Purification of a multimeric complex involved in mRNA export by iTAP.
Using a combination of tandem affinity purification and RNAi (iTAP) in S2 cells, we have shown that human p15 (hsp15) fused to the tandem affinity purification (TAP) tag copurifies with three proteins: RanBP2, NXF1, and RanGAP1 (9
). This tetrameric complex was purified from a polyclonal S2 cell line constitutively expressing TAP-tagged hp15, in which the expression of endogenous Drosophila
p15 was depleted by RNAi. A typical purification is shown in Fig. . When proteins bound to TAP-tagged hsp15 were purified from cells in which expression of RanBP2 was also silenced by RNAi, selection of RanGAP1 was strongly reduced, indicating that binding of NXF1-p15 dimers to RanBP2 was direct, whereas RanGAP1 was tethered to the complex via RanBP2 (data not shown). Similarly, TAP-tagged hsNXF1 transiently expressed in human embryonic kidney (HEK) 293 cells copurifies with RanBP2, RanGAP1, and hsp15 (Fig. ). Hence, this network of protein interactions is conserved.
FIG. 1. RanBP2 forms a stable complex with NXF1-p15 dimers and is required for cell proliferation. (a) S2 cells expressing TAP-tagged hsp15 were depleted of endogenous Drosophila p15 by RNAi. Five days after addition of Drosophila p15 dsRNA, proteins bound to (more ...)
Human RanBP2 contains an amino-terminal 700-residue leucine-rich region (a short part of which is similar to TPR repeats), four RanGTP binding domains related to the one in RanBP1 (RanBP1 homologous domains or RanBD), eight zinc finger motifs, and a carboxy terminus with homology to cyclophilin A (Fig. ). The protein contains several XFXFG repeats scattered along its C-terminal half (37
). We have identified orthologs in all the completely sequenced animal genomes but not in fungi (Fig. ). However, there are considerable differences in the domain architectures among the different species. The ortholog of RanBP2 in Caenorhabditis elegans
and the two recently duplicated paralogs in Fugu rubipens
are considerably shorter and lack structural domains at the N and C termini. The flexibility in the domain composition was also confirmed by the different number of zinc fingers in the human (8
) and mouse (6
) proteins. This difference is not due to alternative splicing patterns between the two species because the zinc finger variable region is encoded by a single exon (see also reference 7
RanBP2 architecture is more similar to the mammalian orthologs than to the other available chordate sequences (F. rubipens
and Ciona intestinalis
), which in turn resemble the C. elegans
ortholog. A phylogenetic tree with the region shared between all sequences supports the expected taxonomy. It is thus likely that several independent domain losses occurred in F. rubipens
, C. intestinalis
, and C. elegans
(Fig. ) but that domains have been gained by the mammalian genes. The additional domains in the mammalian proteins lead to novel functions, as in the case of the region of human RanBP2 which was recently shown to act as an E3 ligase in the sumoylation reaction (21
In Drosophila cells, RanBP2 is an essential nucleoporin required for efficient export of bulk mRNA.
The presence of RanBP2 in a complex including NXF1-p15 dimers suggests a role for this protein in mRNA export. To address this possibility, we analyzed the effect of depleting RanBP2 on cell proliferation and export of bulk mRNA. S2 cells were treated with a dsRNA corresponding to the N terminus of Drosophila RanBP2. A dsRNA corresponding to GFP was used as a control. The efficiency of the depletion was tested by RT-PCR and Western blot analysis (Fig. ). Four days after the addition of RanBP2 dsRNA, the steady-state expression levels of the targeted mRNA were reduced below 30% of the levels detected in untreated cells (Fig. , lane 4 versus lane 1), whereas the protein levels were below 50% of the wild-type levels (Fig. , lane 6 versus lane 2).
Remarkably, cell proliferation was already inhibited 3 days after the addition of RanBP2 dsRNA (Fig. ). This inhibition was comparable to that observed when the essential mRNA export receptor NXF1 was depleted (Fig. ), despite the fact that levels of RanBP2 were reduced by less than 50% (Fig. ). This complete inhibition of cell proliferation indicates that the partial depletion of RanBP2 is not due to a low efficiency of the dsRNA but is likely to reflect a low turnover rate of the protein and the fact that the residual protein is not diluted over time because the cells stop dividing.
