From the Unité de Virologie Humaine, U412 INSERM, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Lyon Cedex 07, France
Received for publication, August 23, 2002, and in revised form, October 3, 2002
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ABSTRACT |
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A striking characteristic of mRNA export
factors is that they shuttle continuously between the cytoplasm and the
nucleus. This shuttling is mediated by specific factors interacting
with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the
Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta,
or SM) localized at the N terminus of the protein between amino acids
61 and 146. We have also found that a previously described double NES
(amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain
is essential for EB2-mediated mRNA export. Accordingly, in
vivo, EB2 is found in complexes containing REF as well as the
cellular factor TAP. However, these interactions are RNase-sensitive,
suggesting that the RNA is an essential component of these complexes.
In cells infected by human herpesviruses, viral mRNAs and
proteins are trafficked through the nuclear pore complex. Several cellular factors that mediate the nucleocytoplasmic transport of
mRNAs have now been identified (1-6). Interestingly, some human
herpesviruses carry, in their genome, genes whose products are also
mRNA export factors, such as
HSV-11
ICP27 (7) and the EBV BMLF1 early
gene product originally called EB2 (8), but later called Mta (9) or SM
(10). Such genes are conserved among all human herpesviruses,
suggesting a conserved function for their products. At least for HSV-1
and EBV, the inactivation of ICP27 (11) or EB2 (12), respectively, abolishes the production of infectious viral particles, demonstrating that ICP27 and EB2 are essential factors for viral mRNA export and
that their function cannot be trans-complemented by cellular factors. Moreover, EB2 appears to have an effect on cellular mRNAs because it has transforming properties when expressed both in established cell lines such as Rat1 and NIH3T3 and in primary rat
fibroblasts (13).
Most of the HSV-1 and EBV early and late mRNAs are transcribed from
intronless genes. However, it is now clearly established that the
nuclear export of mRNAs is dramatically increased when a splicing
event occurs (14). In effect, splicing leads to the deposition on the
mRNA of a multiprotein export complex (called EJC for
exon-exon junction complex),
including REF/Aly (Yra1 in yeast), Y14, RNPS1, SRm160, and Magoh,
20-24 nucleotides upstream of the exon-exon junction (2-5). Such a
complex is thought to export mRNAs by recruiting TAP/Mex67p (15) to
cellular messenger RNPs (16-18). For cellular mRNAs
generated from intronless genes, they are likely to be exported to the
cytoplasm by cellular factors through nonspecific interactions with
mRNA-bound adapters like REF (19) or through sequence-specific
interactions with SRp20 or 9G8 (20) or U2AF (21). It is therefore
tempting to speculate that EB2, like its HSV-1 functional homolog ICP27
(7), is an adapter of viral origin, involved in the export of
intronless early and/or late viral mRNAs.
Interestingly, it has been recently reported that ICP27 recruits the
cellular mRNA export factors REF (Aly) and TAP/NXF1 (7) to viral
mRNAs generated from viral intronless genes, providing these viral
mRNAs with access to the cellular mRNA nuclear export pathway.
However, it was clear that in the absence of viral mRNA, ICP27
nucleocytoplasmic shuttling was TAP- and CRM-1-independent. This
suggests that ICP27-mediated mRNA export and ICP27
nucleocytoplasmic shuttling follow different pathways and are probably
mediated by different ICP27 domains. However, the ICP27
CRM-1-independent nuclear export signal (NES) has not as yet been characterized.
The EBV EB2 protein also shares properties with mRNA export
factors: (i) it exports both intronless and intron-containing RNAs
(22-24); (ii) it shuttles between the cytoplasm and the nucleus (22,
24, 25); and (iii) it is thought to carry two contiguous CRM-1-dependent NES (26). However, the EB2
nucleocytoplasmic shuttling has also been described to be
CRM-1-independent (24). EB2 also appears to homodimerize or multimerize
(27) and binds to RNA in vivo (28), although no EB2-specific
RNA sequences have been identified on EB2 RNA targets.
In this report, we have characterized the EB2 sequences required for
nucleocytoplasmic shuttling and mRNA export. We show that the EB2
domain between amino acids 218 and 236, which have been reported to
carry a CRM-1-dependent double NES (26), can be deleted
without affecting the nucleocytoplasmic shuttling of EB2 and does not
constitute a functional NES. However, we demonstrate that this
designated double NES region binds REF both in vitro and
in vivo and is essential for EB2-mediated mRNA export.
Moreover, we found that the N-terminal region of EB2 carries a
CRM-1-independent NES and two nuclear localization signals (NLS) that
mediate the nucleocytoplasmic shuttling of EB2.
Plasmids--
The following eukaryotic expression plasmids are
all SV40 early promoter-based vectors. When F precedes the name of the
protein, the corresponding protein was tagged at its N terminus with
the FLAG epitope, which can be detected by monoclonal antibody M2 (Sigma). pSG5F.EB2 contains the intronless
BSLF2/BMLF1 cDNA and encodes the
wild-type EB2 protein (23). pSG5F.EB2.dNES expresses an EB2 protein
deleted of amino acids 225-236 in the two putative NES regions, as
described (26). pSG5F-EB2. Cell Lines and Transfections--
HeLa cells and 293T cells
carrying an EBV BMLF1-knockout recombinant
(293BMLF1-KO cells) were grown at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf
serum. HeLa cells were seeded at 8 × 105 cells/100-mm
Petri dish 10 h prior to transfection. Transfections of HeLa cells
were performed by the calcium precipitate method as described
previously (24). Transient transfections of 293BMLF1-KO cells (12) were performed by electroporation (950 microfarads, 220 mV)
using a Bio-Rad electroporator. Plasmids used for transfections were prepared by the alkaline lysis method and purified through two
CsCl gradients.
