formation, because the latter is not necessary for SG formation (Figure 3).

Figure 5.

Figure 5.

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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).

It has not escaped our attention that other cellular processes that inhibit translation, such as some microRNA-mediated suppression may cause targeting of repressed mRNAs to SGs, because some of these are triaged for degradation to PBs. Along these lines, let-7 targeted and translationally inhibited mRNAs localize to PBs (Pillai et al., 2005), structures closely related in location to SGs (Anderson and Kedersha, 2006). As well, argonautes, proteins that play essential roles in RNA silencing, have been reported to localize to SGs (Anderson and Kedersha, 2006). Our study is consistent with a functional role of SGs in mRNA repression and expands our understanding of the stimuli capable of inducing SG formation.

Supplementary Material

[Supplemental Material]
mbc_E06-04-0318_index.html (2.4KB, html)

ACKNOWLEDGMENTS

We are grateful to Drs. Hans Trachsel (University of Berne, Berne, Switzerland), Edward Khandjian (Universite Laval, Quebec, Canada), and J. Lykke-Andersen (University of Colorado, Boulder, CO) for kind gifts of anti-eIF4A, anti-FMRP, and anti-Dcp1α antibodies, respectively. We thank Dr. Nahum Sonenberg (Department of Biochemistry, McGill University, Montreal, Canada) for the gift of anti-eIF4GII antibody. R.M. was supported by a National Cancer Institute of Canada (NCIC) postdoctoral Terry Fox and a Canadian Institute of Health Research (CIHR) postdoctoral fellowship. M.-E.B. was supported by a CIHR Cancer Consortium Training Grant Award and an Fonds de recherche en santé du Québec studentship award. This work was supported by NCIC Grant 014313 (to J.P.), a New Economy Research Fund (NERF) grant from the Foundation of Science, Research and Technology (New Zealand) (to P.T.), a Takeda Science Foundation grant to J.T., and CIHR operating Grant MOP-67026 (to I.G.). J.P. is a CIHR Senior Investigator.

Abbreviations used:

AS

arsenite

eIF

eukaryotic initiation factor

MEF

mouse embryo fibroblast

PABP

poly(A) binding protein

PB

processing body

PI

postinfection

SG

stress granule

siRNA

short interfering RNA

uORF

upstream open reading frame

UTR

untranslated region

wt

wild-type.

Footnotes

Inline graphic The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-04-0318) on July 26, 2006.

