Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Oct 4.
Published in final edited form as: Cell Rep. 2012 Aug 16;2(2):207–215. doi: 10.1016/j.celrep.2012.07.007

The oncogene eIF4E reprograms the nuclear pore complex to promote mRNA export and oncogenic transformation

Biljana Culjkovic-Kraljacic 1, Aurelie Baguet 1, Laurent Volpon 1, Abdellatif Amri 1, Katherine LB Borden 1,*
PMCID: PMC3463940  NIHMSID: NIHMS398117  PMID: 22902403

Abstract

The eukaryotic translation initiation factor eIF4E is a potent oncogene that promotes the nuclear export and translation of specific transcripts. Here, we discovered that eIF4E alters the cytoplasmic face of the nuclear pore complex (NPC) which leads to enhanced mRNA export of eIF4E target mRNAs. Specifically, eIF4E substantially reduces the major component of the cytoplasmic fibrils of the NPC, RanBP2, relocalizes an associated nucleoporin Nup214, and elevates RanBP1 and the RNA export factors, Gle1 and DDX19. Genetic or pharmacological inhibition of eIF4E impedes these effects. RanBP2 overexpression specifically inhibits the eIF4E mRNA export pathway and impairs oncogenic transformation by eIF4E. The RanBP2 cytoplasmic fibrils likely slow the release/recycling of critical export factors to the nucleus. eIF4E overcomes this inhibitory mechanism by indirectly reducing levels of RanBP2. More globally, these studies suggest that reprogramming the NPC is a means by which oncogenes can harness the proliferative capacity of the cell.

Introduction

Nuclear export is a highly regulated process where macromolecular complexes transit to the cytoplasm via the nuclear pore complex (NPC). The NPC consists of the nuclear basket, the central channel spanning the nuclear membrane, and the cytoplasmic face characterized by fibrils that extend into the cytoplasm (Hutten and Kehlenbach, 2007; Kohler and Hurt, 2010; Strambio-De-Castillia et al., 2010; Wente and Rout, 2010). Generally, export complexes are formed by nuclear export signal (NES) containing proteins, chromosome region maintenance protein 1 (CRM1) and RanGTP. These enter the NPC via the nuclear basket, and transit through the central channel (Hutten and Kehlenbach, 2007; Wente and Rout, 2010). Once at the cytoplasmic side, the cargo is released from the export complex by one of two mechanisms. CRM1-cargo-RanGTP complexes associate with soluble RanBP1 in conjunction with RanGAP resulting in RanGTP hydrolysis thereby allowing cargo release from CRM1 (Hutten and Kehlenbach, 2007). Alternatively, the RanBP1 homologous domains of the cytoplasmic fibril protein RanBP2/Nucleoporin (Nup) 358 similarly release CRM1 bound cargo through RanGTP hydrolysis in association with RanGAP (Hutten and Kehlenbach, 2007; Kehlenbach et al., 1999). Interestingly, RanBP2 is absent in yeast while RanBP1 is absent in flies (Hutten and Kehlenbach, 2006; Strambio-De-Castillia et al., 2010).

Although bulk mRNA transits the NPC using the TAP/NXF1 nuclear receptor, the export of some mRNAs is CRM1 mediated including eukaryotic translation initiation factor eIF4E dependent mRNA export (Hutten and Kehlenbach, 2007; Culjkovic et al., 2005, 2006)). Although eIF4E plays a key role in translation, it also acts in the nucleus where it promotes the export of specific mRNAs (Borden and Culjkovic-Kraljacic, 2010). eIF4E’s mRNA export and translation activities both contribute to its oncogenic potential. Indeed, eIF4E is elevated in 30% of cancers including subtypes of acute myeloid leukemia (AML) (Borden and Culjkovic-Kraljacic, 2010). Here, eIF4E is highly elevated and almost entirely nuclear with substantially upregulated mRNA export activity (Assouline et al., 2009; Topisirovic et al., 2003; Topisirovic et al, 2009). A competitive inhibitor of the m7G cap, ribavirin, impairs the activities of eIF4E in translation, mRNA export, and oncogenic transformation (Assouline et al., 2009; Borden and Culjkovic-Kraljacic, 2010; Kentsis et al., 2004; Kentsis et al., 2005). In a Phase II multi-centre clinical trial, ribavirin targeted eIF4E activity, including reducing mRNA export, and this correlated with clinical responses including remissions in some AML patients (Assouline et al., 2009).

In contrast to translation, the mechanics underlying eIF4E dependent mRNA export are only starting to emerge. In contrast to the cytoplasm, eIF4E associates with a subset of nuclear mRNAs and importantly, its export activity is independent of ongoing translation (Culjkovic et al., 2005, 2006). To be an eIF4E export target, mRNAs must be capped and contain a 50-nucleotide structural element in their 3′ UTR known as an eIF4E sensitivity element (4E-SE) (Culjkovic et al., 2005, 2006). eIF4E RNA export targets include c-myc, hdm2, NBS1, ODC, and cyclin D1 amongst others (Culjkovic et al., 2005, 2006). Many target mRNAs have highly structured 5′ UTRs making these also translation targets of eIF4E. In this way, eIF4E coordinately drives the production of constituents of many proliferative and survival signaling pathways (Borden and Culjkovic-Kraljacic, 2010). Thus, when dysregulated, eIF4E is well positioned to drive oncogenesis.