The intracellular distribution of bulk poly(A)+ RNA in control cells and in cells depleted of RanBP2 was investigated by FISH with an oligo(dT) probe. The NPCs were stained with fluorescently labeled WGA. In control cells the oligo(dT) signal was mainly cytoplasmic (Fig. ). Following RanBP2 depletion a significant nuclear accumulation of poly(A)+ RNA was observed (Fig. ). The oligo(dT) signal was widespread within the nucleoplasm but was excluded from the large nucleolus (Fig. ). The nuclear accumulation of poly(A)+ RNA correlated with RanBP2 depletion, as it was detected in about half of the cell population 3 days after the addition of RanBP2 dsRNA and in ca. 90% of the cell population on day 5, in several independent experiments (Fig. and data not shown). This accumulation was, however, not as strong as that observed when NXF1 was depleted (Fig. ).
FIG. 2. Depletion of endogenous RanBP2 causes the accumulation of poly(A)+ RNA within the nucleus. (a to p) S2 cells were treated for 5 days with dsRNAs corresponding to GFP (control) Drosophila RanBP2 or Drosophila NXF1. poly(A)+ RNA was detected (more ...)
The partial inhibition of bulk mRNA export is likely to reflect the inefficient depletion of RanBP2. Since a stronger inhibition was not observed on days 7 and 8 (data not shown), we performed all experiments described below on days 5 and 6, when most cells accumulated poly(A)+
RNA within the nucleus. Notably, the mRNA export block observed in RanBP2-depleted cells is not a direct consequence of the inhibition of cell proliferation, as depletion of the essential protein eIF4G or Cdc37 also results in a strong inhibition of cell proliferation, which is not accompanied by a nuclear accumulation of poly(A)+
; A. Herold, unpublished data). Conversely, the inhibition of cell proliferation may not be a direct consequence of the mRNA export block but could reflect additional functions of this nucleoporin (26
In contrast to the changes in the staining pattern of the oligo(dT) probe, the intensity of the WGA staining was not detectably affected in cells depleted of RanBP2 (Fig. ). Similarly, nuclear envelope staining with the monoclonal antibody mAb414, which recognizes several nucleoporins, was not significantly affected in the RanBP2 knockdown (data not shown), suggesting that the integrity of the NPC is not severely compromised. Our results indicate that RanBP2 is an essential nucleoporin whose depletion leads to the inhibition of both mRNA export and cell proliferation.
RanBP2 functions in the mRNA export pathway.
It was recently shown that Drosophila
cells depleted of essential mRNA export factors such as NXF1, p15, or UAP56 display strikingly similar mRNA expression profiles that represent a specific signature of the mRNA export pathway (15
). In these cells, the total and cytoplasmic levels of most mRNAs are significantly reduced, whereas genes encoding export factors (i.e., NXF1, p15, and REF1) are upregulated as a result of a feedback loop whose underlying mechanism remains to be established (15
We expected that if RanBP2 were acting in the same pathway as NXF1 and p15, its depletion should lead to changes in mRNA expression levels similar to those observed in NXF1-depleted cells. Northern blot analysis of total RNA samples isolated from RanBP2-depleted cells revealed that the levels of nxf1, p15, and ref1 mRNAs were indeed increased (Fig. ). In contrast, the expression levels of rp49 mRNA (encoding ribosomal protein L32) or of hsp83 mRNA were reduced (Fig. , lanes 1 to 3).
FIG. 3. RanBP2 acts in the mRNA export pathway. (a and b) S2 cells were treated with GFP, NXF1, and RanBP2 dsRNAs. Six days later, cells were kept at 25°C or subjected to a 1-h heat shock at 37°C. Total RNA was isolated and analyzed by Northern (more ...)
We also compared the steady-state expression levels of two mRNAs (rp49 and y14 mRNAs) that have been shown to be underrepresented in the cytoplasm of cells depleted of NXF1, p15, or UAP56 (15
). Northern blot analysis revealed that rp49 and y14 mRNAs were underrepresented in the cytoplasm of cells depleted of RanBP2 (Fig. , lanes 4 to 6). The reduction in rp49 or y14 mRNA levels was more pronounced in the cytoplasmic samples than in the total samples, as expected in cells in which mRNA export is inhibited (Fig. , lanes 4 to 6 versus lanes 1 to 3). Overall, except for the nxf1 mRNA, the changes in mRNA expression levels in the RanBP2 knockdown exhibited the same trend as in the NXF1 knockdown but were less pronounced, in agreement with the observation that the inhibition of mRNA export is partial.
