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Molecular and Cellular Biology, July 2000, p. 4562-4571, Vol. 20, No. 13
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
RanGTP-Binding Protein NXT1 Facilitates Nuclear
Export of Different Classes of RNA In Vitro
Batool
Ossareh-Nazari,1
Christèle
Maison,1,
Ben E.
Black,2
Lyne
Lévesque,2
Bryce M.
Paschal,2 and
Catherine
Dargemont1,*
Laboratoire de Transport
Nucléocytoplasmique, Unité Mixte de Recherche 144, Institut
Curie-CNRS, 75248 Paris Cedex 05, France,1 and
Center for Cell Signaling, Department of Biochemistry and
Molecular Genetics, University of Virginia Health Sciences Center,
Charlottesville, Virginia 229082
Received 14 December 1999/Returned for modification 20 January
2000/Accepted 11 April 2000
 |
ABSTRACT |
To better characterize the mechanisms responsible for RNA export
from the nucleus, we developed an in vitro assay based on the use of
permeabilized HeLa cells. This new assay supports nuclear export of U1
snRNA, tRNA, and mRNA in an energy- and Xenopus
extract-dependent manner. U1 snRNA export requires a 5'
monomethylated cap structure, the nuclear export signal
receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does
not require the participation of CRM1. We show here that NXT1, an
NTF2-related protein that binds directly to RanGTP, strongly
stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1
to promote export is dependent on its capacity to bind
RanGTP. These results support the emerging view that NXT1 is a general
export factor, functioning on both CRM1-dependent and CRM1-independent
pathways of RNA export.
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INTRODUCTION |
In eukaryotic cells, molecular
exchanges between the nucleus and the cytoplasm occur through the
nuclear pore complex (NPC). Members of the karyopherin 
family, also
known as importins and exportins, specifically interact with
cargos and mediate their nuclear import and nuclear export,
respectively (33, 52). Nucleocytoplasmic transport
also requires the small GTPase Ran (35, 37). The
compartmentalization of the RanGDP exchange factor RCC1 in
the nucleus (38) and the RanGTPase-activating protein
(RanGAP) in the cytoplasm (17) is thought to provide a steep
gradient of RanGDP (cytoplasmic)/RanGTP (nuclear) across the
nuclear envelope that ensures the directionality of nuclear transport.
More precisely, importins bind their import substrates in the absence
of RanGTP (in the cytoplasm) and release them upon binding to RanGTP
(in the nucleus) (34). In contrast, exportins interact with
export cargos in a RanGTP-dependent manner (in the nucleus), and GTP
hydrolysis on Ran in the cytoplasm triggers dissociation of
exportin-cargo complexes (33).
Nearly all nucleus-encoded RNAs require export to the cytoplasm as part
of their maturation process or biosynthetic function. RNAs are
transported as ribonucleoprotein (RNP) complexes, and considerable
effort has been devoted to understanding the protein- and RNA-based
signals that specify export to the cytoplasm. Mature tRNA are directly
recognized by a karyopherin -like export receptor, exportin t or
Los1p, in a RanGTP-dependent manner (4, 16, 28, 31).
Transport of spliceosomal U snRNAs depends on their 5' monomethylated
cap structure, a cis-acting nuclear export signal (NES) that
interacts with a nuclear cap-binding complex (CBC) composed of CBP20
and CBP80 (15, 20). The leucine-rich nuclear export
receptor, CRM1 or exportin 1 (12, 39, 48), in cooperation with RanGTP is required for the transport of U snRNAs and CBC to the
cytoplasm, but it remains unclear whether CBC, CRM1, and RanGTP
represent the minimal machinery responsible for the nuclear export of U
snRNAs (11). CRM1 is also responsible for the nuclear export
of human immunodeficiency virus (HIV) mRNAs that contain a
Rev-responsive element, recognized by the NES-containing HIV Rev
protein (9-11, 32).
The mechanisms responsible for mRNA export are unclear, but
Ran-dependent pathways involving different soluble proteins likely coexist and converge to the NPC. hnRNP proteins associating with mRNA during or just after transcription could provide NESs.
In particular, hnRNP A1 contains an NES and is thought to stimulate mRNA export (18, 36). Similarly, Npl3, a major
shuttling hnRNP protein in yeast, is thought to be involved in
mRNP export (29). Another factor involved in
mRNA export, the yeast Mex67p protein, was identified by its
genetic interaction with the nucleoporin Nup85p (46). Mex67p
binds directly to mRNA, forms a tight complex with Mtr2p
(24), and interacts with Nup85p via Mtr2p (44). The human homolog of Mex67p, TAP, is required for the nuclear export of
RNA containing a constitutive transport element found in simple type D
retroviruses but could also mediate transport of cellular
mRNA (14, 40). In mammalian cells, TAP physically interacts with a 15-kDa protein termed p15 (25) that is 26% identical to NTF2, a RanGDP-binding protein that mediates
nuclear import of Ran (42, 47, 49). Coexpression of TAP and
p15 partially complements the growth defect of the Mex67p/Mtr2 null mutant, suggesting that these proteins carry out related functions in
mammals and yeast, respectively (25). More recently, p15 (referred to as NXT1) has been shown to stimulate nuclear protein export of protein kinase inhibitor in a pathway that relies upon CRM1
as the receptor (5). It was also shown that NXT1 binds specifically to RanGTP and that it colocalizes with the NPC, suggesting it may play a regulatory and/or targeting function in the CRM1 export
pathway (5).