Quantification of CAT Protein--
To evaluate CAT protein
expression, we used CAT enzyme-linked immunosorbent assay (Roche
Molecular Biochemicals). After transfection, cells were collected in
phosphate-buffered saline. Half of the cells were use for CAT assays
according to the manufacturer's instructions. The other half were used
to monitor protein expression by Western blotting using anti-FLAG
antibody M2.
Heterokaryon Assays--
HeLa cells were transfected in 100-mm
Petri dishes by the calcium precipitate method as previously described
(31). 24 h post-transfection, the precipitate was washed, and
cells were trypsinized. Approximately 2 × 105 HeLa
cells were seeded on glass coverslips with an equal number of NIH3T3
cells in 35-mm dishes. The cells were allowed to grow overnight and
were then treated for 2 h with 100 µg/ml cycloheximide to
inhibit protein synthesis and 25 nM leptomycin B (LMB)
(kindly provided by Dr. B. Wolff) when inhibition of
CRM-1-dependent protein export was required. Subsequently,
cells were washed with phosphate-buffered saline, and heterokaryon
formation was carried out by incubating the coverslips for 2 min in
50% polyethylene glycol 3000-3700 (Sigma) in phosphate-buffered
saline. Following cell fusion, coverslips were washed extensively with
phosphate-buffered saline and returned to fresh medium containing 100 µg/ml cycloheximide and 25 mM LMB when needed. After
2 h at 37 °C, cells were fixed with 4% paraformaldehyde, and
indirect immunofluorescence was performed essentially as described previously (24). For the various immunofluorescence experiments, we
used anti-FLAG monoclonal antibody M2, anti-hnRNP-C monoclonal antibody
4F4 (kindly provided by Dr. G. Dreyfuss) (32), anti-Rev monoclonal
antibody (kindly provided by Dr. B. Wolff) (33), or
anti- Microinjections--
GST fusion proteins (2 mg/ml) were injected
into either the nucleus or the cytoplasm of HeLa cells or HeLa cell
polykaryons generated as described above using an Eppendorf Transjector
5246, an Eppendorf micromanipulator (Injectman), and a Nikon Eclipse TE200 microscope. Texas Red-labeled dextran (70 kDa; Molecular Probes,
Inc.) was systematically added at a concentration of 2 mg/ml to the
microinjection solution so as to mark the injection site.
Microinjections were monitored using a Sony digital camera and a Sony
TV monitor. 5-10 cells or polykaryons on average per experiment were
microinjected, and each experiment was repeated at least twice. After
microinjection, cells were incubated for 1 h at 37 °C and then
fixed with 4% paraformaldehyde. The GST fusion proteins were
visualized by indirect immunofluorescence as described previously (24).
Rabbit anti-GST antibody (a generous gift from Dr. J. J. Diaz) was
used at a 1:500 dilution. Fluorescein isothiocyanate-coupled goat
anti-rabbit antibody (Sigma) was used at a 1:2000 dilution.
In Vitro GST Pull-down Assays--
GST and GST fusion proteins
were expressed in BL21 pLys bacteria and purified with
glutathione-agarose beads following standard procedures.
35S-Labeled F.EB2 and F.EB2 mutants were produced in the
rabbit reticulocyte lysate in vitro
transcription/translation TNT system (Promega). Binding reactions were
carried out in 500 µl of buffer containing 150 mM NaCl,
16 mM Na2HPO4, 4 mM
NaH2PO4, 100 mM EDTA, and 1%
Triton X-100, pH 7.3, for 1 h at 4 °C, and then complexes bound
to the glutathione-agarose beads were washed five times with the same
buffer. Proteins were eluted in SDS-PAGE loading buffer and analyzed by
autoradiography. In some experiments, RNase A (10 µg) and RNase T1
(10 µg) were added to the last wash and incubated for 15 min at room temperature.
Immunoprecipitation of Transfected Cell Extracts--
For
immunoprecipitation, transfected HeLa or 293BMLF1-KO cells
were harvested from 100-mm dishes 48 h post-transfection and lysed
in 300 µl of TNE buffer (10 mM Tris-HCl, pH 8, 100 mM NaCl, and 1 mM EDTA) containing a protease
inhibitor mixture (Roche Molecular Biochemicals). The lysate was then
passed five times through a syringe with a 26-gauge needle, and cell
debris was removed by centrifugation at 13,000 × g.
Cell extracts were incubated with 50 µl of anti-FLAG monoclonal
antibody M2-agarose affinity gel (Sigma) for 4 h in TNE buffer at
4 °C. The precipitates were washed five times with cold buffer A (10 mM Tris-HCl, pH 8, 150 mM NaCl, and 1% Triton
X-100) as described by Koffa et al. (7). Proteins were
eluted by incubation of the immunoprecipitate with a peptide/FLAG
solution at 40 µg/ml in buffer A before loading onto
SDS-polyacrylamide gel. Western blot analysis was performed using
anti-FLAG monoclonal antibody M2 and rabbit polyclonal antibodies to
murine REF (KJ70) (19) and TAP (34) (both a generous gift from Dr. E. Izaurralde). In some experiments, RNase A (10 µg) and RNase T1 (10 µg) were added to the cell extracts prior to immunoprecipitation for
15 min at room temperature.
Both the NLS and NES of EB2 Are Located within a Domain between
Amino Acids 61 and 140--
A computer-assisted search for NLS
sequences indicated that EB2 contains a putative bipartite NLS (KR1 and
KR2) (Fig. 1), which could contribute to
the nucleocytoplasmic shuttling of EB2, together with the double NES
located in peptide D (NES1/NES2) (Fig. 1) previously characterized by
Chen et al. (26). To locate more precisely the EB2 NLS and
NES, we microinjected a series of fusion proteins between GST and the
EB2 peptides represented in Fig. 1 into single nuclei of HeLa cell
polykaryons. Texas Red-labeled dextran was co-injected to mark the
injection site. As shown in Fig.