The association with and dependence of eIF4E ribonuclear particles (RNPs) on CRM1 rather than TAP/NXF1 is a major difference between eIF4E dependent and bulk mRNA export (Culjkovic et al., 2005, 2006; Topisirovic et al., 2009). The eIF4E nuclear RNP consists of eIF4E, the m7G capped 4E-SE mRNA, LRPPRC (a bridging factor binding both 4E-SE and eIF4E), UAP56, hnRNPA1 and others, but not REF/Aly (Topisirovic et al., 2009). Despite these inroads, it is unknown how eIF4E promotes mRNA export. Here, we show that eIF4E alters the NPC. These changes correlate with increased export of 4E-SE mRNAs and increased transformation by eIF4E. These findings suggest that the NPC can be reprogrammed by oncogenes as part of the transformation process.

Results

eIF4E overexpression alters the composition of the NPC

In order to understand the mechanics behind eIF4E dependent mRNA export, we examined the status of NPC components upon eIF4E overexpression including constituents of the nuclear basket, central channel, and cytoplasmic face (Figures 1 & S1). We used two overexpression systems to ensure no construct dependence: a bicistronic construct with untagged eIF4E and GFP virally transduced into U2Os cells, and a stable cell line overexpressing FLAG-eIF4E (Figures 1 &S1). In figure 1, eIF4E overexpression is ~3 fold relative to vector by qRTPCR. Note that M4/M5 AML patients generally have eIF4E overexpression ranging from ~3–8+ fold relative to healthy volunteers (Assouline et al., 2009).

Figure 1. eIF4E overexpression modulates the cytoplasmic face of the NPC.

Figure 1

Open in a new tab

A. Cartoon of the NPC. B, C & D. U2Os cells were examined by western blot (B), or immunofluorescence in conjunction with confocal microscopy (C&D) with indicated antibodies to assess the effects of eIF4E wildtype or mutant expression. Magnification is 200X. DAPI is in blue.

Observed changes were focussed on the cytoplasmic face of the NPC. For instance, eIF4E overexpression leads to ~ 3 fold reduction in RanBP2, the main constituent of cytoplasmic fibrils (Figure 1B). Further, the remaining RanBP2 is less concentrated at the nuclear rim with increased nucleoplasmic staining (Figure 1C & S1). Another component of the cytoplasmic face, Nup 214, had reduced nuclear rim and increased nucleoplasmic staining with no detectable changes in total levels (Figure 1 & S1). Studies of Nup88 (also a constituent of the cytoplasmic face), were confounded by differences in results depending on the antibody used, precluding analysis at this point. Similar results were observed in mouse embryonic fibroblasts (MEFs) as in U2Os cells; thus these findings are not restricted to a single cell line (data not shown). Consistent with previous results, levels of eIF4E targets e.g. cyclin D1, NBS1 and c-Myc, increase by ~2–3 fold (Figure 1B).

We also monitored factors involved in cargo release on the cytoplasmic side of the NPC. Most strikingly, we observed a ~3 fold RanBP1 elevation, with no changes to its localization, upon eIF4E overexpression (Figures 1B & S1). No changes in levels or localization of RanGAP or Ran were observed (Figures 1B & S1). We also observed a ~2–3 fold elevation in an RNA helicase DDX19 and Gle1, its activating protein, with no change in distribution by subcellular fractionation (Figures 1B & S1). However, further analyses are needed to determine the extent of DDX19 and Gle1 binding to the NPC in eIF4E overexpressing cells. DDX19 and Gle1 release bulk mRNA export cargoes at the cytoplasmic face in conjunction with Nup214 or in its absence, hCG1/NLP1 (Hodge et al., 2011; Kendirgi et al., 2005; Noble et al., 2011). We noted no changes in levels or distribution upon eIF4E expression for: bulk mRNA export factors (Nup98, Rae1), nuclear pore components (Nup 153, Nup62, GP210, hCG1/NLP1 and Tpr), CRM1, CAS, Importin β1, Importin α2, Importin α3, LRPPRC, or Lamin A (Figure 1B & S1).

Consistent with the overexpression studies, eIF4E inhibition, via RNAi or ribavirin, lead to RanBP2 elevation, enrichment of RanBP2 and Nup214 at the nuclear rim, and reduction in Gle1, DDX19 and RanBP1 (Figures 2 & S2). eIF4E knockdown substantially increases RanBP2 levels, and increases RanBP2 in the nuclear rim and nucleus, likely due to saturation of NPC binding sites. eIF4E knockdown also leads to enrichment of Nup214 in the nucleus and the rim. Conversely, Gle1, DDX19 and RanBP1 levels were reduced by ~3 fold with no change in distribution by fractionation. Similarly, established eIF4E targets, e.g. cyclin D1, hdm2 and c-myc, were reduced by ~3–10 fold while non-targets e.g. CRM1 or β-actin were unchanged. Ribavirin treatment also leads to RanBP2 elevation, and reduction in RanBP1, Gle1, and DDX19 (by ~2–10 fold) in both control and eIF4E overexpressing cells (Figures 2 & S2), and enrichment of Nup214 and RanBP2 in the perinuclear and nuclear fractions. As expected, ribavirin represses eIF4E dependent mRNA export as observed by reduction of cyclin D1, hdm2 and c-myc levels. Ribavirin did not modulate other factors examined e.g. Nup153, Rae, Ran or Lamin A.