The reduction in mRNA levels observed in RanBP2-depleted cells is unlikely to reflect a general inhibition of transcription as hsp70 and hsp83 mRNAs were strongly induced after shifting the cells to 37°C for 1 h (Fig. , lanes 4 to 6 versus lanes 1 to 3). Moreover, depletion of RanBP2 does not lead to a general block of pre-mRNA splicing, as no accumulation of unspliced hsp83 pre-mRNA was observed in depleted cells at temperatures below 37°C. In contrast, hsp83 pre-mRNA did accumulate in depleted cells as well as in control cells after inhibition of splicing by heat stress at 37°C (Fig. , lanes 4 to 6).
The general inhibition of mRNA export in cells depleted of RanBP2 was confirmed indirectly by metabolic labeling. In cells depleted of RanBP2, the incorporation of [35S]methionine into newly synthesized proteins decreased significantly relative to the control cells (Fig. , lane 3 versus lane 1). Moreover, the expression of HSP70 and HSP83 proteins following heat stress was also reduced (Fig. , lane 6 versus lane 4) despite the fact that the corresponding mRNAs were strongly induced (Fig. ).
To determine whether the inhibition of heat shock protein synthesis in RanBP2-depleted cells was caused by a failure to export the heat shock mRNAs, we analyzed the intracellular distribution of hsp70 mRNA in control cells and cells depleted of RanBP2 after transcriptional induction at 37°C. In control cells, the hsp70 mRNA was detected mainly in the cytoplasm (Fig. ). In contrast, a clear nuclear accumulation of hsp70 mRNA was observed in cells depleted of RanBP2 (Fig. ). In agreement with the results obtained for poly(A)+ RNAs, the nuclear accumulation of hsp70 mRNA was not as strong as that observed when NXF1 was depleted (Fig. ).
FIG. 4. hsp70 mRNA accumulates in the nucleus of cells depleted of RanBP2. (a to o) S2 cells were treated with GFP, NXF1, and RanBP2 dsRNAs. In panels d to o, cells were shifted to 37°C for 1 h, 4 days (NXF1), or 6 days (RanBP2) after the addition of (more ...)
The accumulation of poly(A)+ RNA and hsp70 mRNA within the nucleus of cells depleted of RanBP2 together with the similarities of the effects on mRNA expression levels and protein synthesis observed following RanBP2 or NXF1 depletions indicate that RanBP2 functions in the mRNA export pathway.
RanBP2 provides a major binding site for NXF1-p15 dimers at the NPC.
We next investigated the subcellular localization of NXF1 in cells in which expression of the ranBP2
gene was silenced. We expected that if NXF1-p15 is recruited to the NPC via the interaction with RanBP2, its depletion should result in the dissociation of NXF1-p15 from the NPC. To monitor the efficiency of RanBP2 depletion, cells were labeled with oligo(dT). To visualize the localization of the heterodimer, a polyclonal S2 cell line expressing GFP-NXF1 was used. GFP-NXF1 reflects the subcellular localization of endogenous NXF1 and promotes export of reporter mRNAs in human cells (1
). Moreover, NXF1 derivatives N-terminally fused to GFP restore mRNA export in S2 cells depleted of the endogenous protein (4
), indicating that the GFP tag does not interfere with NXF1 export activity.
In the untreated cell population, GFP-NXF1 was detected within the nucleoplasm and at the nuclear rim in all cells expressing this protein at detectable levels (Fig. ). These represent ca. 40% of the cell population (Fig. ). The nuclear localization of GFP-NXF1 was observed by confocal microscopy (Fig. ) and wide-field fluorescence microscopy (Fig. ), indicating that the protein is predominantly nuclear at steady state. After depletion of RanBP2, the intensity of the GFP signal detected in single optical sections was strongly reduced and only 8% of the cells exhibited clear nuclear staining (Fig. ). Wide-field fluorescence microscopy revealed that the GFP signal was no longer localized at the nuclear rim and within the nucleoplasm but was dispersed throughout the cell with a significant fraction of the protein in the cytoplasm (Fig. ). The nuclear staining of the oligo(dT) probe indicated that RanBP2 was depleted efficiently (Fig. ).