2A, GST diffused passively in
the cytoplasm, but was not imported in the non-injected nuclei,
probably due to the lack of NLS. As expected, GST-peptide C, which
contains the putative bipartite NLS, was restricted to the injected
nucleus. Surprisingly, GST-peptide B was found in all the nuclei of the
injected polykaryons, suggesting that it shuttled between the nucleus
and the cytoplasm. Peptide B may thus contain both an NLS and an NES.
In contrast, GST-peptide D, which contains the putative double NES
mapped by Chen et al. (26), was restricted to the injected
nucleus. A fusion protein between GST and the KNS domain of the hnRNP-K
protein (32), used as a control shuttling protein, was found in all the
nuclei of the injected polykaryons, similar to GST-peptide B.
The GST-peptide B, C, and D fusion proteins were also injected into the
cytoplasm of HeLa cell polykaryons (Fig. 2B). Consistent with the previous results, GST-peptide B was exclusively located in the
nucleoplasm, whereas both GST-peptide C and GST-peptide D stayed at the
injection site, the cytoplasm. It should be noted that in contrast to
GST-peptide D, GST-peptide C, which was expected to be localized in the
nucleus, did not diffuse into the cytoplasm, suggesting that
GST-peptide C might be aggregated at the microinjection site. The
GST-peptide E, F, G, and H fusion proteins (Fig. 1) were also injected
into one single nucleus or into the cytoplasm of HeLa cell polykaryons
and were exclusively localized in the injected cellular compartment,
strongly suggesting that GST-peptide B did not diffuse passively (data
not shown).
Taken together, these results strongly suggest that peptide B carries
an NLS and an NES and that both are transferable. To confirm the
localization of the EB2 NLS sequences, peptides A-H were also fused to
the bacteriophage MS2 coat protein and expressed in COS-7 cells. Only
MS2-peptide B and MS2-peptide C proteins were found to be exclusively
nuclear (Fig. 3), confirming that both
peptides contain NLS sequences. Because peptide B contains only the KR1
motif of the putative bipartite NLS, this suggests that KR1 functions
independently of KR2, rather than being part of a bipartite NLS.
Accordingly (Fig. 4), mutation of the KR1 motif (KRRR Peptide B Contains a Novel CRM-1-independent Transferable
NES--
To ascertain the presence of an NES in peptide B, we wanted
to demonstrate that it could confer nucleocytoplasmic shuttling to a
heterologous non-shuttling protein whose size was incompatible with
passive diffusion between the nucleus and the cytoplasm. Moreover,
because we have previously reported that EB2 shuttles via a
CRM-1-independent pathway (24), nucleocytoplasmic shuttling of the
heterologous protein should be resistant to LMB, a specific inhibitor
of CRM-1. We therefore transferred peptide B to the Deletion of the Putative NES1/NES2 Region Located between Amino
Acids 218 and 236 Does Not Impair the Nucleocytoplasmic Shuttling of
EB2--
It has been previously reported by Chen et al.
(26) that EB2 contains a double NES, called NES1/NES2, located in
peptide D between amino acids 218 and 236 (Fig. 1). Because we found
that peptide B (amino acids 61-140) also harbors a functional NES, we
wanted to re-examine the location of the NES in the full-length EB2
protein. Two mutants with deletions in the NES1/NES2 region (amino
acids 218-236) of EB2 were generated. One mutant, called F.EB2.dNES
(Fig. 6A), has been previously
reported to be a non-shuttling protein (26). However, in our
human-mouse heterokaryon assay, this EB2 mutant shuttled at least as
efficiently, if not more efficiently, than the wild-type F.EB2 protein
(Fig. 6B). Moreover, a EB2 mutant from which the whole
NES1/NES2 region was deleted (F.EB2. The DN Region Is Essential for EB2-mediated mRNA
Export--
The various mutant proteins used in our heterokaryon
assays were then tested in a functional assay for their capacity to
export mRNAs, using the pDM128/PL reporter system (35). In this
assay, unspliced CAT-containing mRNA transcribed from pDM128/PL
(Fig. 7A) was rarely found in
the cytoplasm, whereas the spliced mRNA, which does not contain the
CAT sequence, was efficiently exported from the nucleus to the
cytoplasm. We have previously demonstrated that EB2 can
mediate the nuclear export of unspliced mRNA generated from plasmid
pDM128/PL, and this can be quantified at the level of both
CAT-expressed protein and unspliced CAT mRNA detected in the
cytoplasm (24). Plasmids expressing F.EB2, F.EB2.dNES, F.EB2. The C-terminal RGG-rich Domain of REF Binds to the EB2 DN Region in
Vitro--
As shown above, F.EB2. EB2 Interacts with REF-containing Complexes in Vivo--
The
results described above suggesting that the EB2 DN region is a REF
interaction domain were obtained by in vitro assays. To
demonstrate the existence of protein complexes associating with EB2 and
REF in vivo, F.EB2 or F.EB2.
Because TAP is the REF-interacting factor targeting REF·RNP complexes
to the nuclear pore, we also investigated whether EB2 would
co-immunoprecipitate TAP. As shown in Fig. 9A (second
panel), F.EB2 co-immunoprecipitated TAP, and this
co-immunoprecipitation was also completely abolished by the RNase
treatment. However, contrary to the co-immunoprecipitation of REF by
F.EB2, the deletion of the DN region in F.EB2.
As shown above, the in vivo co-immunoprecipitation of F.EB2
and REF is sensitive to ribonucleases. The HSV-1 EB2 homolog ICP27 has
been co-immunoprecipitated with REF from HSV-1-infected cells, and the
complexes were found to be resistant to RNase treatment (7). We
therefore also evaluated whether FLAG-tagged ICP27 (F.ICP27) transiently expressed in HeLa cells could
co-immunoprecipitate REF. We found that REF was co-immunoprecipitated
with F.ICP27 in this assay. However, RNase treatment also completely
abolished the co-immunoprecipitation of REF with F.ICP27 (Fig.
9A, first panel, lanes 8 and
9), whereas endogenous REF was unaffected by RNase treatment
(fourth panel, lanes 8 and 9).