REFERENCES

  1. Anderson P., Kedersha N. Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation. Cell Stress Chaperones. 2002;7:213–221. doi: 10.1379/1466-1268(2002)007<0213:vstroe>2.0.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson P., Kedersha N. RNA granules. J. Cell Biol. 2006;172:803–808. doi: 10.1083/jcb.200512082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black T. L., Safer B., Hovanessian A., Katze M. G. The cellular 68,000-Mr protein kinase is highly autophosphorylated and activated yet significantly degraded during poliovirus infection: implications for translational regulation. J. Virol. 1989;63:2244–2251. doi: 10.1128/jvi.63.5.2244-2251.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bolten R., Egger D., Gosert R., Schaub G., Landmann L., Bienz K. Intracellular localization of poliovirus plus- and minus-strand RNA visualized by strand-specific fluorescent In situ hybridization. J. Virol. 1998;72:8578–8585. doi: 10.1128/jvi.72.11.8578-8585.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bordeleau M.-E., Matthews J., Wojnar J. M., Lindqvist L., Novac O., Jankowsky E., Sonenberg N., Northcote P., Teesdale-Spittle P., Pelletier J. Stimulation of mammalian translation initiation factor eIF4A. activity by a small molecule inhibitor of eukaryotic translation. Proc. Natl. Acad. Sci. USA. 2005;102:10460–10465. doi: 10.1073/pnas.0504249102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bordeleau M.-E., Mori A., Oberer M., Lindqvist L., Chard L. S., Higa T., Belsham G. J., Wagner G., Tanaka J., Pelletier J. Functional characterization of internal ribosome entry sites by a novel inhibitor of the DEAD box RNA helicase, eIF4A. Nat. Chem. Biol. 2006;2:213–220. doi: 10.1038/nchembio776. [DOI] [PubMed] [Google Scholar]
  7. Conroy S. C., Dever T. E., Owens C. L., Merrick W. C. Characterization of the 46,000-dalton subunit of eIF-4F. Arch. Biochem. Biophys. 1990;282:363–371. doi: 10.1016/0003-9861(90)90130-q. [DOI] [PubMed] [Google Scholar]
  8. Dever T. E. Gene-specific regulation by general translation factors. Cell. 2002;108:545–556. doi: 10.1016/s0092-8674(02)00642-6. [DOI] [PubMed] [Google Scholar]
  9. Duncan R., Milburn S. C., Hershey J. W. Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F. J. Biol. Chem. 1987;262:380–388. [PubMed] [Google Scholar]
  10. Edery I., Humbelin M., Darveau A., Lee K.A.W., Milburn S., Hershey J. W., Trachsel H., Sonenberg N. Involvement of eukaryotic initiation factor 4A in the cap recognition process. J. Biol. Chem. 1983;258:11398–11403. [PubMed] [Google Scholar]
  11. Ferraiuolo M. A., Lee C. S., Ler L. W., Hsu J. L., Costa-Mattioli M., Luo M. J., Reed R., Sonenberg N. A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay. Proc. Natl. Acad. Sci. USA. 2004;101:4118–4123. doi: 10.1073/pnas.0400933101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallouzi I. E., Brennan C. M., Stenberg M. G., Swanson M. S., Eversole A., Maizels N., Steitz J. A. HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc. Natl. Acad. Sci. USA. 2000;97:3073–3078. doi: 10.1073/pnas.97.7.3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gallouzi I. E., Parker F., Chebli K., Maurier F., Labourier E., Barlat I., Capony J. P., Tocque B., Tazi J. A novel phosphorylation-dependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability. Mol. Cell. Biol. 1998;18:3956–3965. doi: 10.1128/mcb.18.7.3956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gradi A., Svitkin Y. V., Imataka H., Sonenberg N. Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection. Proc. Natl. Acad. Sci. USA. 1998;95:11089–11094. doi: 10.1073/pnas.95.19.11089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grifo J. A., Tahara S. M., Morgan M. A., Shatkin A. J., Merrick W. C. New initiation factor activity required for globin mRNA translation. J. Biol. Chem. 1983;258:5804–5810. [PubMed] [Google Scholar]
  16. Kedersha N., Chen S., Gilks N., Li W., Miller I. J., Stahl J., Anderson P. Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol. Biol. Cell. 2002;13:195–210. doi: 10.1091/mbc.01-05-0221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kedersha N., Cho M. R., Li W., Yacono P. W., Chen S., Gilks N., Golan D. E., Anderson P. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. J. Cell Biol. 2000;151:1257–1268. doi: 10.1083/jcb.151.6.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kedersha N., Stoecklin G., Ayodele M., Yacono P., Lykke-Andersen J., Fitzler M. J., Scheuner D., Kaufman R. J., Golan D. E., Anderson P. Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J. Cell Biol. 2005;169:871–884. doi: 10.1083/jcb.200502088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kedersha N. L., Gupta M., Li W., Miller I., Anderson P. RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules. J. Cell Biol. 1999;147:1431–1442. doi: 10.1083/jcb.147.7.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Low W. K., Dang Y., Schneider-Poetsch T., Shi Z., Choi N. S., Merrick W. C., Romo D., Liu J. O. Inhibition of eukaryotic translation initiation by the marine natural product pateamine A. Mol. Cell. 2005;20:709–722. doi: 10.1016/j.molcel.2005.10.008. [DOI] [PubMed] [Google Scholar]