FIG. 5. RanBP2 provides a major binding site for NXF1 at the NPC. (a to j) S2 cells constitutively expressing GFP-NXF1 (green) were treated with RanBP2 dsRNA for 5 days as indicated. poly(A)+ RNA was detected by FISH with an oligo(dT) probe (red). All (more ...)
The expression levels of GFP-NXF1 in the RanBP2 knockdown were similar to those detected in control cells, as judged by Western blot analysis (Fig. , lane 8 versus lane 7), indicating that the detection of the GFP signal in the cytoplasm reflects a change in its subcellular localization. To estimate the shift in steady-state distribution of GFP-NXF1, we quantitated the fluorescence signal in the nucleus and cytoplasm of at least 18 representative control or depleted cells. The cytoplasmic fraction of NXF1 increased from 28% ± 9% to 42% ± 12% after depletion of RanBP2 (Fig. ).
Similar results were obtained when the localization of a derivative of NXF1 having two UBA-like domains but no NTF2-like domain was investigated (Fig. S1 in the supplemental material [http://www.embl.de/ExternalInfo/izaurral/ranbp2
]). Although GFP-NXF1-2× UBA does not interact with p15, it localizes at the nuclear rim (Fig. S1c and e in the supplemental material [http://www.embl.de/ExternalInfo/izaurral/ranbp2
), and this nuclear rim staining was strongly reduced in RanBP2-depleted cells (Fig. S1h and j in the supplemental material [http://www.embl.de/ExternalInfo/izaurral/ranbp2
]), indicating that NXF1-2× UBA binds to RanBP2 without p15. Thus, the interaction of NXF1-p15 with RanBP2 is mediated by NXF1. This conclusion is fully consistent with available data showing that NXF1, but not p15, binds directly to nucleoporin FG repeats and is responsible for the localization of the heterodimer at the NPC (1
To summarize, in cells depleted of RanBP2, the association of NXF1 (or NXF1-2× UBA) with the NPC at steady state is strongly reduced and a larger fraction of the protein is detected in the cytoplasm.
The selectivity of the NPC is not altered in RanBP2-depleted cells.
In the RanBP2 knockdown, NXF1 partially localizes to the cytoplasm. This observation led us to investigate whether the general selectivity of the NPC was altered so that many nuclear or cytoplasmic proteins redistributed between the two compartments. Moreover, since there is no RanBP1 homolog in Drosophila, RanBP2 is probably the only Ran binding protein that, together with RanGAP1, activates GTP hydrolysis by Ran. It was therefore of particular relevance to address whether the partial depletion of RanBP2 resulted in perturbations of the Ran system and, consequently, of nucleocytoplasmic transport. To this end, we analyzed the subcellular localization of proteins that reside either in the nucleoplasm or in the cytoplasm at steady state.
We found that in the RanBP2 knockdown, the nuclear localization of endogenous REF1 was unchanged, even in cells strongly accumulating poly(A)+
RNA within the nucleus (Fig. ). Similarly, the distribution of Y14 was not changed in cells depleted of RanBP2 (Fig. ). These results indicate that not all nuclear proteins redistribute to the cytoplasm in the RanBP2 knockdown. Moreover, the cytoplasmic RNA-associated proteins YPS and Exuperantia were excluded from the nuclear compartment in cells depleted of RanBP2 (Fig. and data not shown). We conclude that the selectivity of the NPC was not compromised following depletion of RanBP2. These results are consistent with those reported by Walther et al. (34
), which show that nuclei assembled in vitro in Xenopus
egg extracts depleted of RanBP2 lack cytoplasmic filaments but exclude nonimport cargoes and are still functional for protein import mediated by importin α/β or by transportin. Nevertheless, the possibility that RanBP2 depletion affects the rate of import or export of the analyzed proteins without changing their subcellular localizations at steady state cannot be ruled out (see below).
FIG. 6. Depletion of RanBP2 does not alter the selectivity of the NPC. (A to C) S2 cells were fixed 5 days after the addition of RanBP2 dsRNA and labeled by FISH with an oligo(dT) probe [poly(A)+ RNA, red]. The nuclear envelope (WGA) was stained with (more ...) CRM1-mediated protein export is not detectably affected by partial depletion of RanBP2.