Comparable amounts of F.ICP27 were efficiently immunoprecipitated with
and without RNase treatment (third panel, lanes 8 and 9).
As the interaction between ICP27 and REF has previously
been reported to be RNase-insensitive in the context of HSV-1-infected cells, we then asked whether the interaction between EB2 and REF might
also be resistant to RNase in the context of an EBV productive cycle.
For this, we used 293BMLF1-KO cells. When these cells are transfected with an expression vector for the EB1 transactivator to
induce the EBV productive cycle, no infectious viral particles are
produced. However, when these cells are cotransfected with expression
plasmids for both EB1 and EB2 to trans-complement the missing gene product, infectious viral particles are produced (12).
These cells were thus transfected with expression plasmids for EB1
alone, for both EB1 and F.EB2, or for EB1 and F.ICP27, and
immunoprecipitation from whole cell extracts was carried out using
anti-FLAG antibody M2. As shown in Fig. 9B, REF
co-immunoprecipitated with both F.EB2 and F.ICP27 (upper
panel, lanes 2 and 3, respectively), but
this co-immunoprecipitation was sensitive to RNase treatment (lanes 5 and 6, respectively). Thus, even in the
context of the viral infection, EB2 interacts with the cellular REF
protein in a way that is dependent upon the presence of RNA.
We have investigated the signals directing the nucleocytoplasmic
shuttling of the EBV mRNA export factor EB2. We have identified the
NLS sequences as two KR-rich motifs acting independently and a new
CRM-1-independent NES, located 90 amino acids upstream of the putative
CRM-1-dependent double NES (NES1/NES2) previously described
by Chen et al. (26). In our assay, this NES1/NES2 region
could be deleted without affecting the nucleocytoplasmic shuttling of
EB2. Moreover, we show that this domain is in fact a REF-binding
domain, essential for EB2-mediated mRNA export.
We have previously reported that EB2 shuttles between the nucleus and
the cytoplasm in an LMB-insensitive manner (24), which suggests that
the EB2 NES is CRM-1-independent, whereas others have reported that EB2
carries two contiguous CRM-1-dependent NES (NES1/NES2)
(26). Our results clearly show that this double NES, which we now call
the DN region, does not direct alone the nuclear export of EB2 because
deletion of the DN region did not impair EB2 shuttling. Moreover, a
nuclear EB2 protein lacking the N-terminal 185 amino acids, but still
carrying the DN domain (F.NLS.EB2.Cter), did not shuttle in a
heterokaryon assay. In fact, the shuttling appeared to be even more
efficient when the DN region was deleted. This could be explained by a
change in the conformation of the protein, which could make the NES
located in the N-terminal region of EB2 more accessible. Alternatively, the DN region could overlap with a nuclear retention domain. Taken together, our results suggest that (i) the DN region does not seem to
be implicated at all in the shuttling of the EB2 protein; and (ii) the
nucleocytoplasmic shuttling function is provided by the N-terminal
region of the protein, probably via a motif contained in an EB2 peptide
of 80 amino acids called peptide B and located between amino acids 61 and 140 (Fig. 1). In effect, when peptide B was transferred to a
nuclear Although not necessary for the nucleocytoplasmic shuttling of the
protein, the DN region appears to be important for EB2-mediated mRNA export, through its capacity to bind the cellular REF export factor. In effect, our data demonstrate that in vitro
synthesized EB2 interacts with bacterially purified GST-REF
independently of RNA. They also show that the REF C-terminal RGG repeat
region makes contact with the EB2 DN region in vitro. In
this respect, EB2 differs from HSV-1 ICP27, which interacts with the
RNA-binding domain of REF (7). These results therefore suggest a direct interaction between REF and EB2. However, although RNase treatment did
not abolish the in vitro EB2/REF interaction, it completely abolished the EB2/REF co-immunoprecipitation from both extracts of
transfected HeLa cells and extracts of EBV-infected cells in which a
productive cycle was induced. The discrepancy between the in
vivo co-immunoprecipitation and the in vitro GST
pull-down assays is probably due to the high amount of GST-REF used in
the latter, which displaces the equilibrium of the reaction toward an
RNA-independent interaction. Moreover, although ICP27
co-immunoprecipitation of REF has previously been shown to be resistant
to RNase when performed with cells infected with HSV-1 (7), we found
that this interaction was also completely abolished by RNase treatment when we used extracts from transfected HeLa cells. Thus, our results suggest that EB2 recruits REF to mRNAs and that this recruitment is
stabilized by RNA.
EB2 also co-immunoprecipitated TAP, a cellular RNA export receptor
known to interact with REF (15). However, in transfected HeLa cells,
this complex, which was also completely abolished by RNase treatment,
was not affected by deletion of the EB2 DN region. These results
suggest that TAP is not recruited in the complex by REF, but more
likely by RNA-bound EB2 or by other RNA-bound proteins. It is
interesting to note that co-immunoprecipitation of TAP by the EJC
factor Upf3/3X is also RNase-sensitive (39). Finally, it is also
possible that EB2 binds to messenger RNPs, but exports them
independently of REF and TAP.
Although we could not demonstrate a direct interaction between EB2 and
REF in vivo, the REF interaction DN domain is important for
EB2-mediated mRNA export. Indeed, we have previously reported that
EB2 exports unspliced mRNAs transcribed from pDM128/PL (24), a reporter plasmid widely used and designed to identify factors with
mRNA export potential. Using this reporter gene, we have now shown
that EB2 deleted of the REF-binding motif does not export unspliced
pDM128/PL mRNAs. Moreover, we have recently produced an EBV virus
whose genome is deleted for the EB2 gene. Infectious virus production
by cells harboring such a viral genome is only effective when EB2 is
provided in trans. In this assay, EB2 deleted of the
REF-binding site did not trans-complement the inactivated wild-type EB2 function (12). This suggests that the REF-binding motif
(and probably REF itself) is required both for mRNA export in
transient expression assays and for production of infectious virions.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DN expresses an EB2 protein with the two
putative NES (amino acids 218-236) entirely deleted. These two
plasmids were obtained by site-directed mutagenesis of pSG5F.EB2 using
the QuikChange site-directed mutagenesis kit (Stratagene) and the
oligonucleotide primer
5'-CTCCAAGATTACATTTGCGGCCGCGGAGCCCATCCAAGAC-3' and its
complement on the opposite strand for pSG5F.EB2.dNES, 5'-GACATGAGTCTGGTTAAGGAGCCCATCCAAGAC-3', and its complement on the opposite strand for pSG5F.EB2.