Figure 2. Effects of eIF4E inhibition on the NPC.

Figure 2

Open in a new tab

A. & B. Western blot analyses of total U2Os cell lysates upon RNAi mediated knockdown of eIF4E (si4E), or luciferase control (siLuc) or with 20μM Ribavirin. C& D. Subcellular distribution of RanBP2 and Nup214 upon si4E or siLuc treatments using immunofluorescence and confocal microscopy. DAPI is also shown. Magnification is 200X.

RanBP2 specifically impairs 4E-SE mRNA export

To investigate if RanBP2 inhibits eIF4E dependent mRNA export, we used the zinc fingers (ZF) of RanBP2 which binds CRM1 (a.a.1314 to 1963) and Nup-4 (a.a. 2500–3224) a C-terminal RanBP2 fragment which does not bind CRM1, (Singh et al., 1999; Yaseen and Blobel, 1999). Nup-4 contains cyclophilin homology and RanBP1 homology 4 domains. We analyzed nuclear and cytoplasmic RNA content in eIF4E overexpressing cells, and compared the effects of RanBP2-ZF overexpression on lacZ and lacZ-4E-SE mRNA export, where only the latter is eIF4E dependent (Figure 3). We also monitored the export of c-myc, an endogenous eIF4E target. tRNAlys and U6 snRNA served as controls for the cytoplasmic and nuclear fractions respectively (Figure S3). As previously shown, lacZ-4E-SE mRNA export and protein levels are higher compared to LacZ only in eIF4E overexpressing cells. Note that eIF4E overexpression does not change the nuclear stability, or total RNA levels of eIF4E targets (Figure 3 & S3, (Culjkovic et al., 2006)).

Figure 3. RanBP2 suppresses eIF4E dependent mRNA export and transformation.

Figure 3

Open in a new tab

A&B. Western blot analysis of protein levels in U2Os cells stably expressing 2FLAG-eIF4E and lacZ or lacZ-4E-SE (Xpress tag) upon RanBP2-ZF (A.) or Nup-4 (B.) overexpression; end. indicates endogenous eIF4E. The cytoplasmic to nuclear ratio (C/N) of lacZ (+/− 4E-SE) or endogenous c-myc target RNAs was determined using qRTPCR. Averaged values (+/− standard deviation) were normalized to β-Actin and to vector control (set to 1). C. Foci assays in U2Os cells stably transfected as indicated. D. Values are number of foci +/− SD. E. Western analysis as indicated with β-Actin as a loading control. Foci assays were carried out in triplicate three independent times.

RanBP2-ZF impaired the export of lacZ-4E-SE mRNAs by ~3 fold with no effect on lacZ (Figure 3). Consistently, lacZ-4E-SE protein levels were reduced by over 2 fold in RanBP2-ZF cells relative to vector. RanBP2-ZF impeded c-myc mRNA export up to 5 fold relative to vector in both lacZ and lacZ-4E-SE expressing cells, and lowered c-myc protein levels ~3 fold. RanBP2-ZF overexpression did not alter eIF4E levels. In contrast, Nup-4 had no effect on eIF4E dependent or bulk mRNA export (Figure 3). Thus RanBP2-ZF specifically impairs eIF4E dependent mRNA export.

RanBP2 reduction increases eIF4E dependent mRNA export

We examined if reduction in RanBP2 levels increased eIF4E dependent mRNA export using RNAi mediated knockdown of RanBP2, or by comparing wildtype and RanBP2 hypomorph MEFs (Figure S4). In either case, RanBP2 reduction resulted in increased mRNA export for lacZ-4E-SE and endogenous eIF4E target mRNAs, compared to controls. Consistently, lacZ-4E-SE and endogenous target protein levels were elevated. Importantly, we observed no difference in eIF4E levels or localization as a function of RanBP2 expression (data not shown). However, the effects are more modest here (~1.5–2 fold) relative to eIF4E overexpression. Thus, all of the effects of eIF4E on the NPC, in their totality, likely contribute to its mRNA export phenotype.

No changes were observed in RanBP2 mRNA export or total mRNA levels upon eIF4E overexpression (Figure S5 and data not shown). Consistent with RanBP2 protein stability studies (Um et al., 2006), RanBP2 protein levels are stabilized in vector controls treated with the proteosomal inhibitor MG132 (Figure S5). However, eIF4E overexpression abrogated the stabilizing effects of MG132. Thus eIF4E appears to indirectly reduce the protein stability of RanBP2.