To define the roles of individual proteins in the transport of
RNA, we set up an assay that reconstitutes RNA export in vitro. We used
the assay to show that nuclear export of U1 snRNA and that of
tRNA and mRNA proceed through CRM1-dependent and
-independent pathways, respectively. Nuclear export of these different
classes of RNA relies on NXT1 as a cofactor. Mutations in NXT1
that reduce binding to RanGTP also reduce its ability to stimulate U1
snRNA and tRNA export, indicating that an interaction with RanGTP is required for NXT1-dependent stimulation of RNA export.
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MATERIALS AND METHODS |
In vitro transcription.
cDNA encoding U3 snRNA (gift of
J. P. Bachellerie, Centre National de la Recherche Scientifique,
[CNRS], Toulouse, France), was subcloned in pGEM1 in positive
orientation relative to the promoter for T7 polymerase. The plasmid was
linearized with BamHI. Expression vectors encoding U1
Sm
snRNA, Rab11, and tRNAMet were provided by I. Mattaj
(European Molecular Biology Laboratory, Heidelberg, Germany), B. Goud
(CNRS, Paris, France), and E. Lund (Madison, Wis.) and linearized with
BamH1, HindIII, and MvaI, respectively.
For in vitro transcription, linearized plasmid (3 µg) was incubated
for 60 min at 37°C in a 30-µl transcription mixture containing transcription buffer (Promega), 10 mM dithiothreitol (DTT), 0.1 mg of
bovine serum albumin (BSA) per ml, 0.8 mM ATP, CTP, and UTP, 0.2 mM
GTP, 0.8 mM m7G(5')ppp(5')G cap analog except for tRNA
(Boehringer Mannheim), 40 U of RNasin (Promega), 50 µCi of
[
-32P]GTP (Amersham), and 60 U of T7 polymerase
(Promega). DNA was then digested by 4 U of RNase-free DNase I (Promega)
for 15 min at 37°C, RNAs were extracted with phenol-chloroform, and
unincorporated nucleotides were removed with a Sephadex G-50 column.
RNA was precipitated and resuspended in 25 µl of H2O.
Fluorescently labeled RNA transcripts were generated by the same
procedure but with different concentrations of nucleotides: 1 mM ATP
and CTP, 0.25 mM GTP, 1 mM m7G(5')ppp(5')G cap analog, 1 mM
aminoallyl-UTP-UTP (1:15; Sigma). Resulting RNA was supplemented with
15 mM 5 (and 6)-carboxy-X-rhodamine (succinimidyl ester; Molecular
Probes) dissolved in dimethyl formamide (Sigma) and 37.5 mM bicarbonate
buffer (pH 9). The labeling reaction was performed at room temperature
for 1 h. Unincorporated fluorochromes were removed by gel
filtration on a Sephadex G-50 column. RNA was precipitated and
resuspended in 20 µl of H2O.
Preparation of Xenopus egg extracts and
membranes.
Female Xenopus laevis frogs were purchased
from the Elevage de Xenopes du CNRS (Montpellier, France). A
10,000 × g crude extract was prepared from
unfertilized Xenopus eggs resuspended in lysis buffer
consisting of 250 mM sucrose, 70 mM KCl, 2.5 mM MgCl2, and
20 mM PIPES-KOH (pH 7.5), supplemented with 1 mM DTT, 5 µg of
cytochalasin B per ml, 10 µg each of aprotinin, leupeptin, and
pepstatin per ml, and 200 µg of 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; Sigma) per ml. This crude extract was then separated into supernatant, membrane phase, and pellet fraction by
ultracentrifugation at 120,000 × g for 60 min at
4°C. The supernatant was recentrifuged at 200,000 × g for 30 min at 4°C, complemented with glycerol 5% (vol/vol),
aliquoted, frozen in liquid nitrogen, and stored at 
80°C until use
(Xenopus extracts). Membranes were collected, diluted in 10 volumes of cold lysis buffer, and centrifuged at 40,000 × g for 30 min at 4°C. Pelleted membranes were resuspended, diluted in 10 volumes of cold lysis buffer, and spun through a cushion
of lysis buffer containing 0.5 M sucrose (10,000 × g, 20 min, 4°C). Membranes were recovered in the minimal volume of cold
lysis buffer, aliquoted, frozen in liquid nitrogen, and stored at
80°C until use.
Cell permeabilization.
HeLa cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum and
penicillin-streptomycin. Cells were dissociated with cell dissociation
solution (Sigma), washed twice in phosphate-buffered saline (PBS), and
resuspended at 106 cells/ml in ice-cold
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer containing 50 mM PIPES-KOH (pH 7.4), 50 mM KCl, 5 mM
MgCl2, 2 mM EGTA, 1 mM DTT, 1 µg each of aprotinin,
leupeptin, and pepstatin per ml, and 200 µg of AEBSF (Sigma) per ml,
supplemented with 50 µg of digitonin (Sigma) per ml at 4°C.