DN. Mutations in the putative EB2
NLS sequences were introduced by site-directed mutagenesis using the
QuikChange kit. For the KR1 mutation
(127KRRR130
AAAA), the oligonucleotide
primer 5'-CACCAGAGGCCACGCAGCGGCAGCCGGAGAGGTCCATGG-3' and
its complement on the opposite strand were used. For the KR2 mutation
(143KRR145
AAA), the oligonucleotide primer
5'-GATGAAAGTTATGGCGCGGCCGCACACCTGCCCCC-3' and its complement on the
opposite strand were used. pSG5F.EB2.Cter has been described elsewhere
(29). The NLS sequence from the SV40 T antigen (APKKKRKV) was
introduced between the FLAG and the EB2 C-terminal DNA sequences
to generate pSG5F.NLS.EB2.Cter. Seven partially overlapping EB2 DNA
fragments (fragments B-H) were cloned into the prokaryotic expression
vector pGEX-4T-2 (Amersham Biosciences) to produce GST proteins fused
to EB2 peptides B-H (see Fig. 1). EB2 DNA fragments B-H were
generated by PCR using the following oligonucleotide primers:
5'-CGGGATCCGATGAAGATCCAACT-3' and 5'-CCGCTCGAGACTTTCATCGGTGCA-3'
for fragment B, 5'-CGGGATCCTCTTACACCAGAGGC-3' and
5'-CCGCTCGAGGCGTTCTTGCCTCGC-3' for fragment C,
5'-CGGGATCCCCCCGGTCAGAATCT-3' and 5'-CGGCTCGAGCTGCCAGGCTCCAAT-3' for
fragment D, 5'-CGGGATCCGACCCGTTCCTACAG-3' and
5'-CCGCTCGAGGTAGGTGATCTCCTG-3' for fragment E,
5'-CGGGATCCCTCTGCACCCTGGTG-3' and 5'-CCGCTCGAGCTTGTTTTGACGGGC-3' for
fragment F, 5'-CGGGATTCCGACTACAACTTTGTG-3' and
5'-CCGCTCGAGCTCTGTCAAAAGGGA-3' for fragment G, and
5'-CGGGATCCTTCCTGGGCCACTAC-3' and
5'-CCGCTCGAGTTGATTTAATCCAGG-3' for fragment H. These
PCR fragments were subsequently digested with BamHI and
XhoI for insertion between the BamHI and
XhoI sites of pGEX-4T-2. The same EB2 DNA fragments (B-H)
were also ligated to a DNA sequence encoding the bacteriophage MS2 coat
protein in the eukaryotic expression plasmid pCMV-MS2 (a generous gift
from Dr. B. R. Cullen) (30) to produce MS2-EB2 peptide fusion
proteins. Fragments B-H were obtained by PCR amplification using
oligonucleotides similar to those listed above, except for the
substitution of the BamHI site present in each upper strand oligonucleotide with an EcoRI site. Fragment A was
obtained by PCR amplification using the following two oligonucleotides:
5'-CGGAATTCATGGTTCCTTCTCAG-3' and
5'-CGGAATTCGATGAAGATCCAACT-3'. The PCR fragments were
subsequently cut with EcoRI and XhoI for
insertion between the EcoRI and XhoI sites of
pCMV-MS2. The hnRNP-K NLS/NES DNA sequence was generated by PCR using
oligonucleotide primers 5'-CGGGATCCTATGACAGAAGAGGGAGA-3' and
5'-CCGCTCGAGATAAGCCATCTGCCATTC-3', cut with BamHI and
XhoI for insertion into pGEX-4T-2 to express GST-KNS (where
KNS is the K nuclear shuttling
domain). Plasmid pCMV.NLS.
gal was constructed by inserting the SV40
T antigen NLS into plasmid pCMV
(Clontech) by
site-directed mutagenesis (Stratagene kit) using oligonucleotide 5'-GAGCTGCTCAAGCGCGATGCTAGCGGGCCTAAGAAGAAGCGCAAAGTCAGATCTGTCGTTTTACAACGTCGTG-3' and its complement on the opposite strand. pCMV.NLS.B.
gal was then
constructed by inserting a PCR-amplified fragment generated from EB2
using the following two oligonucleotides: 5'-CGGGATCCGATGAAGATCCAACT-3' and 5'-CCGGATCCACTTTCATCGGTGCA-3'. PCR DNA fragment B was then cut with
BamHI and inserted downstream of the SV40 T antigen NLS into
a BglII site of pCMV.NLS.
gal introduced by site-directed mutagenesis concomitantly with the SV40 T antigen NLS. The REF constructs used in this study have been described elsewhere (19) and
were a generous gift from Dr. E. Izaurralde. The reporter plasmid
pDM128/PL has been described by Yang et al. (30) and was a
generous gift from Dr. B. R. Cullen.
-galactosidase antibody (Roche Molecular Biochemicals) as the
primary antibody. An Alexa Fluor-conjugated goat anti-mouse IgG
(H+L; Interchim) was used as the secondary antibody, and the nuclei of
the cells were stained by incubation with a Hoechst 33258 solution
(Sigma) at 5 µg/ml.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Location of the various EB2 peptides and
mutants used in this study on the EB2 amino acid sequence. The
80-amino acid long EB2 peptides A-H used in this study are represented
by double-headed arrows above the EB2 sequence. The two
KR-rich motifs (KR1 and KR2) are identified. The N terminus of the
truncated EB2.Cter protein is shown at position 185. The locations of
the two putative NES (NES1 and NES2) reported previously by Chen
et al. (26) are indicated, as are the sites of the deleted
sequences in F.EB2.dNES and F.EB2. NES (dotted line above
and thin line below the sequence, respectively).