RanBP1, Gle1 and DDX19 are mRNA export targets of eIF4E

We examined whether RanBP1, Gle1 and DDX19 are direct targets of eIF4E dependent mRNA export (Figure 4). RNA immunoprecipitations (RIP) from nuclear fractions indicate that endogenous eIF4E RIPs with Gle1, DDX19 and RanBP1 transcripts with 5–10 fold enrichment relative to IgG using qRTPCR or sqPCR (Figure 3 & S4). To assess if nuclear RIP correlated with export, the cytoplasmic to nuclear (C/N) ratio of mRNAs was examined by qRTPCR upon eIF4E overexpression (Figure 4). eIF4E promoted export up to 3 fold for all three mRNAs relative to vector. The C/N ratio for non-eIF4E export targets e.g. VEGF and ubiquitin was not altered. Consistently, increased mRNA export corresponded to increased protein levels with no changes in total mRNA levels (Figure 4 & S5). Bioinformatics analysis identified putative 4E-SE elements in the 3′ UTR of these mRNAs (data not shown). Thus RanBP1, Gle1 and DDX19 mRNAs are direct export targets of eIF4E.

Figure 4. Link between NPC, mRNA export and oncogenic transformation.

Figure 4

Open in a new tab

A&B. RNAs isolated by RIP with an anti-FLAG antibody from nuclear (N) or cytoplasmic (C) fractions of U2Os cells stably expressing with indicated constructs and analyzed using qRTPCR. Values represent relative fold difference +/− SD. C. Western blot analysis of IP efficiency. D. The C/N ratio of target transcripts as a function of wildtype or mutant eIF4E overexpression relative to β-Actin. E. Western blot analysis of the effects of eIF4E mutants on the NPC. Note that mutants are expressed to similar levels.

Linking NPC reprograming, mRNA export and oncogenic activities of eIF4E

To dissect features of eIF4E required for modulating the NPC, we monitored the effects of two previously described eIF4E mutants, W56A and W73A (Cohen et al., 2001; Culjkovic et al., 2005, 2006, 2008) and of a third mutant that we further characterize here, S53A. The W56A mutation impairs cap binding and thus renders eIF4E inactive in mRNA export, translation, transformation and apoptotic rescue. The W73A mutant is active in mRNA export, transformation and apoptotic rescue, but does not promote translation due to impaired eIF4G binding. The S53A mutant is active in translation (Kaufman et al., 1993; Zhang et al., 1995) but does not transform cells (Lazaris-Karatzas et al., 1990).

First, we examined the effects of the S53A mutant on mRNA export (Figure 4). This mutant does not increase export of endogenous eIF4E targets e.g. cyclin D1, nbs1, hdm2 or c-myc. In contrast, overexpression of wildtype eIF4E, or the W73A mutant, increases mRNA export and protein levels of targets (by up to 3 fold). Consistent with c-myc being both a translation and export target of eIF4E, its levels are slightly elevated in S53A cells relative to vector or W56A cells, but not to the same extent as in wildtype or W73A cells. Importantly, all constructs expressed eIF4E to similar levels and did not affect total RNA levels for targets (Figures 4 & S5). Thus, the S53A mutant is impaired in mRNA export.

Given these findings, we determined if the S53A mutant formed a nuclear RNP (Figure 4). RIP studies indicate that wildtype eIF4E binds specific mRNAs in the nucleus as observed previously (Culjkovic et al., 2006). For instance, wildtype eIF4E binds ODC mRNA in the nucleus and cytoplasm while it only binds VEGF in the cytoplasm. In contrast, the S53A mutant does not bind mRNAs in the nucleus but does bind mRNAs, including VEGF, in the cytoplasm with similar efficiency to wildtype eIF4E (Figure 4), consistent with its ability to act in translation. FLAG IPs were of equivalent efficiency for S53A and wildtype eIF4E (Figure 4). Thus, the S53A mutant does not bind mRNAs in the nucleus but does so in the cytoplasm.

Cap binding and localization were not altered by the S53A mutation (Figure S6). 1H-15N HSQC NMR studies indicated that the S53A mutant has a fold and cap binding activity indistinguishable from wildtype eIF4E. Further, the subcellular localization of the S53A mutant was not altered relative to wildtype eIF4E (data not shown).

We next examined the effects of these mutants on the NPC (Figures 1 & 4). The W73A-export competent mutant mirrors wildtype eIF4E: reducing RanBP2 levels, altering Nup214 and RanBP2 localization, and increasing mRNA export of Gle1, DDX19 and RanBP1. In contrast, the S53A and W56A mutants are completely impaired in these activities. These data suggest a functional link between remodelling the NPC and eIF4E dependent mRNA export.