Digitonin-permeabilized cells were then pelleted, washed twice, finally
resuspended at 107 cells/ml in PIPES buffer containing 5%
dimethyl sulfoxide, and frozen in 100-µl aliquots at 80°C.
Freshly thawed digitonin-treated HeLa cells (10
6 cells)
were permeabilized with 0.0725% decanoyl-
N-methylglucamide
(MEGA-10;
Sigma) in a final volume of 3 ml PIPES buffer for 6 min at
4°C
with occasional gentle mixing. Reaction was stopped by addition
of 4% nuclease-free BSA (Sigma) in PIPES buffer. Permeabilized
nuclei
were pelleted by centrifugation at 210 ×
g for 5 min
and
washed twice in 1 ml of PIPES buffer. Pelleted nuclei were
resuspended
to 10
7 nuclei/ml and used
immediately.
Nuclear envelope repair.
MEGA-10-permeabilized nuclei were
repaired by incubation with membranes and extracts prepared from
Xenopus eggs. Typically, 10 µl of nuclei (105
nuclei) was mixed with an equal volume of membranes and supplemented with 5.5 µl of Xenopus extracts and an energy-regenerating
system (1 mM ATP, 1 mM GTP, 6 mM creatine phosphate, 4.8 U of creatine phosphokinase per ml). The samples were incubated at 20°C for up to
60 min. Routinely, aliquots were taken and assayed for nuclear exclusion of fluorescein isothiocyanate (FITC)-labeled immunoglobulin G.
In vitro nuclear export assay.
Typically, 105
permeabilized nuclei in PIPES buffer were incubated for 5 min at room
temperature with 1.5 pmol of labeled RNA, 0.5 mM m7GTP
(except for tRNA), and 20 U of RNasin and then repaired as described
previously. Repaired nuclei were washed three times in PIPES buffer by
centrifugation at 210 × g for 5 min at room temperature and resuspended in 10 µl of PIPES buffer (104
nuclei/µl). Export of labeled RNA was followed under different conditions as described in Results. A standard 20-µl assay was performed in PIPES buffer containing an energy-regenerating system, FITC-labeled BSA coupled to the simian virus 40 large T antigen nuclear
localization signal (BSA-NLS-FITC) (20 µg/ml) (13), 10 µl of Xenopus extracts (50%, final concentration), and
105 repaired nuclei. Transport was allowed to proceed for
45 to 90 min at 20°C.
When radiolabeled RNAs were introduced into nuclei, export reactions
were separated into nuclear and supernatant fractions
by centrifugation
at 210 ×
g for 5 min at room temperature; both
fractions were supplemented with 200 µl of homomedium buffer (50
mM
Tris-HCl [pH 7.5], 5 mM EDTA, 1.5% sodium dodecyl sulfate,
300 mM
NaCl, 1.5 mg of proteinase K per ml), incubated for 45
min at 50°C,
and extracted twice with phenol-chloroform. RNAs
were precipitated,
resuspended in 5 µl of H
2O, and analyzed on
an 8%
polyacrylamide-7 M urea gel. When indicated, results were
quantified
using the Bioprint acquisition system and Bioprofil
program (Vilbert
Lourmat).
When rhodamine-labeled snRNAs were introduced into nuclei, samples were
diluted to 200 µl with 20 mM PIPES-KOH (pH 7.4)-70
mM KCl-2.5 mM
MgCl
2-250 mM sucrose and fixed by addition of 200
µl of
6% paraformaldehyde-100 mM PIPES-KOH (pH 7.5) for 5 min
on ice;
nuclei were spun through a 1-ml sucrose cushion (30% [wt/vol]
in
PIPES buffer) onto polylysine-coated coverslips (1,891 ×
g,
5 min). Coverslips were briefly washed in water, incubated with
DAPI (4',6-diamidino-2-phenylindole) dye for 1 min, and mounted
in PBS
containing 50% glycerol. Confocal laser scanning microscopy
and
fluorescence analysis were performed with a TCS4D confocal
microscope
based on a DM microscope interfaced with a mixed-gas
argon-krypton
laser (Leica Laser
Technik).
Indirect immunofluorescence.
Digitonin-permeabilized cells
(105) or repaired nuclei samples were diluted in 200 µl
with 20 mM PIPES-KOH (pH 7.4)-70 mM KCl-2.5 mM MgCl2-250
mM sucrose and fixed by addition of 200 µl of 6% paraformaldehyde-100 mM PIPES-KOH (pH 7.5) for 5 min on ice; nuclei were spun through a 1-ml sucrose cushion (30% [wt/vol] in PIPES buffer) onto polylysine-coated coverslips (1,891 × g,
5 min). Coverslips were briefly washed in PBS and treated with 0.1%
Triton X-100 for 5 min. Primary antibodies were applied for 30 min
followed by a 30-min incubation with FITC- or Texas red-conjugated
secondary antibodies (Jackson ImmunoResearch Laboratories). Coverslips
were then incubated with DAPI dye for 1 min and mounted in PBS
containing 50% glycerol. Rabbit polyclonal antibodies against gp210
and Nup84 were a gift from J. C. Courvalin and R. Bastos,
respectively. Mouse monoclonal antibody (MAb) to Nup153 was a gift from
B. Burke, and MAb 414 was obtained from (BAbCo, Richmond, Calif.).
Electron microscopy.
