Plasmids and protein expression in E. coli

Human eIF4E2 cDNA, amplified from pcDNA3-HA-eIF4E2 vector (gift from Nahum Sonenberg) by PCR was subcloned into pEGFP-C1 (EcoRI/XmaI restriction sites) and yeast vectors, pGADT7 and pGBKT7 (BD Biosciences-Clontech), using NdeI/BamHI sites. Similarly, human 4E–T cDNA in the pEYFP-C1 vector (gift from Rheinhard Luhrmann [22]) was subcloned into pEGFP-C1 (KpnI/XmaI) and yeast vectors (NdeI/BamHI). eIF4E2 cDNA C-terminally truncated by 69 bp was amplified by PCR and recloned into pcDNA3-HA vector (BamHI/XhoI). FLAG-MS2-4E-T was constructed by inserting 3xFLAG epitope DNA into the NcoI site of the MS2-4E-T plasmid [27] using Pfu DNA polymerase (Stratagene), and similarly for the control FLAG-MS2 control construct. Mutagenesis of 4E-T was carried out in a two step process. For the human protein, Y30A and Y55A were mutated first, followed by LL35/36 AA and VW60/61 AA respectively, and similarly in the case of Xenopus 4E-T (Y28,Y53, LL33/34 AA, VW58/59 AA respectively). All cloning and mutagenesis, with the indicated oligonucleotides (Table S1), was carried out using Stratagene QuikChange site-directed mutagenesis kit and verified by sequencing.

Recombinant His-4E-T was prepared by the Novagen autoinduction system and denaturing purification on NTA-agarose in 8M urea and elution with pH 4.5 buffer, according to Qiagen. Recombinant eIF4E1 and eIF4E2 proteins were expressed as inclusion bodies in Rosetta2 (DE3), then purified under denaturing conditions (4-6 M guanidinium hydrochloride) and refolded by one step dialysis as described in [16]. Additional purification by anion-exchange chromatography gave homogenous fractions of proteins.

Protein quantitation

Quantitative analysis of endogenous eIF4E1, eIF4E2 and 4E-T was performed by comparison of a concentration series of protein lysates obtained from HeLa and HEK293 cells with known amounts of purified recombinant protein counterparts loaded on the same gel. Cultures of HeLa and HEK293 cells were prepared in the same size 6-well plates and incubated in parallel. Cells from both cultures were counted using a hemocytometer, and lysates were prepared in RIPA buffer, whose protein concentration was determined by the Bradford assay. Western blots with a concentration series of recombinant eIF4E1, eIF4E2 and 4E-T proteins, quantitated with known amounts of BSA, alongside lysates from HeLa and HEK293 cells, were developed with ECL.

Y2H double transformations and selection

A yeast two-hybrid assay was performed using the GAL4-based two-hybrid system adopted from Clontech. pGBK-protein constructs were transformed into AH109 cells on selective medium (minimal SD base supplemented with 2% glucose and the required dropout solution, -Trp in this case). Singly transformed strains were used as a yeast stock in which to transform the pGAD-protein constructs. Double transformed cells were selected on -Leu/-Trp dropout medium suitable for pGBKT7 and pGADT7 vectors containing TRP1 and LEU2 nutritional markers, respectively. pGBKT7 constructs were verified that they did not autonomously activate reporter genes and interaction of proteins was checked on high stringency -Ade/-His/-Leu/-Trp dropout medium. Lamin C (pGBKT7-Lamin) and SV40 large T-antigen (pGADT7-T) co-transformants were used as negative controls.

Cell culture, transfection and drug treatments

HeLa and HEK293 cells were routinely grown in DMEM supplemented with 10% fetal calf serum. Transient transfections were performed in 24-well plates using 1 μl of Lipofectamine 2000 (Invitrogen) and 0.5 μg of plasmid constructs, or in 6-well plates using 3 μl of Lipofectamine 2000 and 3 µg of siRNA. The 4E-T siRNA was 5’ CAGUCGAGUGGAGUGUACAUUGUdTdT, purchased from Thermo Scientific, and the control β-globin siRNA was described in [35]. Cells were fixed and stained with the appropriate antibody 24 hrs after plasmid transfection or 48 hrs after siRNA transfection.