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Fig. 2.
EB2 peptide B contains both an NLS and an
NES. A single nucleus of HeLa cell polykaryons
(A) or the cytoplasm of HeLa cells or HeLa cell polykaryons
(B) was microinjected with the indicated GST fusion proteins
together with Texas Red-labeled dextran (70 kDa) as a marker of the
injection site. After a 1-h incubation at 37 °C, cells were fixed
and analyzed for the localization of the GST fusion proteins using
rabbit anti-GST antibody as the primary antibody and fluorescein
isothiocyanate (FITC)-coupled goat anti-rabbit antibody.
Cell nuclei were labeled by Hoechst staining.
AAAA in F.EB2.M1) in the full-length F.EB2 protein only
slightly impaired the nuclear localization of EB2, whereas mutation of
the KR2 motif (KRR
AAA in F.EB2.M2) was silent. However, mutation
of both KR1 and KR2 (mutant F.EB2.M1.2) almost completely abrogated
nuclear translocation. This demonstrates the presence of two
independent functional NLS regions, suggesting that EB2 contains two
independent functional NLS domains.
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Fig. 3.
Both peptides B and C contain functional
NLS. COS-7 cells were transfected with plasmids expressing the
hemagglutinin (HA)-tagged MS2 fusion proteins as indicated.
The cells were immunostained using anti-hemagglutinin monoclonal
antibody (Roche Molecular Biochemicals) as the primary antibody and the
Alexa Fluor-conjugated goat anti-mouse IgG as the secondary antibody to
determine the localization of the proteins and stained with Hoechst dye
to visualize the nuclei.
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Fig. 4.
The EB2 NLS is composed of two functionally
independent KR-rich motifs. HeLa cells were transfected with
plasmids expressing FLAG-tagged mutant EB2 proteins as indicated.
Mutants F.EB2.M1 and F.EB2.M2 have their KR1 or KR2 motif (see Fig. 1)
mutated, respectively. Mutant F.EB2.M1.2 has both motifs mutated, and
F.EB2.Cter is deleted of the first 184 amino acids. The cells were
immunostained using anti-FLAG monoclonal antibody M2 as the primary
antibody and Alexa Fluor-conjugated goat anti-mouse IgG as the
secondary antibody to determine the localization of the proteins and
stained with Hoechst dye to visualize the nuclei.
-galactosidase
carrying the SV40 T antigen NLS motif (NLS.B.
gal) (Fig.
5A) and tested this fusion
protein in a human-mouse heterokaryon assay in the presence of
cycloheximide to suppress de novo protein synthesis. In this
assay (Fig. 5B), NLS.B.
gal was found both in the
transfected HeLa nuclei and in the murine nuclei of heterokaryons (panels e and f), suggesting that this protein
shuttles between the nucleus and the cytoplasm. NLS.
gal (used as a
control) was exclusively nuclear and, as expected, was found only in
the HeLa cell nuclei of heterokaryons (panels c and
d). Most interestingly, shuttling of NLS.B.
gal was
insensitive to LMB (panels k and l), similar to
that of F.EB2 (compare panels g and h with
panels a and b). As a control for the
functionality of LMB, the HIV Rev protein was expressed in HeLa cells
(Fig. 5C). As already reported (33), in transfected HeLa
cells, Rev is located in the nucleolus and the cytoplasm; but in the
presence of LMB, which inhibits Rev nuclear export, Rev is located
mainly in the nucleolus. This is what we also observed (Fig. 5),
demonstrating that LMB is active. These results strongly suggest that
peptide B contains a CRM-1-independent transferable NES.
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Fig. 5.
EB2 peptide B contains a CRM-1-independent
transferable NES. A, shown is a schematic
representation of the NLS. gal and NLS.B.
gal proteins. The
N-terminal gray boxes correspond to a partial sequence of
the Drosophila melanogaster alcohol dehydrogenase providing
the eukaryotic translation/initiation signals. The black
boxes indicate the position of the NLS T antigen SV40 sequence.
The peptide B and
-galactosidase sequence localization is indicated.
B, expression vectors for F.EB2, NLS.
gal, and
NLS.B.
gal were transfected into HeLa cells. After 48 h, HeLa
cells were fused with NIH3T3 cells to form heterokaryons and incubated
for 2 h with medium containing cycloheximide and leptomycin B
where indicated as described under "Experimental Procedures." The
cells were immunostained using either anti-FLAG monoclonal antibody M2
or anti-hemagglutinin monoclonal antibody as the primary antibody and
Alexa Fluor-conjugated goat anti-mouse IgG as the secondary antibody
and then stained with Hoechst dye, which allows differentiation between
the HeLa and NIH3T3 nuclei in the heterokaryons. Arrows
identify the NIH3T3 cell nuclei. Numbers indicate the number
of heterokaryons observed in which the protein is shuttling/the total
number of heterokaryons with positive HeLa nuclei scanned.
C, an expression vector for the HIV Rev protein was
transfected into HeLa cells, and the cells were treated exactly as
indicated for B. The cells were immunostained using
anti-Rev monoclonal antibody as the primary antibody and Alexa
Fluor-conjugated goat anti-mouse IgG as the secondary antibody and then
stained with Hoechst dye. IF, immunofluorescence.