Cells treated in different conditions
were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)
for 2 h at 4°C. They were washed in the same buffer and then
postfixed for 30 min at 4°C with 2% osmic acid (OsO4) in
water. They were dehydrated in ethanol before embedding in Epon.
Ultrathin sections were counterstained with uranyl acetate and viewed
with an electron microscope (CM120TEM; Philips, Eindoven, The Netherlands).
Expression of recombinant proteins.
Expression plasmid for
glutathione S-transferase (GST)-Ran was a gift from M. Dasso (National Institutes of Health, Bethesda, Md.), and wild-type Ran
(Ran wt) was purified as described previously (8). RanQ69L
was expressed and purified essentially as described elsewhere
(27), using an expression vector provided by C. Dingwall (Stony Brook, N.Y.). His-tagged NTF2 expression and purification were
performed as described elsewhere (37a), using the NTF2
expression vector provided by G. Blobel (The Rockefeller Institute, New
York, N.Y.). Expression plasmid for GST-M9 was a gift from G. Dreyfuss (University of Pennsylvania, Philadelphia); GST-M9 and -M9 mutant (GST-M9mut) were purified as described previously (41).
Untagged NXT1 proteins were expressed in
Escherichia coli
(BL21) and purified by column chromatography as described elsewhere
(
5). Mutations of the NXT1 open reading frame were generated
on the bacterial expression vector by using the QuickChange
site-directed
mutagenesis system (Stratagene, La Jolla, Calif.) and
confirmed
by
sequencing.
RanGTP binding assay.
Solid-phase binding assays were
performed as described elsewhere (5). Ran preloaded with
[
-32P]GTP was incubated in wells containing the
indicated recombinant proteins. The bound Ran was eluted and
quantitated by scintillation counting. Binding to wells containing BSA
alone was subtracted from binding to wells containing NXT1 proteins.
Binding assays were performed in duplicate, and the values shown
represent the mean ± standard deviation.
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RESULTS |
Development of an in vitro RNA export assay.
To characterize
the molecular mechanisms responsible for nuclear export of RNA, an in
vitro assay was developed (Fig. 1). Our
assay is based on the use of semipermeabilized cells and was adapted
from a previously described cell-free system for DNA replication (30). HeLa cells were first permeabilized with digitonin to remove the endogenous cytosol without affecting the integrity of the
nuclear envelope (1). Treatment of the resulting
permeabilized cells with the nonionic detergent MEGA-10 resulted in the
permeabilization of the nuclear envelope. At this stage, radiolabeled
or fluorescently labeled RNAs were introduced into nuclei by diffusion.
The nuclear envelope was subsequently resealed upon addition of
Xenopus egg membranes in the presence of the nucleotides ATP
and GTP and Xenopus egg extracts. After extensive washing,
RNA-loaded nuclei were incubated under different conditions, and
nuclear export of RNA was analyzed either by direct fluorescence or by
RNA extraction and gel electrophoresis.
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FIG. 1.
Schematic representation of the in vitro nuclear export
system. HeLa cells were permeabilized first with digitonin and then
with the nonionic detergent MEGA-10, resulting in permeabilization of
the nuclear envelope. At this stage, radiolabeled or fluorescently
labeled RNAs were introduced into nuclei by free diffusion. The nuclear
envelope was resealed upon addition of Xenopus egg membranes
in the presence of the nucleotides ATP and GTP and Xenopus
egg extracts. After extensive washing, RNA-loaded nuclei were submitted
to different incubation conditions, and nuclear export of RNA was
analyzed either by direct fluorescence or by RNA extraction and gel
electrophoresis.
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The morphological integrity of the nuclear envelope and NPC was
examined at each step by both electron microscopy analysis
(Fig.
2A) and indirect immunofluorescence with
different antinucleoporin
antibodies (Fig.
2B). As
previously described, nuclei from digitonin-treated
cells displayed an
intact nuclear envelope, and rim-like staining
was observed using
antibodies directed against nucleoporins localized
in the transmembrane
domain (gp210), at the cytoplasmic face (Nup84),
or at the nuclear face
(Nup153) of the NPC or recognizing members
of the O-linked family of
nucleoporins (p62, Nup214, Nup153) (MAb
414). In contrast, the
characteristic of the double membrane of
the nuclear envelope from
MEGA-10-permeabilized nuclei appeared
completely disorganized and
formed vesicles. Resealing with
Xenopus egg membranes
restored a normal-looking nuclear envelope with
inner and outer
membranes and NPC. Immunofluorescence analysis
using
antinucleoporin antibodies revealed that the normal repertoire
of
proteins was present at the pore. Moreover, NPC staining observed
with
the anti-Nup153 antibody which does not recognize the
Xenopus Nup153 indicated that NPC of repaired nuclei
contained human nucleoporins.
However, the presence of
Xenopus nucleoporins in NPC of repaired
nuclei cannot be
formally excluded.