We examined whether activities in mRNA export and NPC reprogramming were linked to the oncogenic potential of eIF4E. eIF4E overexpression leads to the formation of anchorage dependent foci due to the loss of contact inhibition, a hallmark of transformation (Lazaris-Karatzas et al., 1990), Importantly, the S53A mutant is devoid of the ability to form these foci (Figure S5; Lazaris-Karatzas et al., 1990). Further we observe that RanBP2-ZF, which repressed eIF4E dependent mRNA export, represses foci formation in eIF4E overexpressing cells, with a 60% reduction versus controls (Figure 3). Thus, RanBP2-ZF inhibits both eIF4E dependent mRNA export and transformation. Together, these findings functionally link the mRNA export, NPC reprograming and transformation activities of eIF4E.

Discussion

We demonstrate that eIF4E overexpression leads to major alterations to the cytoplasmic face of the NPC, and this is linked to both its mRNA export and oncogenic activities. These effects on the NPC are due to both indirect effects on Nup214 and RanBP2, as well as direct effects on RanBP1, Gle1 and DDX19 mRNAs. Moreover, RanBP2 is a potent and specific inhibitor of the mRNA export function of eIF4E. While RanBP2 depletion leads to a loss of cytoplasmic fibrils, it does not lead to substantial defects in the export of specific NES containing cargoes or bulk mRNA export (Dawlaty et al., 2008; Hutten and Kehlenbach, 2006; Walther et al., 2002). In fact, and consistent with the eIF4E phenotype, RanBP2 hypomorph mice have substantially higher rates of spontaneous tumour formation than littermates (Dawlaty et al., 2008). In contrast, deletion of RanBP2 is lethal and RanBP2−/− MEFs harbor substantial bulk mRNA export defects (Hamada et al., 2011). Importantly, the regions of RanBP2 involved in eIF4E dependent and bulk mRNA export are distinct, involving the zinc fingers and leucine rich domains respectively (Figure 3, Hamada et al., 2011).

Why reduce RanBP2 and increase RanBP1 levels given they play similar roles in cargo disassembly? RanBP2 at 358 kDa forms the cytoplasmic fibrils of the NPC while RanBP1 is a soluble 23 kDa protein. RanBP2 may provide docking sites for the sequestration of 4E-SE specific export factors. Reduction in RanBP2 may enable faster release/recycling of specific rate limiting factors back to the nucleus, leading to increased export of eIF4E sensitive mRNAs (Figure S7). Here, elevated RanBP1 compensates for reduced RanBP2. Consistently, studies in RanBP2−/− cells suggest that docking of CRM1 to RanBP2 is not critical for CRM1 cargo dissassembly (Hamada et al., 2011), presumably due to RanBP1. In summary, eIF4E overexpression favors a RanBP1 release pathway. These changes likely enhance export by promoting release and/or recycling of export complexes (Figure S7).

Why deplete Nup214 and RanBP2 from the nuclear rim? Importantly, Nup 214 localization is independent of RanBP2, and vice versa (Hutten and Kehlenbach, 2006; Walther et al., 2002). This suggests that the re-localization of Nup214 and RanBP2 upon eIF4E overexpression is a targeted event. The factors controlling their localization, and thus the associated mechanism, are unknown. Notably, Nup214 is found on nuclear and cytoplasmic sides of the NPC and may bind multiple sites within the NPC (Boer et al., 1997). Mechanistically, Nup214 directly binds CRM1 and forms a GTPase resistant Nup214-CRM1-RanGTP complex (Hutten and Kehlenbach, 2006; Walther et al., 2002). Thus reducing Nup214 at the NPC may enhance release of 4ESE RNA cargoes. In the absence of Nup214, hCG1/NLP1 controls the localization of Gle1 and enables mRNA export (Kendirgi et al., 2005). Importantly, hCG1 also directly binds CRM1, RanGTP and cargoes, and here, RanBP1 dissociates this complex (Kendirgi et al., 2005; Waldmann et al. 2012). Given that hCG1 does not change upon eIF4E overexpression (Figure 1 & S1), it is tempting to speculate that perhaps a hCG1, CRM1, Ran, 4E-SE cargo (plus accessory factors) complex forms. Additionally, it is possible that modulation of Nup214 or RanBP2 enables unknown functions that promote export of eIF4E target mRNAs.

eIF4E overexpression may not only change CRM1 dependent export of 4E-SE RNAs but also modulate the export of a subset of mRNAs using the TAP/NXF1-Gle1-DDX19 pathway. Thus, there could be cross-talk between these RNA export pathways. Alternatively, the RNA helicase activity of DDX19 may be important for remodeling 4E-SE export RNPs. Additionally, DDX19 and Gle1 play independent roles in the initiation and termination of translation for some mRNAs (Alcazar-Roman et al.; Bolger et al., 2008; Gross et al., 2007) and it may be these activities that are relevant. Thus, in addition to its direct effects on translation, eIF4E could impact on translation indirectly via its effects on DDX19 and Gle1. In all, these findings strongly suggest that substantial interplay exists between the bulk and eIF4E dependent mRNA export machinery and perhaps, between these mRNA export and translation pathways.