To induce oxidative stress, cells were incubated in media supplemented with 0.5 mM sodium arsenite (Sigma) for 30 min, then allowed to recover for 30 min in the absence of arsenite. For transcription inhibition of RNA polymerase I and II, 4 µM Actinomycin D (ActD) (in ethanol) was used. To inhibit Crm1-dependent export, Leptomycin B (LMB) (in methanol) (Sigma) was used at a final concentration of 10 nM for 5 hrs. The reported outcomes of these treatments were seen in all cells. Addition of the same volume of methanol or ethanol to the cells was used as a control for these inhibitor experiments. Cells were scraped in phosphate-buffered saline (PBS) and resuspended in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% NaDOC, 0.1% SDS) or NET buffer with non-ionic detergent for immunoprecipitation assays (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.25% gelatin) and incubated on ice 20 min. Buffers for lysis were supplemented with Complete Protease Inhibitor cocktail (Roche). Soluble proteins were recovered after centrifugation at 10 000 rpm at 4^°C for 10 min and quantitated by the Bradford method.

Immunofluorescence

Cells were plated on 13 mm glass coverslips in 24-well plates. Cells were fixed in 4% paraformaldehyde for 20 min, washed three times with phosphate buffered saline (PBS) and permeabilized in PBS with 0.5% Triton X-100 for 4 min 30 sec followed by three PBS washes. Cells were incubated with primary antibodies for 1 hr using chicken eIF4EL3/eIF4E2 (1:200; Novus Biologicals), mouse eIF4E1 (1:1000; Santa Cruz), goat 4E-T (1:200; Abcam), rabbit Pat1b (1:100), rabbit rck/p54 (1:1000; Bethyl Laboratories), rat HA (1:200; Roche), mouse G3BP (1:200; BD Biosciences), goat TIAR (1:500; Santa Cruz). The cells were then washed three times in PBS, followed by the incubation of the secondary antibodies conjugated to Rhodamine Red-X RRX, Alexa Fluor 488 or Cumarin AMCA fluorescent probes used at a 1:1000 dilution (Jackson ImmunoResearch Laboratories) for 1 hr. After rinsing three times with PBS, cells were stained with DAPI (1.25 μg/ml) for 10 sec. The coverslips were mounted in Citifluor (Citifluor Labs). All steps were performed at room temperature. Cells were observed under a Zeiss Axioimager M1 fluorescent microscope with a Plan-Apochromat 100/1.4 Oil DIC objective. Where indicated, >100 cells from several independent experiments were counted to assess the number of cells with P-bodies containing eIF4E2 and 4E-T (or as stated). We did not distinguish between small or large foci, or count the number of P-bodies per cell.

GFP-Trap pull-down assays

GFP-Trap® Magnetic beads (Chromotek) (25 µl per sample) were re-suspended in 250 µl ice-cold NET buffer in hydrophobic tubes. Washed beads were incubated with 1 mg lysate protein, prepared from GFP-4E-T transfected HEK293-T cells, in 1 ml NET buffer supplemented with Protease Inhibitor cocktail, for 1.25 h at 4°C with constant rotation. Supernatants were then removed, beads washed and associated proteins eluted in SDS sample buffer (2% SDS, 80mM Tris-HCl pH 6.8, 10% glycerol, 5% β-mercaptoethanol, 0.0001% bromophenol blue) prior to gel electrophoresis and western blotting.

Immunoprecipitation of eIF4E2

Chicken IgY Precipitating Resin (GenScript) consisting of agarose beads coupled to Goat Anti-Chicken IgY was used for immunoprecipitation of human eIF4E2. First, 10 µl of beads were incubated with 0.5 to 2 µl of chicken α-eIF4E2 (no antibody for control) in 1 ml NET buffer for 2 hrs at 4^°C with constant rotation. The beads were then washed before incubation with 1 mg of protein lysate prepared from HeLa cells transfected with HA-eIF4E2 plasmid in 1 ml NET buffer supplemented with Protease Inhibitor cocktail, using the same conditions. Following extensive washing of the beads, associated proteins were eluted with 10 µl of SDS sample buffer.