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FIG. 2.
The nuclear envelope is morphologically and functionally
intact after permeabilization and resealing. (A) Samples corresponding
to each step of the procedure used were fixed with glutaraldehyde and
processed for thin sectioning and transmission electron microscopy. N,
nucleus. Arrows point to NPCs. (B) Indirect immunofluorescence (IF)
labeling of digitonin-permeabilized HeLa cells or repaired nuclei,
using antibodies against O-linked nucleoporins (MAb 414), Nup153,
Nup84, or gp210. (C) Digitonin-permeabilized HeLa cells or repaired
nuclei were incubated for 45 min with 50% Xenopus extracts
and FITC-BSA-NLS (20 µg/ml), FITC-BSA-SLN (20 µg/ml), FITC-GST-M9
(50 µg/ml), or FITC-GST-M9mut (50 µg/ml) at 20°C with an
energy-regenerating system. After incubation, cells were processed for
direct fluorescence.
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The functional integrity of the nuclear envelope was analyzed by
testing nuclear import of two different cargos, FITC-BSA-NLS
and
FITC-GST fused to the M9 sequence of hnRNPA1 (GST-M9)
(
23,
41), which are imported by the importins
/
and
transportin-mediated
pathways, respectively (Fig.
2C). Nuclei from
digitonin-treated
cells accumulated BSA-NLS and GST-M9 after incubation
at 20°C
in the presence of
Xenopus egg extracts and
energy. In agreement
with the drastic morphological change observed
after MEGA-10 treatment,
permeabilized nuclei were not able to import
BSA-NLS (data not
shown). In contrast, nuclear import of both BSA-NLS
and GST-M9
was restored after nuclear envelope repair. No import was
observed
using BSA coupled to a reverse NLS peptide (BSA-SLN) or GST
fused
to a mutant M9 (G274
A). These data indicate that the
permeabilization
and resealing procedure gave rise to nuclei competent
for protein
import.
We examined the capacity of the in vitro assay to support nuclear
export of U snRNAs. For this purpose, we used both U1
Sm
snRNA (U1),
a U1 snRNA mutant lacking the Sm binding site that
is therefore unable
to be reimported in the nucleus after its
export (
21,
22),
and U3 small nucleolar RNA (U3), an RNA species
that is retained in the
nucleus in vivo (
50). For this purpose,
radiolabeled or
rhodamine-labeled U1 and U3 were synthesized in
vitro and introduced
into permeabilized nuclei by free diffusion.
Following nuclear envelope
repair, the RNA-loaded nuclei were
extensively washed to remove RNA
background from the cytosol.
Nuclei were then incubated for 90 min with
BSA-NLS in transport
buffer at 20°C or with 50%
Xenopus
extracts in the presence of
an energy-regenerating system at 20 or
4°C or with 50%
Xenopus extracts in the presence of
apyrase at 20°C. Nuclei were then
fixed and observed by direct
fluorescence using confocal microscopy
(Fig.
3A). Alternatively, incubation reactions
were centrifuged
to separate nuclei from incubation medium containing
exported
substrates; RNAs were extracted from both fractions and
analyzed
by denaturing gel electrophoresis and autoradiography (Fig.
3B).
Prior to initiating the transport reaction (time zero
[
t0]), both
U1 and U3 were detected
exclusively in the nucleus. RNA appeared
mainly localized in nuclear
dots, with a small fraction in the
nucleoplasm. Incubation of U1- or
U3-containing nuclei in the
absence of extracts, at 4°C or in the
presence of apyrase, allowed
neither BSA-NLS nuclear import nor nuclear
export of RNAs which
remained intact in the nucleus. In contrast,
addition of
X. laevis egg extracts and ATP at 20°C led to
the nuclear accumulation of
BSA-NLS and a significant decrease in the
nuclear content of rhodamine-labeled
U1. RNA extraction and
electrophoretic analysis indicated that
radiolabeled U1 was exported
out of the nucleus in a full-length
form (52% ± 8% of loaded RNA was
exported). Damage of repaired
nuclei appeared after 90 min and
prevented export at longer incubation
times from being measured. No
export of U3 (7% ± 6%) was observed
under the same experimental
conditions. Thus, our assay reconstitutes
nuclear export of U1 in a
cytosol- and energy-dependent manner.
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FIG. 3.
Nuclear export of U1 and U3 in vitro. Radiolabeled or
rhodamine-labeled U1 and U3 were synthesized in vitro and introduced by
diffusion into permeabilized nuclei. The nuclear envelope was then
repaired (t0), and nuclei were incubated for 90 min with FITC-BSA-NLS and 10% gelatin in transport buffer at 20°C,
with 50% Xenopus extracts in the presence of an
energy-regenerating system at 20 or 4°C, or with 50%
Xenopus extracts in the presence of apyrase at 20°C, as
indicated. (A) After incubation under different conditions, nuclei were
processed for direct fluorescence and visualized by confocal
microscopy. (B) Radiolabeled U1 and U3 were extracted from nuclear
fractions (N) and from incubation buffer (S) and analyzed by denaturing
gel electrophoresis.