Our studies provide a link between eIF4E’s ability to reprogram the NPC, promote mRNA export and transform cells. The S53A mutant is impaired in reducing and relocalizing RanBP2, increasing RanBP1, DDX19 and Gle1, relocalizing Nup214; and, in mRNA export and transformation but not in translation. Although the S53A mutant rescues yeast null in eIF4E and acts in global translation (Kaufman et al., 1993; Zhang et al., 1995), we cannot rule out the possibility that some mRNAs are more sensitive to this mutation and this reduces the oncogenic activity of eIF4E, without altering viability.

Interesting parallels can be drawn between targeting the NPC by the oncogene eIF4E and by viruses. For instance, vesicular stomatitis virus inhibits cellular mRNA export by interaction of the viral M protein with Nup98 and Rae1 (Enninga et al., 2002; Faria et al., 2005). Poliovirus impairs cellular nuclear import by degrading Nup153 and Nup62 (Gustin and Sarnow, 2001). In some cancers, CRM1 and Nup88 are overexpressed (Kohler and Hurt, 2010) but whether this drives transformation, or is a side product of it, is not known. eIF4E targets the NPC in a distinct manner from those described above. These observations suggest that there will not be a single oncogenic NPC phenotype, but rather that just as viruses co-opt the NPC using different strategies, oncogenes will modulate the NPC in specific fashions.

In conclusion, these studies are the first to show that an oncogene alters the composition of the NPC. Here, drastic modification of the cytoplasmic face of the NPC leads to enhanced mRNA export and increased oncogenic potential. We hypothesize that this activity will not be restricted to eIF4E so other oncogenes likely reprogram the NPC to subvert normal growth control.

Experimental Procedures

Reagents and constructs

pcDNA2Flag-eIF4E, MSCV-pgk-GFP-eIF4E (or mutants), pcDNA3.1-lacZ, lacZ-4E-SE (from cyclin D1) and bacterial expression constructs are from (Culjkovic et al., 2006). Human RanBP2-ZF and Nup358-4 (Yaseen and Blobel, 1999) were subcloned into pKH and confirmed by sequencing. MG132 was obtained from Sigma and Ribavirin from Kemprotec Ltd.

Cell culture

U2OS cells (ATCC) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen). 2Flag-eIF4E wildtype or mutant and LacZ/LacZ-4E-SE +/− 2Flag-eIF4E U2OS cell lines were generated as in (Culjkovic 2006). MSCV-pgk-GFP-eIF4E wildtype or mutants were used for retroviral transduction of U2Os cells as in (Culjkovic et al., 2008). For siRNA experiments, U2Os cells were transfected with Lipofectamine 2000 (Invitrogen) and 20 nM siRNA duplexes and analyzed 96 h after transfection. Transient transfections were carried out using TransIT®-LT1 Transfection Reagent (Mirus). 50–70% confluent cells were used for protein and RNA preparation. See supplement for antibodies, primers, siRNAs and subcellular fractionation.

Anti-FLAG® M2 agarose affinity beads were used to purify 2FLAG eIF4E as in (Culjkovic et al., 2005). For RIP, complexes were heated to 95°C for 5min in Tris-EDTA containing 1% SDS and isolated using Trizol. DNAse treated RNA samples (TurboDNase, Ambion) were reverse transcribed using MMLV reverse transcriptase (Invitrogen). qRTPCR analyses were performed using EXPRESS SYBR® GreenER™ qPCR SuperMix (Invitrogen) in AB StepOne thermal cycler using the relative standard curve method (Applied Biosystems User Bulletin #2).

Anchorage dependent foci assays

500 cells were seeded per 10cm plate for 14 days, then stained with Giemsa (Sigma).

Immunofluorescence, and laser-scanning confocal microscopy

U2Os cells were grown on coverslips, fixed in 2% paraformaldehyde for 15 minutes at RT, washed 3 times with PBS and permeabilized with 0.2% (v/v) Triton X-100 for 10 minutes at RT. Upon permeabilization, cells were washed 3 times, blocked for 1h, and incubated with 1° antibodies (1:1000 dilution) overnight at 4°C, followed by 3 washes in blocking solution. Cells were then incubated with 2° donkey anti-rabbit IgG-TexasRed antibody (Jackson Immunolaboratories, diluted 1:150 in blocking solution), washed 4 times with 1xPBS (7.4) and mounted in Vectashield with DAPI (Vector laboratories). Analysis was carried out using a laser-scanning confocal microscope (LSM510; Carl Zeiss, Inc.), exciting 405 and 543nm with a 100x objective and 2x digital magnification. Channels were detected separately, with no cross talk observed. Confocal micrographs represent single sections through the plane of the cell.

Supplementary Material

01

Highlights.