Immunoprecipitation of Xenopus 4E-T

In vitro transcribed mRNAs encoding FLAG-MS2-x4E-T fusion proteins (50 nl at ~500 ng/μl) were injected into stage VI oocytes, as described previously [27,28], with 150 oocytes injected for each mRNA. Oocytes were incubated for 36 h at 18^oC before harvesting. Extracts were made by addition of 1 ml NET buffer and clarification by centrifugation at 10,000 g, 2 x 10 min, 4 ^oC. To each extract were added 20 μl of Protein-G-Sepharose beads and 5 μl mouse monoclonal α-FLAG (Sigma; 4.2 mg/ml) antibody. The mixture was incubated for 4 h at 4 ^oC, with constant rotation, and then centrifuged at 800 g, 2 min, 4 ^oC, and the supernatant discarded. The beads were washed 3 x 1 ml NET buffer, and bound proteins eluted in 40 µl of PBS containing 250 µg/ml FLAG peptide. The eluate was concentrated in a micro-spin column (Pierce) and centrifuged at 13,000 rpm for 1min, before denaturation in SDS-Sample buffer and western blotting.

Protein gel electrophoresis and western blotting

SDS-PAGE gels and western blotting were performed as described previously [28, 35]. Typically, 40 μg protein per lane was loaded. Primary antibodies and dilutions used were as follows: chicken eIF4E2 (1:2000; Novus Biologicals), rabbit eIF4E1 (1:15000; gift from Simon Morley), goat 4E-T (1:500; Abcam), mouse GFP (1:2000; Santa Cruz), rabbit p54/rck (Bethyl Labs; 1:2000), mouse CPEB [36] and rabbit MS2 antibodies [62]. Western blots were subsequently detected by enhanced chemiluminescence (ECL). Magic Mark XP Standard Western proteins (Invitrogen) were used as molecular weight markers.

Expression and quantitation of eIF4E1, eIF4E2 and 4E-T in HeLa and HEK293 cells

To examine the expression of eIF4E2, we first identified an eIF4E2-specific antibody. Indeed, we found several commercial antibodies failing to detect this protein, with the exception of the chicken anti-eIF4EL3 antibody, which, as Figure 1A shows, detects both GFP-eIF4E2, HA-eIF4E2 and an endogenous protein of the predicted size of 28 kDa in HeLa cells. We compared the expression of eIF4E1, eIF4E2 and 4E-T in HeLa and HEK293 cells, probably the two most commonly used mammalian cell lines (Figure 1B and 1C). Levels of eIF4E1 and eIF4E2 are similar in both cell lines, with HeLa cells possessing slightly higher levels of eIF4E1 than HEK293 cells, though 4E-T was significantly more expressed in HEK293 than in HeLa cells. We also noted that in HEK293 cells 4E–T migrates solely as the lower band of the doublet observed in HeLa cells, possibly indicating differential phosphorylation in the two cell lines (Figure 1B).

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Figure 1. Quantitation of eIF4E1, eIF4E2 and 4E-T levels.

A. Verification of chicken eIF4EL3/eIF4E2 antibody using untransfected Hela cells (1), and cells overexpressing GFP-eIF4E2 (2) and HA-eIF4E2 (3). Top panel developed with eIF4E2 antibody, bottom panels with eIF4E1 and 4E-T antibodies. Molecular weight standards are indicated in kDa. B. Comparison of eIF4E1, eIF4E2 and 4E-T levels in HeLa and HEK293 cells analysed by western blotting with indicated antibodies. Increasing amounts of lysate from HeLa cells (20, 40 and 80 µg total protein) and HEK293 cells (10, 20 and 40 µg) approximating to 5.7, 11.4, 17 and 23 x 10⁴ cells. C. Table comparing eIF4E1, eIF4E2 and 4E-T levels (protein copies/per cell) in HeLa and HEK293 determined by western blotting (top), to those reported in genome-wide mass spectrometry studies in HeLa [35], U20S [34] and NIH3T3 [36] cells.