The results described above indicate that bulk protein import proceeds despite RanBP2 depletion so that no change in the steady-state subcellular distribution of the analyzed proteins (with the exception of NXF1) was detected. Under the same conditions (i.e., when the levels of RanBP2 were reduced to ca. 50% of the wild-type levels), bulk mRNA export was significantly impaired, so it was of interest to investigate whether other export pathways were affected. To this end, we analyzed the subcellular localization of a reporter protein whose export is mediated by CRM1. The PYM protein is the uncharacterized product of the wibg
). At steady state, PYM was excluded from the nucleus (Fig. ). In cells treated with leptomycin B (LMB), PYM partially accumulated within the nucleoplasm, indicating that its nuclear exclusion is a consequence of being actively exported by CRM1 (Fig. ). LMB is a cytotoxic drug that binds to CRM1 and prevents its association with RanGTP and the export cargo (10
FIG. 7. CRM1-mediated export is not inhibited in RanBP2-depleted cells. (a to f) S2 cells expressing a TAP-tagged form of Drosophila PYM were treated with LMB (Sigma) as indicated. Three hours after the addition of LMB (final concentration, 20 ng/ml), cells were (more ...)
In cells depleted of RanBP2 [and consequently showing a nuclear accumulation of poly(A)+ RNA], PYM remained entirely cytoplasmic (Fig. ). This cytoplasmic localization was not due to the inhibition of its nuclear import because when RanBP2-depleted cells were treated with LMB, PYM accumulated within the nucleus, as in control cells (Fig. ). Thus, neither import nor CRM1-mediated export of PYM was severely compromised in the RanBP2 knockdown, indicating that the translocation of receptor-cargo complexes across the NPC is not apparently affected by the depletion. Similar results were obtained when the subcellular localization of the endogenous protein Extradenticle, which is exported by CRM1, was analyzed (data not shown).
The experiments described above showed that RanBP2 depletion did not affect the steady-state subcellular localization of several nuclear and cytoplasmic proteins but did not address whether import or export rates were affected. To investigate this issue, we analyzed the import of PYM in living cells by FLIP. Cells expressing GFP-PYM were repetitively bleached in the nucleoplasm (Fig. ), and loss of fluorescence in the cytoplasm was monitored over time (Fig. ). In both control and RanBP2-depleted cells, the fluorescent signal in the cytoplasm could be depleted to near background levels, showing that PYM import proceeded despite RanBP2 depletion. The half-time of depletion of the cytoplasmic signal was found to be 19 ± 5 s in control cells and 27 ± 8 s in RanBP2-depleted cells (Fig. ), indicating that the import rate of PYM was slightly reduced in RanBP2-depleted cells. We conclude that although nuclear import and export proceed in RanBP2-depleted cells, the transport rates are affected for at least a subset of receptor-cargo complexes. Nevertheless, these effects may not always alter the subcellular distribution of the cargo at steady state.
Nuclear import of NXF1 is not significantly inhibited by RanBP2 depletion.
In contrast to the proteins analyzed above, the steady-state subcellular localization of NXF1 is significantly changed in RanBP2-depleted cells. This change could be due to a decrease of its import rate, an increased export, or both. To discriminate between these possibilities, we first analyzed the import of NXF1 in wild-type cells and cells treated with RanBP2 dsRNA by FRAP. In the experiment whose results are shown in Fig. , the nucleoplasmic GFP-NXF1 signal was bleached in control and knockdown cells, and the recovery of the nuclear fluorescence was measured over time. Immediately after bleaching a region of the nucleus (Fig. ), no discernible bleached zone could be seen (Fig. ). Instead, there was an immediate decrease in fluorescence intensity through the entire nucleus, indicating that NXF1 diffuses rapidly within the nucleus.
FIG. 8. Import of NXF1 is not significantly affected in RanBP2 depleted cells. (a to f) Control and RanBP2-depleted cells expressing GFP-NXF1 were imaged immediately before photobleaching a defined zone in the nucleus (white circle) and every 1.5 s afterwards. (more ...)
After the bleach, the total nucleoplasmic fluorescence partially recovered, the total cytoplasmic fluorescence decreased by a similar amount (Fig. ), and the whole-cell fluorescence remained constant (data not shown), indicating that the recovery of the nuclear signal is due to the import of the cytoplasmic pool of the protein. To compare NXF1 import rates in control and depleted cells, the recovery of the nucleoplasmic signal was measured in representative cells and normalized to the intensity of the signal when recovery was complete (160 s) (Fig. ). The nucleoplasmic signal of NXF1 recovered with similar kinetics in control and RanBP2-depleted cells (with an error of less than 20% of the rate). Simulations indicated that a strong decrease in the NXF1 import rate (>20%), which would account for the change in the steady-state distribution of GFP-NXF1 in RanBP2-depleted cells, could not fit the rate of nuclear fluorescence recovery (Fig. S2 in the supplemental material [http://www.embl.de/ExternalInfo/izaurral/ranbp2
Depletion of RanBP2 affects the release of NXF1 into the cytoplasm.