DN) (Fig. 6A), also
shuttled (Fig. 6B). However, it should be noted that the
fluorescence detected in the murine nuclei was always more intense in
the case of the mutant EB2 proteins than in the case of wild-type EB2,
suggesting a more efficient shuttling of the mutant proteins. It
therefore seemed either that the NES1/NES2 region (from now on referred
to as the DN region) was not contributing to the EB2 nucleocytoplasmic
shuttling or that EB2 contained two independent functional NES, one in
peptide B that is CRM-1-independent, as suggested by our results, and a
second one in peptide D, as suggested by Chen et al. (26).
To distinguish between these two hypotheses, we used an EB2 mutant with
a deletion of the N-terminal part of the protein, including the peptide
B region. Because we have shown that the NLS of EB2 was also located in
the N-terminal region of the protein, we generated an N-terminally
truncated mutant to which we fused the SV40 T antigen NLS
(F.NLS.EB2.Cter) (Fig. 6A). This protein is expected to be
nuclear, but if it lacks a functional NES, it should not shuttle in our
heterokaryon assay. This is exactly what we observed (Fig.
6B), strongly suggesting that EB2 contains only one
functional NES located in the N-terminal part of the protein. The above
results were validated by the observation that in our human-mouse
heterokaryon assay, the non-shuttling hnRNP-C protein was not
transported from the human to the mouse nucleus.
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Fig. 6.
The N-terminal part of EB2 contains an
NES. A, shown is a schematic representation of F.EB2,
F.EB2.dNES, F.EB2. DN, and F.NLS.EB2.Cter proteins. The
black box in the F.EB2.dNES diagram indicates the
presence of three alanines in place of the missing EB2 sequence. + or
indicates the capacity of the protein to shuttle or not
between the nucleus and the cytoplasm as determined in B. B, expression vectors for F.EB2, F.EB2.dNES, F.EB2.
DN,
and F.NLS.EB2.Cter were transfected into HeLa cells. After 48 h, HeLa cells were fused with NIH3T3 cells to form heterokaryons and
incubated for 2 h with medium containing cycloheximide as
described under "Experimental Procedures." The cells were
immunostained using either anti-FLAG (M2) or anti-hnRNP-C (4F4)
monoclonal antibody as the primary antibody and Alexa Fluor-conjugated
goat anti-mouse IgG as the secondary antibody and then stained with
Hoechst dye, which differentiates between HeLa and NIH3T3 nuclei in
heterokaryons. Arrows identify the NIH3T3 cell nuclei.
Numbers indicate the number of heterokaryons observed in
which the protein is shuttling/the total number of heterokaryons with
positive HeLa nuclei scanned. IF,
immunofluorescence.
DN and
F.NLS.EB2.Cter were thus transfected into HeLa cells together with the
pDM128/PL reporter construct, and the amount of CAT protein expressed
was quantified by CAT enzyme-linked immunosorbent assay. As expected,
F.EB2 very efficiently increased the level of CAT protein expressed
from pDM128/PL (Fig. 7, B and C, compare bars 1 and 2), whereas the F.NLS.EB2.Cter
protein, which did not shuttle, was completely inactive (Fig.
7B, bar 3). Moreover, F.EB2.dNES and, more
importantly, F.EB2.
DN, although not completely inactive, were much
less efficient than F.EB2 in this functional assay (bars 3 and 4, respectively), suggesting an important role for the
DN region (amino acids 218-236) in mRNA nuclear export.
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Fig. 7.
Both the N-terminal region of EB2 and amino
acids 218-236 (DN region) are required for efficient export of
mRNA. A, shown is a schematic representation of the
pDM128/PL reporter plasmid. The CAT coding sequence is inserted
into intronic sequences. The positions of the 5'- and 3'-donor site
sequences (SS) are indicated. B and
C, expression plasmids for F.EB2, F.NLS.EB2.Cter,
F.EB2.dNES, and F.EB2. NES were transfected into HeLa cells together
with the pDM128/PL reporter plasmid. CAT protein was quantified by CAT
enzyme-linked immunosorbent assay. The results are expressed as
relative amount of CAT protein, with a fixed value of 100 given
to the amount of CAT protein expressed in the presence of wild-type
F.EB2.
DN shuttled between the nucleus
and the cytoplasm. However, this EB2 mutant did not export mRNA
efficiently. We therefore tested whether EB2 could bind known cellular
export factors via the DN region. We first tested whether REF/Aly, a promiscuous mRNA export factor, would interact with EB2. To do so,
in vitro translated F.EB2 (Fig.
8B, lane 1) was
incubated with GST-REF1-II or GST-REF2-II (Fig. 8A)
immobilized on glutathione-agarose beads. F.EB2 bound both GST-REF1-II
and GST-REF2-II (Fig. 8B, lanes 4 and
6), and this interaction was not RNA-dependent
(lanes 5 and 7). To map the EB2-binding site in
REF, F.EB2 was incubated with various REF1-II subfragments fused to GST
and immobilized on glutathione-agarose beads. F.EB2 interacted with
both subdomains at amino acids 14-163 and 103-163 of REF1-II
(lanes 10-13), but not with the subregions at amino acids
14-102 and 129-163 (lanes 8, 9, 14,
and 15). Again, the interactions between F.EB2 and these subregions of REF were RNase-resistant (lanes 11 and
13). These results suggest that there is a direct
interaction between EB2 and REF in vitro and that the
EB2-binding site in REF is located in the RGG-rich C-terminal
variable region of the protein. We then asked whether the REF-binding
site in EB2 is located in the DN region. As compared with F.EB2 (Fig.
8C, lanes 1-5), F.EB2.
DN did not bind to
GST-REF1-II (lanes 6-10). The DN region is thus likely to
be an interaction domain with the cellular REF export protein.
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Fig. 8.
EB2 interacts with REF in
vitro. A, a schematic representation of
REF2-II and REF1-II as described by Rodrigues et al. (19).