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U1 nuclear export requires GTP hydrolysis by Ran.
Exportins
have been shown to bind their substrates in a RanGTP-dependent manner
in the nucleus and release their cargo in the cytoplasm upon GTP
hydrolysis (33). However, the translocation step of
substrates like NES-containing proteins or tRNA does not require GTP
hydrolysis by nuclear Ran (19, 43). To analyze whether the
nuclear export of U1 in vitro depends on GTP hydrolysis by Ran, we
compared the effects of Ran wt and Ran Q69L, added either in extracts
or in nuclei, on both BSA-NLS import and U1 export. Addition of 10 µM Ran Q69L to the extracts resulted in the inhibition of both
BSA-NLS import and U1 export (12.8% ± 3%) compared to what observed
in the presence of 10 µM Ran wt (46% ± 2%) (Fig.
4A and C, left panel). Addition of the
importin -binding site of importin in extracts also blocked
nuclear import of BSA-NLS as well as U1 export (data not shown).
The effect of cytoplasmic RanQ69L on U1 export was therefore likely due
to protein import defect rather than an inhibition of
U1-CRM1-RanQ69L dissociation after translocation.
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FIG. 4.
Nuclear export of U1 in vitro requires GTP hydrolysis by
Ran. (A) Effect of cytoplasmic and nuclear RanQ69L on U1 export. To
analyze the effect of cytoplasmic RanQ69L, nuclear export of U1 was
performed in 50% Xenopus extracts and energy (control) or
supplemented with 10 µM RanQ69L (RanQ69L, extracts) or 10 µM Ran wt
(Ran wt, extracts). To analyze the effect of nuclear RanQ69L, repaired
nuclei containing radiolabeled U1 were incubated with either 28 µM
RanQ69L (RanQ69L, nucleus) or 28 µM Ran wt (Ran wt, nucleus). After a
20-min incubation at room temperature, nuclei were washed, resuspended
in 50% Xenopus extracts, and processed for nuclear export.
After 60 min of incubation, U1 was then extracted from nuclear
fractions (N) and from incubation buffer (S) and analyzed by gel
electrophoresis. (B) Expression of Ran in Xenopus extracts
(extract), in MEGA-10-permeabilized (perm.) nuclei, in repaired nuclei,
and in nuclei incubated for 20 min at room temperature with 28 µM
RanQ69L, 28 µM Ran wt, or PIPES buffer was analyzed by Western
blotting using an anti-Ran MAb. (C) Effect of cytoplasmic and nuclear
RanQ69L on protein import. The procedure was the same as for panel A. The import of FITC-BSA-NLS present in the export reaction was followed
by direct fluorescence.
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To analyze whether U1 nuclear export would require nuclear GTP
hydrolysis by Ran, U1-loaded nuclei were repaired and then
incubated
with 28 µM RanQ69L or Ran wt before being washed and
processed for
nuclear export reaction. In contrast to permeabilized
or repaired
nuclei that did not express any Ran protein detectable
by Western
blotting, nuclei incubated in the presence of Ran wt
or mutant retained
a significant amount of Ran protein (approximately
5 to 10 µM; Fig.
4B). Nuclear export of U1 was strongly affected
in RanQ69L-loaded
nuclei (6.5% ± 4%), whereas Ran wt-containing
nuclei did not display
any defect in U1 export (32% ± 3.1%) (Fig.
4A). These experimental
conditions did not affect the ability
of nuclei loaded with Ran wt or
RanQ69L to import BSA-NLS (Fig.
4C, right panel) indicating that the
export inhibition by nuclear
RanQ69L did not result from an import
defect.
These data are consistent with previous data showing that U1 export in
Xenopus oocytes is inhibited by the nuclear injection
of
RanQ69L and indicate that GTP hydrolysis by Ran is necessary
for
efficient export of this RNA (
19).
5' monomethylated cap structure and CRM1 are required for U1 export
in vitro.
Two specific factors have been shown to efficiently
stimulate nuclear export of U1 snRNA: CBC (15, 20), which
directly recognizes the monomethylated cap structure
m7GpppG of U snRNAs, and CRM1, the receptor for
leucine-rich NESs (11). To analyze whether U1 was exported
by a cap-dependent pathway in vitro, radiolabeled U1 snRNA capped with
a m7GpppG cap or an unmethylated ApppG cap was introduced
into permeabilized nuclei, and export was analyzed following nuclear
envelope repair (Fig. 5A). After 40 min
of incubation, 31% ± 12% of m7GpppG-capped U1 was
exported, while only 7% ± 6% of ApppG-capped U1 had left the nucleus
during the same period of time. The m7GpppG cap structure
therefore strongly increases the export rate of U1 in vitro, as
previously shown by microinjection into Xenopus oocyte
nuclei (15, 51). This result indicates that interaction between the cap structure and CBC is required for nuclear export of U1
in this reconstitution export assay.
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|
FIG. 5.