Next, we investigated the relative export rates of GFP-NXF1 in control and depleted cells by FLIP. To compare the export rate of NXF1 quantitatively in these cells, the entire cytoplasmic signal was bleached in a single pulse at time zero (Fig. ). Subsequently, cells were repetitively bleached in the cytoplasm (Fig. ), and loss of fluorescence in the nucleus was monitored over time (Fig. ). In both control and RanBP2-depleted cells, the fluorescence signal in the nucleoplasm could be depleted to near background levels, showing that the protein was exported efficiently from the nucleus.
FIG. 9. RanBP2 depletion increases the release of NXF1 in the cytoplasm. (a) Representative control and RanBP2-depleted cells expressing GFP-NXF1 were selected. A cytoplasmic region (delineated in red) was photobleached at time zero to reduce the cytoplasmic (more ...)
To compare the export rates, the loss of the nuclear signal was measured in control or knockdown cells and normalized to the intensity of the nuclear signal before bleaching (t = 0) (Fig. ). The half-time of depletion of the nucleoplasmic signal, measured by FLIP in two independent experiments, was found to be 39 ± 11 s in control cells and 27 ± 7 s in depleted cells. Assuming that NXF1 export is limited by a single rate, these results indicate that, in RanBP2-depleted cells, NXF1 is released in the cytoplasm 1.4-fold more rapidly than in wild-type cells. Note that since the cytoplasmic fraction of the protein increased from 28 to 42% after RanBP2 depletion, but we could not detect a corresponding decrease of the import rate (Fig. ), a 1.5-fold stimulation of cytoplasmic release was expected.
In summary, in RanBP2-depleted cells, NXF1 shuttles as in wild-type cells, but a higher fraction of the protein is detected in the cytoplasm. Our results indicate that this is due mainly to an increased release of the protein into the cytoplasm.
The release of NXF1 in the cytoplasm is a specific effect of RanBP2 depletion.
The faster kinetics of NXF1 release in the cytoplasm could be a specific effect of RanBP2 depletion or alternatively due to a more general effect of depleting nucleoporins localized at the cytoplasmic face of the NPC. In particular, it was of interest to determine whether depletion of CAN (which also localizes at the cytoplasmic face of the NPC) could lead to a similar phenotype. CAN, like many nucleoporins possessing FG repeats, interacts with NXF1 and together with its yeast counterpart Nup159p has been implicated in mRNA export (1
Depletion of CAN inhibited cell proliferation and led to a significant nuclear accumulation of poly(A)+
RNA (Fig. S3 in the supplemental material [http://www.embl.de/ExternalInfo/izaurral/ranbp2
] and Fig. ) (see also reference 31
). These inhibitory effects on cell proliferation and mRNA export were comparable to the inhibitory effects observed when RanBP2 was depleted (Fig. S3 in the supplemental material [http://www.embl.de/ExternalInfo/izaurral/ranbp2
] and Fig. versus Fig. ). As observed in the RanBP2 knockdown, depletion of CAN did not significantly affect NPC staining with fluorescently labeled WGA (Fig. ).
FIG. 10. Depletion of CAN inhibits mRNA export. (a to m) S2 cells were treated with a dsRNA corresponding to Drosophila CAN. poly(A)+ RNA was detected by FISH with an oligo(dT) probe (red). The nuclear envelope (WGA) was stained with Alexa 488-WGA conjugates (more ...)
In contrast to RanBP2 depletion, CAN depletion did not alter the steady-state subcellular localization of NXF1. Indeed, when cells expressing GFP-NXF1 were depleted of CAN, the GFP signal was detected at the nuclear rim and within the nucleus (Fig. ), even in cells exhibiting a strong nuclear accumulation of poly(A)+ RNA (Fig. ). Moreover, the fraction of the protein in the cytoplasm was not detectably changed despite the inhibition of mRNA export (Fig. ). These results highlight the specific role of RanBP2 in the recruitment of NXF1 to the NPC.