  • eIF4E remodels the cytoplasmic face of the nuclear pore targeting RanBP2 and Nup214

  • eIF4E requires its mRNA export and NPC remodelling functions for transformation

  • RanBP2 suppresses eIF4E dependent mRNA export and oncogenic transformation

  • Oncogenes can harness the proliferative capacity of the cell by targeting the NPC

Acknowledgments

We are grateful for RanBP2 constructs, hypomorphs and wildtype cells from Jan Van Deursen and Nabeel Yaseen, and for helpful discussions with Susan Wente and Beatriz Fontoura. This work was supported by NIH RO1 80728. AB held a Cole Foundation fellowship. K.L.B.B. holds a Canada Research Chair.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errorsmaybe discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Alcazar-Roman AR, Bolger TA, Wente SR. Control of mRNA export and translation termination by inositol hexakisphosphate requires specific interaction with Gle1. J Biol Chem. 285:16683–16692. doi: 10.1074/jbc.M109.082370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Assouline S, Culjkovic B, Cocolakis E, Rousseau C, Beslu N, Amri A, Caplan S, Leber B, Roy DC, Miller WH, Jr, et al. Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin. Blood. 2009;114:257–260. doi: 10.1182/blood-2009-02-205153. [DOI] [PubMed] [Google Scholar]
  3. Boer JM, van Deursen JM, Croes HJ, Fransen JA, Grosveld GC. The nucleoporin CAN/Nup214 binds to both the cytoplasmic and the nucleoplasmic sides of the nuclear pore complex in overexpressing cells. Exp Cell Res. 1997;232:182–185. doi: 10.1006/excr.1997.3502. [DOI] [PubMed] [Google Scholar]
  4. Bolger TA, Folkmann AW, Tran EJ, Wente SR. The mRNA export factor Gle1 and inositol hexakisphosphate regulate distinct stages of translation. Cell. 2008;134:624–633. doi: 10.1016/j.cell.2008.06.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borden KL, Culjkovic-Kraljacic B. Ribavirin as an anti-cancer therapy: Acute Myeloid Leukemia and beyond? Leukemia and ymphoma. 2010;51:1805–1815. doi: 10.3109/10428194.2010.496506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen N, Sharma M, Kentsis A, Perez JM, Strudwick S, Borden KL. PML RING suppresses oncogenic transformation by reducing the affinity of eIF4E for mRNA. Embo J. 2001;20:4547–4559. doi: 10.1093/emboj/20.16.4547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Culjkovic B, Tan K, Orolicki S, Amri A, Meloche S, Borden KL. The eIF4E RNA regulon promotes the Akt signaling pathway. J Cell Biol. 2008;181:51–63. doi: 10.1083/jcb.200707018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KL. eIF4E promotes nuclear export of cyclin D1 mRNAs via an element in the 3′UTR. J Cell Biol. 2005;169:245–256. doi: 10.1083/jcb.200501019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Culjkovic B, Topisirovic I, Skrabanek L, Ruiz-Gutierrez M, Borden KL. eIF4E is a central node of an RNA regulon that governs cellular proliferation. J Cell Biol. 2006;175:415–426. doi: 10.1083/jcb.200607020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dawlaty MM, Malureanu L, Jeganathan KB, Kao E, Sustmann C, Tahk S, Shuai K, Grosschedl R, van Deursen JM. Resolution of sister centromeres requires RanBP2-mediated SUMOylation of topoisomerase IIalpha. Cell. 2008;133:103–115. doi: 10.1016/j.cell.2008.01.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Enninga J, Levy DE, Blobel G, Fontoura BM. Role of nucleoporin induction in releasing an mRNA nuclear export block. Science. 2002;295:1523–1525. doi: 10.1126/science.1067861. [DOI] [PubMed] [Google Scholar]
  12. Faria PA, Chakraborty P, Levay A, Barber GN, Ezelle HJ, Enninga J, Arana C, van Deursen J, Fontoura BM. VSV disrupts the Rae1/mrnp41 mRNA nuclear export pathway. Mol Cell. 2005;17:93–102. doi: 10.1016/j.molcel.2004.11.023. [DOI] [PubMed] [Google Scholar]
  13. Gross T, Siepmann A, Sturm D, Windgassen M, Scarcelli JJ, Seedorf M, Cole CN, Krebber H. The DEAD-box RNA helicase Dbp5 functions in translation termination. Science. 2007;315:646–649. doi: 10.1126/science.1134641. [DOI] [PubMed] [Google Scholar]
  14. Gustin KE, Sarnow P. Effects of poliovirus infection on nucleocytoplasmic trafficking and nuclear pore complex composition. EMBO J. 2001;20:240–249. doi: 10.1093/emboj/20.1.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hamada M, Haeger A, Jeganathan KB, van Ree JH, Malureanu L, Walde S, Joseph J, Kehlenbach RH, van Deursen JM. Ran-dependent docking of importin-beta to RanBP2/Nup358 filaments is essential for protein import and cell viability. J Cell Biol. 2011;194:597–612. doi: 10.1083/jcb.201102018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hodge CA, Tran EJ, Noble KN, Alcazar-Roman AR, Ben-Yishay R, Scarcelli JJ, Folkmann AW, Shav-Tal Y, Wente SR, Cole CN. The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1. Genes Dev. 2011;25:1052–1064. doi: 10.1101/gad.2041611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hutten S, Kehlenbach RH. Nup214 is required for CRM1-dependent nuclear protein export in vivo. Mol Cell Biol. 2006;26:6772–6785. doi: 10.1128/MCB.00342-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hutten S, Kehlenbach RH. CRM1-mediated nuclear export: to the pore and beyond. Trends Cell Biol. 2007;17:193–201. doi: 10.1016/j.tcb.2007.02.003. [DOI] [PubMed] [Google Scholar]
