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

Abstract

The eukaryotic translation initiation factor eIF4E is a potent oncogene that promotes the nuclear export and translation of specific transcripts. Here, we have 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 most likely slow the release and/or recycling of critical export factors to the nucleus. eIF4E overcomes this inhibitory mechanism by indirectly reducing levels of RanBP2. More generally, these results suggest that reprogramming the NPC is a means by which oncogenes can harness the proliferative capacity of the cell.

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

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.

Figure 2. Effects of eIF4E inhibition on the NPC

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.

Figure 3. RanBP2 suppresses eIF4E dependent mRNA export and transformation

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.

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

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.