https://doi.org/10.1371/journal.pone.0072761.g001

Using a concentration series of recombinant human eIF4E1, eIF4E2 and 4E-T proteins made in E. coli alongside lysates prepared from HeLa and HEK293 cells in western blotting (data not shown), we estimated their levels in terms of protein copies per cell (summarised in Figure 1C). While these levels are higher than those obtained from recent proteome mass spectrometry determinations in U2OS, HeLa and NIH 3T3 cells and there are indeed considerable differences between these proteomic studies themselves [37–39], the following conclusions using all the data can be made. First, eIF4E1 levels are consistently higher than those of eIF4E2 (with the exception of U2OS cells where they are equal), and second, 4E-T levels are significantly much lower than either eIF4E protein. Our quantitation data show that eIF4E1:eIF4E2 ratios range from approx 8:1 to 5:1 in HeLa and HEK293 cells respectively, while ratios of eIF4E1:4E-T range from 50:1 to 15:1, and those of eIF4E2:4E-T vary from 6:1 to 3:1 (Figure 1C). We also considered the levels of eIF4G1+3 and 4E-BP1+2 in the proteomic studies [37–39], and concluded that 4E-T is the least expressed of these eIF4E-binding proteins in mammalian cells. Interestingly, too, none of these studies reported significant levels of eIF4E3. Altogether, these data indicate that eIF4E1 is the principal eIF4E protein in mammalian cells, and that eIF4E:4E-T ratios can vary from 3:1 to 50:1 depending on the cell and the cap-binding protein.

eIF4E2 binds 4E-T via YX₄LL and a nearby downstream sequence

eIF4E1 binding to 4E-T via its N-terminal YX ₄LL sequence has been reported previously, and shown to prevent interaction with eIF4G, reducing translation initiation of capped mRNAs [20,21,23,27]. Large scale yeast protein–protein interaction screens suggested 4E-T also interacts with eIF4E2 [40]. First, we verified that eIF4E2 binds 4E-T in the yeast two hybrid system. AH109 cells were transformed with pGADT7-4E-T and pGBKT7-4E2 plasmids and grown in -Leu/-Trp dropout medium to select for both plasmids. Stocks made from single colonies of double transformants were re-streaked on both -Leu/-Trp and -Ade/-His/-Leu/-Trp dropout plates to screen for plasmid retention and high-stringency protein–protein interactions, respectively. Yeast growth in stringent conditions was supported by 4E-T co-expressed with eIF4E2, with no growth in the case of controls (Figure S1A). The interaction was next assessed in HeLa cells over-expressing GFP-4E-T. In contrast to untransfected cells in which cellular eIF4E2 is essentially homogeneously distributed in the cytoplasm, ectopic 4E-T recruits eIF4E2 to P-bodies (Figure 2A), as was reported for tagged and cellular eIF4E1 ( [20,23], and Figure S2). We found that eIF4E2 co-localised with GFP-4E-T in P-bodies in approx. 70% of cells, relative to untransfected cells where it only co-localised with 4E-T P-bodies in 20% of cells, as verified by immunostaining. We confirmed this redistribution from the cytoplasm to P-bodies with HA-tagged eIF4E2, where it co-localises with GFP-4E-T in approx. 75% of transfected cells (Figure 2A). Moreover, we show that eIF4E2 does not localise to P-bodies containing GFP-4E-T with the mutations in the critical tyrosine and leucine residues of the Y³⁰X₄LL motif (Figure 2B and Figure S2), indicating that eIF4E2 associates with 4E-T in a similar manner as eIF4E1.

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Figure 2. eIF4E2 is recruited to P-bodies by ectopic 4E-T.