  19. Kaufman RJ, Murtha-Riel P, Pittman DD, Davies MV. Characterization of wild-type and Ser53 mutant eukaryotic initiation factor 4E overexpression in mammalian cells. J Biol Chem. 1993;268:11902–11909. [PubMed] [Google Scholar]
  20. Kehlenbach RH, Dickmanns A, Kehlenbach A, Guan T, Gerace L. A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export. J Cell Biol. 1999;145:645–657. doi: 10.1083/jcb.145.4.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kendirgi F, Rexer DJ, Alcazar-Roman AR, Onishko HM, Wente SR. Interaction between the shuttling mRNA export factor Gle1 and the nucleoporin hCG1: a conserved mechanism in the export of Hsp70 mRNA. Mol Biol Cell. 2005;16:4304–4315. doi: 10.1091/mbc.E04-11-0998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kentsis A, Topisirovic I, Culjkovic B, Shao L, Borden KL. Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap. Proc Natl Acad Sci U S A. 2004;101:18105–18110. doi: 10.1073/pnas.0406927102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kentsis A, Volpon L, Topisirovic I, Soll CE, Culjkovic B, Shao L, Borden KL. Further evidence that ribavirin interacts with eIF4E. Rna. 2005;11:1762–1766. doi: 10.1261/rna.2238705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kohler A, Hurt E. Gene regulation by nucleoporins and links to cancer. Mol Cell. 2010;38:6–15. doi: 10.1016/j.molcel.2010.01.040. [DOI] [PubMed] [Google Scholar]
  25. Lazaris-Karatzas A, Montine KS, Sonenberg N. Malignant transformation by a eukaryotic initiation factor subunit that binds to mRNA 5′ cap. Nature. 1990;345:544–547. doi: 10.1038/345544a0. [DOI] [PubMed] [Google Scholar]
  26. Noble KN, Tran EJ, Alcazar-Roman AR, Hodge CA, Cole CN, Wente SR. The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1. Genes Dev. 2011;25:1065–1077. doi: 10.1101/gad.2040611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Singh BB, Patel HH, Roepman R, Schick D, Ferreira PA. The zinc finger cluster domain of RanBP2 is a specific docking site for the nuclear export factor, exportin-1. J Biol Chem. 1999;274:37370–37378. doi: 10.1074/jbc.274.52.37370. [DOI] [PubMed] [Google Scholar]
  28. Strambio-De-Castillia C, Niepel M, Rout MP. The nuclear pore complex: bridging nuclear transport and gene regulation. Nat Rev Mol Cell Biol. 2010;11:490–501. doi: 10.1038/nrm2928. [DOI] [PubMed] [Google Scholar]
  29. Topisirovic I, Guzman ML, McConnell MJ, Licht JD, Culjkovic B, Neering SJ, Jordan CT, Borden KL. Aberrant eukaryotic translation initiation factor 4E-dependent mRNA transport impedes hematopoietic differentiation and contributes to leukemogenesis. Mol Cell Biol. 2003;23:8992–9002. doi: 10.1128/MCB.23.24.8992-9002.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Topisirovic I, Siddiqui N, Lapointe VL, Trost M, Thibault P, Bangeranye C, Pinol-Roma S, Borden KL. Molecular dissection of the eukaryotic initiation factor 4E (eIF4E) export-competent RNP. EMBO J. 2009 doi: 10.1038/emboj.2009.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Um JW, Min DS, Rhim H, Kim J, Paik SR, Chung KC. Parkin ubiquitinates and promotes the degradation of RanBP2. J Biol Chem. 2006;281:3595–3603. doi: 10.1074/jbc.M504994200. [DOI] [PubMed] [Google Scholar]
  32. Waldmann I, Spillner C, Kehlenbach RH. The nucleoporin-like protein NLP1 (hCG1) promotes CRM1-dependent nuclear protein export. J Cell Sci. 125:144–154. doi: 10.1242/jcs.090316. [DOI] [PubMed] [Google Scholar]
  33. Walther TC, Pickersgill HS, Cordes VC, Goldberg MW, Allen TD, Mattaj IW, Fornerod M. The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import. J Cell Biol. 2002;158:63–77. doi: 10.1083/jcb.200202088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wente SR, Rout MP. The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol. 2010;2:a000562. doi: 10.1101/cshperspect.a000562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yaseen NR, Blobel G. GTP hydrolysis links initiation and termination of nuclear import on the nucleoporin nup358. J Biol Chem. 1999;274:26493–26502. doi: 10.1074/jbc.274.37.26493. [DOI] [PubMed] [Google Scholar]
  36. Zhang Y, Klein HL, Schneider RJ. Role of Ser-53 phosphorylation in the activity of human translation initiation factor eIF-4E in mammalian and yeast cells. Gene. 1995;163:283–288. doi: 10.1016/0378-1119(95)00302-m. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS398117-supplement-01.pdf (1.5MB, pdf)

RESOURCES