N-vr and C-vr, N- and C-terminal variable
regions, respectively; RBD, RNA-binding domain. REF
conserved N- and C-terminal domains are indicated by gray
boxes. Numbers indicate the positions of the different
domains in amino acids. B, EB2 interacts with a domain in
REF1-II located between amino acids 102 and 129. [35S]Methionine-labeled F.EB2 synthesized in rabbit
reticulocyte lysate (lane 1) was incubated with beads coated
with GST (lanes 2 and 3), GST-REF (lanes
4-7), or GST-REF1-II truncations (lanes 8-15). The
input (lane 1) and the bound fractions (lanes
2-15) were analyzed by SDS-PAGE and visualized by
autoradiography. RNase was added to the incubation as indicated.
C, the EB2 DN region is required for in vitro
interaction with REF1-II. [35S]Methionine-labeled F.EB2
(lane 1) and F.EB2. DN (lane 6) synthesized in
rabbit reticulocyte lysate were incubated with GST beads (lanes
2, 3, 7, and 8) or with
GST-REF1-II beads (lanes 4, 5, 9, and
10). The inputs (lanes 1 and 6) and
the bound fractions (lanes 4, 5, 9,
and 10) were analyzed by SDS-PAGE and visualized by
autoradiography. RNase was added to the incubation as indicated.
DN was transiently expressed in HeLa cells, and immunoprecipitation from whole cell extracts was
carried out with anti-FLAG monoclonal antibody M2. The amount of
co-immunoprecipitated REF was then evaluated by Western blot analysis
using anti-REF polyclonal antibody. We found that F.EB2 co-immunoprecipitated REF from the transfected HeLa cell extracts much more efficiently than the mutant F.EB2.
DN protein (Fig. 9A, first panel,
compare lanes 2 and 6). Most importantly, RNase treatment completely abolished the co-immunoprecipitation of REF by
F.EB2 (lane 3), whereas endogenous REF was present at
comparable levels in every cell extract and was unaffected by RNase
treatment (fourth panel). Comparable amounts of F.EB2 and
F.EB2.
DN were efficiently immunoprecipitated by anti-FLAG antibody
M2 (third panel).
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Fig. 9.
F.EB2 interacts with REF and TAP in
vivo. A, EB2 forms a complex with REF, TAP,
and RNA in vivo. HeLa cells were transfected with plasmids
expressing the FLAG-tagged proteins EB2, EB2. DN, and
EB2.Cter, and immunoprecipitation (IP) was performed with
agarose beads coated with anti-FLAG antibody M2. Western blot analyses
were performed using either anti-FLAG monoclonal antibody M2 or rabbit
anti-REF (KJ70) or anti-TAP antibody. RNase was added to the extract
prior to immunoprecipitation as indicated. The amount of REF protein in
the input was analyzed by Western blotting using anti-REF antibody
(REF Input). B, EB2 interaction with REF in the
context of an EBV productive cycle. 293BMLF1-KO cells were
transfected with plasmids expressing EB1 and the FLAG-tagged EB2
and ICP27 proteins as indicated, and immunoprecipitation was performed
using agarose beads coated with anti-FLAG antibody M2. Western blot
analyses were performed using either anti-FLAG monoclonal antibody M2
or rabbit anti-REF antibody KJ70. RNase was added to the extract prior
to immunoprecipitation as indicated. The amount of REF protein in the
input was analyzed by Western blotting using anti-REF antibody
(REF Input).
DN did not affect the
co-immunoprecipitation of TAP (lane 6). Finally, it should
be noted that the cytoplasmic EB2 mutant F.EB2.Cter
co-immunoprecipitated much less REF and TAP compared with F.EB2
(lanes 4 and 5). This latter result is in
accordance with the published observations that REF dissociates from
messenger RNPs as they travel from the nucleus to the cytoplasm (18)
and strongly suggests that the co-immunoprecipitation of REF and TAP by
F.EB2 and F.EB2.
DN results mainly from interactions occurring in the
nuclear compartment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase, the fusion protein shuttled between the
nucleus and the cytoplasm (Fig. 5B). Moreover, we found that
this shuttling was CRM-1-independent, which is in agreement with our
previous data showing that shuttling of the entire EB2 protein is
CRM-1-independent (24). It is possible that the inhibition of EB2
shuttling by LMB observed previously by others (25, 26) was due to
nonspecific effects of the drug, i.e. because of too long
incubation periods or use of too high LMB concentrations. Similarly,
the HSV-1 mRNA export factor ICP27 was first proposed to be
exported out of the nucleus via a leucine-rich CRM-1-dependent NES (36, 37). However, a recent report
demonstrated that neither nucleocytoplasmic shuttling of ICP27 in the
absence of viral RNA nor ICP27-dependent viral mRNA
export is CRM-1-dependent (7). It should be noted that
there are now three functionally conserved human herpes proteins, HSV-1
ICP27, EBV EB2, and human cytomegalovirus UL69 (38), that
express proteins containing a CRM-1-independent NES with, however, no
obvious sequence homology.
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ACKNOWLEDGEMENTS |
---|
We thank B. R. Cullen for providing plasmids pCMV-MS2, pDM128/PL, and pc-Rev; E. Izaurralde for the generous gift of the REF constructs as well as antibodies against REF and TAP; B. Wolff for providing anti-Rev antibody and LMB; and G. Dreyfuss for anti-hnRNP-C monoclonal antibody. We thank R. Buckland for reading the manuscript, S. Ansieau for helpful discussions, and G. Gourru-Lesimple for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by INSERM and by Grant 4357 from the Association pour la Recherche contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Ministère de l'Education Nationale de
la Recherche et des Technologies (MENRT) fellowship.
§ CNRS Scientist.
¶ To whom correspondence should be addressed. Tel.: 33-472-728-176; Fax: 33-472-728-777; E-mail: emanet@ens-lyon.fr.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M208656200
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ABBREVIATIONS |
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The abbreviations used are: HSV-1, herpes simplex virus-1; EBV, Epstein-Barr virus; NES, nuclear export signal(s); NLS, nuclear localization signal(s); GST, glutathione S-transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; CAT, chloramphenicol acetyltransferase; LMB, leptomycin B.
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