A. Cellular eIF4E2 distribution in HeLa cells was assessed by indirect immunofluorescence in the absence and presence of GFP-4E-T, and similarly for HA-tagged eIF4E2 co-transfected or not with GFP-4E-T. B. Wild-type but not mutant GFP-4E-T recruits eIF4E2 to P-bodies. Mutations in the 4E-T eIF4E-binding site residues Y³⁰X₄LL to alanine are indicated. Cells were also stained with DAPI. Scale bar, 10 µm.

https://doi.org/10.1371/journal.pone.0072761.g002

Previously characterised protein partners of eIF4E2/4EHP in fly and mammalian cells, including Bicoid [17], Prep1 [18] and GIGYF2 [41], were shown to interact via a slightly extended N-terminal binding sequence of YX ₆Lϕ or YXYX ₄Lϕ (Figure 3A). We thus examined whether 4E-T proteins possess this extended sequence. Interestingly, with the exception of several (but not all) fish 4E-T sequences (Danio rerio, Oreochromis niloticus and Oryzias latipes, though not Takifugu rubripes), which contained an additional tyrosine at -2 position relative to the consensus tyrosine, all other vertebrates lacked this upstream residue. Examining the sequence of 4E-T, we noted a possible nearby downstream eIF4E-binding site Y⁵⁵X₄VW, that is conserved in vertebrates, including Xenopus (Figure 3A). While this putative additional sequence is not strictly speaking a consensus sequence, we were intrigued by the potential of sequences downstream of the consensus site in enhancing eIF4E-binding, noted in both 4E-BP and eIF4G [42,43].

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Figure 3. The eIF4E-binding site in 4E-T.

A. Top. The sequences of the eIF4E2-binding site in Dm Bicoid [17], Mm Prep1 [18], Hs GIGYF2 [19] and Hs 4E-T ( [20]. Bottom. Alignments of five indicated vertebrate 4E-T sequences using ClustalW2. In bold are indicated the eIF4E-binding site residues, note the upstream tyrosine in Dr 4ET, bold and underlined. Underlined are the residues of a potential conserved second eIF4E-binding site. B. HEK293 cells were transfected with wild type or mutant GFP-4E-T as indicated and lysates were analysed by GFP-Trap and western blotting with eIF4E1, eIF4E2 and 4E-T antibodies. C. mRNAs encoding FLAG-MS2 and FLAG-MS2-X4E-T proteins were injected into Xenopus oocytes. After 16 hours, lysates were prepared and immunoprecipitated with FLAG antibodies, and the FLAG peptide eluates were analysed by western blotting with indicated antibodies. L lane indicates uninjected oocyte lysate. The mutations in human and Xenopus 4E-T sequences are indicated in B and C.

https://doi.org/10.1371/journal.pone.0072761.g003

To assess this possibility, we employed a pull-down assay with lysates from HEK293 cells overexpressing GFP-4E-T using the GFP-Trap [44], as a more sensitive method to detect interactions in mammalian cells than the P-body recruitment approach. First, we confirmed that eIF4E2 binds GFP-4E-T, and comparison of input and immunoprecipitated levels indicates that eIF4E2 binds with apparent lower affinity to 4E-T than does eIF4E1 (Figure 3B, lanes 2 and 9). Furthermore, in a reciprocal immunoprecipitation, HA-eIF4E2 brings down endogenous 4E-T (Figure S1B). Therefore in mammalian cells, 4E-T interacts with eIF4E2 as well as eIF4E1. Moreover, mutation of the critical conserved tyrosine and leucines in Y³⁰X₄LL or just the tyrosine on its own to alanine abrogated the interaction between GFP-4E-T and eIF4E2, in agreement with the HeLa P-body data (Figure 2B), as well as between eIF4E1 and GFP-4E-T (Figure 3B, lanes 10 and 12). We next tested whether the Y⁵⁵X₄VW binding site contributes to eIF4E binding. Mutation of Y⁵⁵ to alanine reduced binding of eIF4E1 to 4E-T, and all interaction was lost with the triple mutation of Y⁵⁵VW^60/61 positions, while no eIF4E2 binding was detected with either mutant 4E–T protein (Figure 3B, lanes 11 and 13). In agreement with these pull-down results, the single tyrosine mutation (Y⁵⁵ to A) in GFP-4E-T expressed in HeLa cells largely unaffected eIF4E1 localisation in P-bodies (wt and Y⁵⁵ both approx. 90% of cells with P-bodies) while preventing that of eIF4E2 (wt and Y⁵⁵ approx. 70 and 10 % of cells respectively with P-bodies; Figure S2).