Nuclear export of U1 in vitro requires a 5'
monomethylated cap structure and is inhibited by LMB. (A) In vitro
nuclear export of U1 is facilitated by the presence of a cap guanosine
structure. Radiolabeled U1 containing m7GpppG cap (m7G
capped U1) or ApppG cap (A capped U1) was synthesized in vitro and
introduced by diffusion into permeabilized nuclei. The nuclear envelope
was then repaired (t0), and nuclei were
incubated with 50% Xenopus extracts in the presence of an
energy-regenerating system at 20°C for different times. U1 was then
extracted from nuclear fractions (N) and from incubation buffer (S) and
analyzed by gel electrophoresis. (B) In vitro nuclear export of U1 is
inhibited by LMB. Radiolabeled U1 was introduced by diffusion into
permeabilized nuclei. The nuclear envelope was then repaired
(t0), and nuclei were resuspended either in 50%
Xenopus extracts (control) or in 50% Xenopus
extracts pretreated with 0.1 or 1 µM LMB (45 min, room temperature).
After 60 min of incubation at 20°C, U1 was extracted from nuclear
fractions (N) and from incubation buffer (S) and analyzed by gel
electrophoresis.
|
|
To determine whether CRM1 is responsible for the nuclear export of U1
in the in vitro system, U1 export was analyzed upon
addition of
leptomycin B (LMB), a cytotoxin that specifically
binds CRM1 and
inhibits its export receptor function (
11,
39).
As shown in
Fig.
5B, U1 export was already strongly reduced upon
treatment of both
nuclei and extracts with 0.1 µM LMB and almost
completely inhibited
in the presence of 1 µM LMB (13% ± 4% of
exported RNA compared to
42% ± 5% in the control), strongly suggesting
that U1 is exported by
a CRM1-dependent
pathway.
NXT1 stimulates nuclear export of U1 in vitro.
NXT1, a
mammalian 15-kDa protein related to NTF2, has been recently identified
to bind the mRNA export factor TAP (25) and to
specifically interact with RanGTP with nanomolar affinity
(5). Moreover, NXT1 has been found to facilitate
CRM1-dependent nuclear export of both PKI (5) and HIV type 1 Rev (J. M. Holaska and B. M. Paschal, unpublished
observations). To determine whether NXT1 promotes CRM1-dependent
nuclear export of U1, we supplemented RNA export assay with
purified recombinant NXT1. Addition of NXT1 to a transport
reaction containing 50% Xenopus extract had no effect (Fig.
6A). Since NXT1 is likely to be present in the Xenopus extract, we performed transport reactions in the presence of a subsaturating level of extract (15%) in the absence and presence of
NXT1 protein. NXT1 induced a clear stimulation of U1 export, and the
effect was even more pronounced at the higher concentration of NXT1
tested (Fig. 6A). In contrast, NXT1 was
unable to induce nuclear export of U3, an RNA restricted to the nucleus
(Fig. 6B). Moreover, NXT1 had no effect on nuclear import of BSA-NLS
(data not shown). U1 export rate was not affected upon addition of NTF2 (Fig. 6C), indicating that the stimulatory effect of NXT1 on U1 export
was specific. Finally, addition of NXT1 to a transport reaction without
extracts did not promote any U1 export (Fig. 6C). The
NXT1-stimulated export of U1 was inhibited by LMB (data not
shown), confirming that NXT1 is involved in an export pathway for
which CRM1 is the receptor. We note that LMB also blocks
NXT1-stimulated export of PKI in permeabilized cells as well
(5).
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FIG. 6.
NXT1 facilitates nuclear export of U1 in vitro. (A)
Radiolabeled U1 was introduced by diffusion into permeabilized nuclei.
The nuclear envelope was then repaired (t0), and
nuclei were incubated either in 50% Xenopus extracts alone
or supplemented with NXT1 (100 µg/ml) or in 15% Xenopus
extracts alone or supplemented with NXT1 (100 or 250 µg/ml), as
indicated. (B) Radiolabeled U3 was introduced by diffusion into
permeabilized nuclei. The nuclear envelope was then repaired
(t0), and nuclei were incubated in 50%
Xenopus extracts or in 15% Xenopus extracts
alone or supplemented with NXT1 (250 µg/ml). (C) Radiolabeled U1 was
introduced by diffusion into permeabilized nuclei. The nuclear envelope
was then repaired (t0), and nuclei were
incubated either in 50% Xenopus extracts or in 15%
Xenopus extracts alone or supplemented with NXT1 (250 µg/ml) or NTF2 (250 µg/ml), as indicated; alternatively, nuclei
were incubated with NXT1 (250 µg/ml) in the absence of extracts
(NXT1). After 60 min of incubation at 20°C, U1 was extracted from
nuclear fractions (N) and from incubation buffer (S) and analyzed by
gel electrophoresis.
|
|
We predicted that NXT1 stimulation of U1 export relies upon its ability
to bind RanGTP. To test this, we generated NXT1 mutants
by changing
residues that are adjacent to its Ran-binding domain.
These include two
asparagines (N48 and N50) and a glutamate (E102)
that are predicted to
surround the hydrophobic pocket of NXT1
where RanGTP is thought to bind
(
5). The interaction of the
NN48,50KK mutant with RanGTP was
reduced twofold, as measured
in a solid-phase binding assay (Fig.
7A). We found that the NXT1
NN48,50KK
mutant is 2.5-fold less effective than NXT1 wt in its
ability to
stimulate U1 export (Fig.