  21. Mazroui R., Huot M. E., Tremblay S., Filion C., Labelle Y., Khandjian E. W. Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression. Hum. Mol. Genet. 2002;11:3007–3017. doi: 10.1093/hmg/11.24.3007. [DOI] [PubMed] [Google Scholar]
  22. McEwen E., Kedersha N., Song B., Scheuner D., Gilks N., Han A., Chen J. J., Anderson P., Kaufman R. J. Heme-regulated inhibitor kinase-mediated phosphorylation of eukaryotic translation initiation factor 2 inhibits translation, induces stress granule formation, and mediates survival upon arsenite exposure. J. Biol. Chem. 2005;280:16925–16933. doi: 10.1074/jbc.M412882200. [DOI] [PubMed] [Google Scholar]
  23. McInerney G. M., Kedersha N. L., Kaufman R. J., Anderson P., Liljestrom P. Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation. Mol. Biol. Cell. 2005;16:3753–3763. doi: 10.1091/mbc.E05-02-0124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nover L., Scharf K. D., Neumann D. Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs. Mol. Cell. Biol. 1989;9:1298–1308. doi: 10.1128/mcb.9.3.1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. O’Neill R. E., Racaniello V. R. Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2. J. Virol. 1989;63:5069–5075. doi: 10.1128/jvi.63.12.5069-5075.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992;11:2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pelham H. R. Hsp70 accelerates the recovery of nucleolar morphology after heat shock. EMBO J. 1984;3:3095–3100. doi: 10.1002/j.1460-2075.1984.tb02264.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pelletier J., Peltz S. W. Therapeutic opportunities in translation. In: Mathews M. B., Sonenberg N., Hershey J.W.B., editors. Translational Control in Biology and Medicine. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2006. (in press) [Google Scholar]
  29. Pillai R. S., Bhattacharyya S. N., Artus C. G., Zoller T., Cougot N., Basyuk E., Bertrand E., Filipowicz W. Inhibition of translational initiation by Let-7 microRNA in human cells. Science. 2005;309:1573–1576. doi: 10.1126/science.1115079. [DOI] [PubMed] [Google Scholar]
  30. Pincus S. E., Wimmer E. Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity. J. Virol. 1986;60:793–796. doi: 10.1128/jvi.60.2.793-796.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rogers G. W., Jr, Komar A. A., Merrick W. C. eIF4A: the godfather of the DEAD box helicases. Prog. Nucleic Acid Res. Mol. Biol. 2002;72:307–331. doi: 10.1016/s0079-6603(02)72073-4. [DOI] [PubMed] [Google Scholar]
  32. Rogers G. W., Jr, Richter N. J., Merrick W. C. Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A. J. Biol. Chem. 1999;274:12236–12244. doi: 10.1074/jbc.274.18.12236. [DOI] [PubMed] [Google Scholar]
  33. Scheuner D., Song B., McEwen E., Liu C., Laybutt R., Gillespie P., Saunders T., Bonner-Weir S., Kaufman R. J. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol. Cell. 2001;7:1165–1176. doi: 10.1016/s1097-2765(01)00265-9. [DOI] [PubMed] [Google Scholar]
  34. Sheth U., Parker R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science. 2003;300:805–808. doi: 10.1126/science.1082320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tourriere H., Chebli K., Zekri L., Courselaud B., Blanchard J. M., Bertrand E., Tazi J. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J. Cell Biol. 2003;160:823–831. doi: 10.1083/jcb.200212128. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  36. Tourriere H., Gallouzi I. E., Chebi K., Capony J. P., Mouaikel J., van der Geer P., Tazi J. RasGAP-associated endoribonuclease G3BP: selective RNA degradation and phosphorylation-dependent localization. Mol. Cell. Biol. 2001;21:7747–7760. doi: 10.1128/MCB.21.22.7747-7760.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yoder-Hill J., Pause A., Sonenberg N., Merrick W. C. The p46 subunit of eukaryotic initiation factor (eIF)-4F. exchanges with eIF-4A. J. Biol. Chem. 1993;268:5566–5573. [PubMed] [Google Scholar]

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