Altogether, our data thus suggests that while both eIF4E1 and eIF4E2 interact with sequences downstream of Y³⁰X₄LL of 4E-T, the contribution of the downstream sequence is less important in the case of eIF4E1, indicating that despite the participation of the same region of 4E-T, formation of 4E-T:eIF4E1/2 complexes is differentially stabilised.

In Xenopus oocytes, eIF4E1 binding to 4E-T is also sensitive to the same downstream mutations

We next examined the interaction between 4E-T and eIF4E proteins in Xenopus oocytes. FLAG-MS2-tagged x4E-T and the control FLAG-MS2 protein were expressed from in vitro transcribed mRNA microinjected into stage VI oocytes, and co-precipitation of endogenous eIF4E1 proteins by FLAG antibodies was assessed by western blotting. Unlike endogenous 4E-T, ectopic 4E-T can bind both short and long forms of eIF4E1 (differing in an 18 amino acid repeat) [27], though it preferentially interacts with eIF4E1b, as shown by comparing the input and the wild-type protein bound lanes (Figure 3C, lanes 1 and 3). Mutation of the consensus eIF4E-binding site, Y²⁸LL^33/34 to AAA in FLAG-MS2-4E-T abolishes all eIF4E1a binding as does mutation of the second putative site Y⁵³VW^58/59 to AAA, while both sets of mutations also abolish most, though not all, eIF4E1b binding (Figure 3C, lanes 4 and 5). Neither set of mutations abrogates 4E-T co-immunoprecipitating CPEB or Xp54 helicase, demonstrating that they do not result in extensive misfolding. We could not assess Xenopus 4E-T binding to eIF4E2, since the chicken eIF4EL3 antibody does not detect an oocyte protein of the appropriate size, though we suspect eIF4E2 to be maternally expressed, as it is in mice [18].

Altogether we conclude that sequences downstream of the canonical eIF4E-binding site in human or Xenopus 4E-T, including the YX ₄VW motif, strongly influence 4E–T interaction with eIF4E1 or eIF4E2 isoforms and may regulate their differential binding. The exact delineation of the additional sequences is beyond the scope of this study, and clearly warrants further investigation, ideally at the structural level. We note also that the previously described downstream eIF4E-interacting sequences in human 4E-BP and eIF4G proteins [42,43], located approx. 25 away from the consensus sequence, are not conserved in sequence, and are not present in 4E-T. Moreover, the downstream sequences may not necessarily interact directly with eIF4E, but could enhance the ability of the canonical consensus site to form a stable α-helix [45]. Indeed, secondary structure predictions for human or Xenopus 4E-T (using Jpred3 server: http://www.compbio.dundee.ac.uk/www-jpred/) suggests that only the canonical binding site Y(T/S) KEELL has the potential to form an α-helix, not YDSDGVW, though this does not exclude it from directly binding eIF4E. Interestingly, the D. melanogaster protein Cup, related to human 4E–T, has two nearby eIF4E-binding sites, the high-affinity one which conforms to the consensus, being Y³⁴²TRSRLM, with a second lower affinity and non-consensus one, ELEGRLR, some 30 residues downstream [34]. The second site binds laterally and perpendicularly in α-helix form to the eIF4E β-sheet [46], and plays a role in the stabilisation of associated mRNA [47].

Unlike eIF4E1, eIF4E2 does not re-localise to stress granules in arsenite-treated cells

In addition to its native localisation in P-bodies, eIF4E1 is found in stress granules in mammalian cells treated briefly with arsenite, resulting in oxidative stress [48]. Stress granules are large cytoplasmic granules, considerably larger than P-bodies, which contain stalled translation initiation complexes including eIF4E, eIF4A, eIF4G and PABP, non-translating mRNA and RNA-binding proteins such as G3BP and TIAR [36,48,49]. Like P-bodies, stress granules are dynamic complexes whose assembly is dependent on the pool of nontranslating mRNAs, and indeed the two types of mRNP granules are frequently found juxtaposed or overlapping [49,50]. Arsenite-treated HeLa cells contain typical TIAR and G3BP-containing stress granules, to which eIF4E1 co-localises (Figure 4A), as shown by immunostaining. Interestingly, endogenous p54/rck remains in P-bodies adjacent to stress granules in these cells, as observed previously, though RFP-p54/rck can relocate to stress granules from the the diffuse cytoplasmic pool [48,50]. In contrast to eIF4E1, eIF4E2 remains homogenously distributed in the cytoplasm in arsenite-treated cells (Figure 4B), and 4E-T remains partly cytoplasmic and partly enriched in P-bodies, as in untreated cells (Figure 4C). In other words, localisation of eIF4E2 and 4E-T is insensitive to arsenite, unlike eIF4E1, implying distinct functions to translation initiation.

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Figure 4. eIF4E1 and eIF4E2 distribution in arsenite-treated cells.

Control HeLa cells (- Ars) and cells treated with arsenite (+ Ars) to induce stress granules were immunostained with eIF4E1 (A), eIF4E2 (B) and 4E-T (C) antibodies, and counter-stained with antibodies against p54/rck (P-body marker), and TIAR or G3BP (stress granule markers) as indicated. Scale bar, 10 µm.

https://doi.org/10.1371/journal.pone.0072761.g004

Inhibition of RNA transcription does not impact on eIF4E2 distribution, but enhances eIF4E1 localisation to P-bodies

We recently showed that in HeLa cells treated with Actinomycin D for 5 hours to inhibit new RNA synthesis, the P-body factors Pat1b, p54/rck and 4E-T were localised in fewer but bigger P-bodies than in untreated HeLa cells [51]. Particularly striking was the loss of small P-body foci upon treatment, suggesting that in the absence of new transcripts, smaller foci aggregate into larger ones. Moreover, P-bodies in Trypanosoma brucei also enlarge upon Actinomycin D treatment [52]. The cause of this increase is not clear but may be due to the reduced levels of newly synthesised mRNAs resulting in an inhibition of protein synthesis and thus an increase in P-bodies. Similarly, using Pat1b immunostaining as a control, we note that eIF4E1 redistributes to P-bodies in ActD-treated HeLa cells, even in the absence of ectopic 4E-T (Figure 5A). In contrast, eIF4E2 localisation in the cell is unaffected by this treatment (Figure 5B), additionally highlighting the differences between eIF4E1 and eIF4E2.

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Figure 5. eIF4E1 and eIF4E2 distribution in Actinomycin D-treated cells.

Control HeLa cells (- ActD) and cells treated with Actinomycin D (+ ActD) to inhibit RNA synthesis were immunostained with eIF4E1 (A) and eIF4E2 (B) antibodies, and counter-stained with Pat1b antibodies. Note that the apparent nuclear staining in the case of Pat1b antibody was previously shown to be insensitive to Pat1b siRNA [49]. Cells were also stained with DAPI. Scale bar, 10 µm.

https://doi.org/10.1371/journal.pone.0072761.g005

eIF4E2 is a 4E-T-independent nucleocytoplasmic shuttling protein

Previously, 4E-T was shown to transport eIF4E1 into nuclei in cells treated with Leptomycin B (LMB), which inhibits the Crm1 receptor [53], demonstrated with tagged proteins [20]. Indeed, these studies defined 4E-T as an eIF(4E-T) ransport protein. Here we demonstrate that while 4E-T (both cellular and ectopic) is nuclear in treated cells attesting to the LMB addition, eIF4E1 is unresponsive to LMB in untransfected HeLa cells, and only becomes sequestered within nuclei in the presence of both GFP-4E-T and LMB (Figure 6A and B). 4E-T is a nucleocytoplasmic shuttling protein, which does not require eIF4E-binding to translocate to nuclei in LMB-treated cells ( [20,51], Figure S3), whereas eIF4E1 requires additional 4E-T protein to visibly accumulate in nuclei in these conditions (Figure 6). This observation presumably reflects the relatively low levels of 4E-T to eIF4E1 in HeLa cells (Figure 1).

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Figure 6. eIF4E2 is a nucleocytoplasmic shuttling protein.

Hela cells were treated with Leptomycin B for 5 hrs (+ LMB) or with methanol as control (- LMB), without (A and C) and with ectopic GFP-4E-T (B and D), and immunostained with eIF4E1 (A and B), eIF4E2 (C and D) and 4E-T antibodies, while GFP-4E-T fluorescence was detected directly in transfected cells. Scale bar, 10 µm.

https://doi.org/10.1371/journal.pone.0072761.g006

In contrast, eIF4E2 becomes partially nuclear in LMB-treated cells, in the absence of ectopic 4E-T, and overexpression of 4E-T does not enhance this nuclear accumulation (Figure 6C and D). To confirm this finding, 4E-T was depleted from cells using RNAi, which were subsequently treated with LMB and immunostained for eIF4E1, eIF4E2 and 4E-T. 4E-T siRNA was shown to be effective in depleting cellular 4E-T, unlike the control β-globin siRNA (lacking a target in HeLa cells). Nevertheless, eIF4E2 continues to accumulate in nuclei in LMB-treated 4E-T depleted cells, and as predicted from above, eIF4E1 remains cytoplasmic (Figure 7 and Figure S4. Thus, 4E-T is a transporter for eIF4E1 only, and not for eIF4E2, though 4E-T binds both proteins, and ectopic 4E-T relocalises eIF4E2 to P-bodies (Figures 2 and 3; Figures S1 and S2). Previously we reported that in approx. 33% of transfected and LMB-treated cells GFP-4E-T localises in numerous foci, overlapping or close to PML bodies [49]. Here we show that the Y³⁰A mutation, which in such cells would only prevent eIF4E1 import and not that of eIF4E2, strikingly enhances the number and brightness of such GFP-4E-T foci, though the reasons for this are not known (Figure S3).

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Figure 7. eIF4E2 shuttling in the presence of LMB is 4E-T independent.

Untransfected Hela cells (no siRNA), or cells transfected with 4E-T siRNA or control β-globin siRNA were treated with LMB and immunostained with eIF4E2 and 4E-T antibodies as indicated. Cells were also stained with DAPI. Scale bar, 10 µm.

https://doi.org/10.1371/journal.pone.0072761.g007

How eIF4E2 enters and exits nuclei remains to be investigated, whether on its own or with a shuttling partner protein. We noted that mammalian eIF4E2 proteins possess short N- and C-terminal extensions relative to eIF4E1 proteins, and that its C-terminal region (in human protein) contains five conserved leucines, possibly related to the nuclear export signals (NES) in Crm1-dependent shuttling proteins. Most NES sequences conform to the consensus ϕ-(x)_2-3-ϕ-(x)_2-3-ϕ-x-ϕ, where x is an amino acid that is preferentially charged, small or polar, and ϕ is hydrophobic [54]. However the spacing of the leucines in the eIF4E2 C-terminal portion does not follow the consensus, and they are not conserved in vertebrates (Figure 8A and B). More importantly, truncation of HA-tagged eIF4E2 at amino acid 222 did not lead to eIF4E2 accumulation in nuclei in the absence of LMB (Figure 8C), demonstrating that they do not act as an NES. eIF4E1 localisation can also be dictated by 4E-BPs [55], and this may be the case for eIF4E2 as well. Irrespective of the shuttling mechanism, it is interesting to note yet another difference between eIF4E1 and eIF4E2, and to speculate on their nuclear functions, perhaps in specific mRNA export [56].

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Figure 8. The leucine-rich C-terminus of eIF4E2 does not act as a NES.

A. Sequence alignment of human eIF4E1 and eIF4E2 performed with ClustalW2. Secondary structural elements of α-helices (H0-H4) and β-strands (S1-S7) were shown above the alignment according to the crystal structure of human eIF4E2 in complex with m⁷ GTP (PDB id: 2JGB) [15]. Identities are shaded in grey. With blue and red are marked hydrophobic residues that typically form the NES. The leucine-rich C-terminus of eIF4E2 is underlined in red. B. ClustalW alignment of the C-terminal regions of indicated eIF4E2 proteins. C. HeLa cells transfected with HA-eIF4E2 full-length protein or HA-eIF4E2ΔC (truncated at residue 222) were treated with LMB or methanol (-) for 5 hrs and immunostained with HA and 4E-T antibodies as indicated. Cells were also stained with DAPI. Scale bar, 10 µm.

https://doi.org/10.1371/journal.pone.0072761.g008