7B). In contrast, the E102N
mutation did not
affect U1 export or RanGTP binding (Fig.
7).
Our results indicate
that NXT1 modulates CRM1-dependent export
of U1 snRNA through a
mechanism that involves RanGTP.
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|
FIG. 7.
The interaction of NXT1 with RanGTP is required to
stimulate U1 export. (A) Ran preloaded with [-32P]GTP
was incubated in wells containing the indicated immobilized NXT1
proteins. NXT1 wt binds RanGTP, as previously demonstrated
(5). The NXT1 NN48,50KK mutant is deficient in binding
RanGTP; however, this binding is unhindered for the NXT1 E102N mutant.
(B) Radiolabeled U1 was introduced by diffusion into permeabilized
nuclei. The nuclear envelope was then repaired
(t0), and nuclei were resuspended either in 50%
Xenopus extracts or in 15% Xenopus extracts
alone or supplemented with NXT1 wt (250 µg/ml), NXT1 NN48,50KK, or
NXT1 E102N. After a 60-min incubation at 20°C, U1 was extracted from
nuclear fractions (N) and from incubation buffer (S) and analyzed by
gel electrophoresis. (C) Quantification of results from three
independent experiments. The value of nuclear export observed at
t0 was subtracted from each condition, and
results were expressed as a percentage of nuclear export occurring in
the presence of 50% extracts.
|
|
NXT1 stimulates nuclear export of tRNA and mRNA in
vitro.
To determine whether NXT1 function is restricted to the
CRM1-dependent nuclear export pathway or is involved more generally in
other export routes, we tested the ability of recombinant NXT1 to
stimulate tRNA and mRNA export in vitro. For this purpose, tRNAMet (31) and mRNA encoding Rab11
were in vitro transcribed and introduced in permeabilized nuclei, and
their export was analyzed in different incubation conditions following
nuclear envelope repair. As for U1, the in vitro assay allowed the
reconstitution of a cytosol and energy-dependent nuclear export of both
tRNA and Rab11 mRNA (Fig. 8A
and 9A). In contrast, treatment of nuclei and extracts with LMB did not affect the transport of this
mRNA, indicating that Rab11 mRNA was not exported
through a CRM1-dependent pathway in vitro (Fig. 9B).
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FIG. 8.
NXT1 facilitates nuclear export of tRNA in vitro. (A)
Radiolabeled tRNAMet was synthesized in vitro and
introduced by diffusion into permeabilized nuclei. The nuclear envelope
was then repaired (t0), and nuclei were
incubated for 30 min with 10% gelatin in transport buffer at 20°C
(buffer), with 50% Xenopus extracts in the presence of an
energy-regenerating system at 20 or 4°C, or with 50%
Xenopus extracts in the presence of apyrase at 20°C, as
indicated. tRNAs were then extracted from nuclear fractions (N) and
from incubation buffer (S) and analyzed by denaturing gel
electrophoresis. (B) Radiolabeled tRNA was introduced by diffusion into
permeabilized nuclei. The nuclear envelope was then repaired
(t0), and nuclei were resuspended either in 50%
Xenopus extracts alone or supplemented with NXT1 (100 µg/ml) or in 15% Xenopus extracts alone or supplemented
with NXT1 wt (250 µg/ml) or NXT1 NN48,50KK. After a 30-min
incubation at 20°C, tRNA was extracted from nuclear fractions (N) and
from incubation buffer (S) and analyzed by gel electrophoresis. The
arrowhead indicates the band corresponding to tRNA before maturation of
its pppGGG-5' end by RNaseP. (C) Results from three independent
experiments were quantified. The value of nuclear export observed at
t0 was subtracted from each condition, and
results were expressed as percentage of nuclear export occurring in the
presence of 50% extracts.
|
|
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|
FIG. 9.
NXT1 facilitates nuclear export of mRNA in
vitro. (A) Radiolabeled Rab11 mRNA was synthesized in vitro
and introduced by diffusion into permeabilized nuclei. The nuclear
envelope was then repaired (t0), and nuclei were
incubated for 90 min with 10% gelatin in transport buffer at 20°C
(buffer), with 50% Xenopus extracts in the presence of an
energy-regenerating system at 20 or 4°C, or with 50%
Xenopus extracts in the presence of apyrase at 20°C, as
indicated. mRNAs were then extracted from nuclear fractions
(N) and from incubation buffer (S) and analyzed by denaturing gel
electrophoresis. (B) Radiolabeled Rab11 mRNA was introduced
by diffusion into permeabilized nuclei. The nuclear envelope was then
repaired (t0), and nuclei were resuspended
either in 50% Xenopus extracts (control) or in 50%
Xenopus extracts pretreated with 0.1 or 1 µM LMB (45 min,
room temperature). After 60 min of incubation at 20°C,
mRNAs were extracted from nuclear fractions (N) and from
incubation buffer (S) and analyzed by gel electrophoresis. (C)
Radiolabeled Rab11 mRNA was introduced by diffusion into
permeabilized nuclei. The nuclear envelope was then repaired
(t0), and nuclei were resuspended either in 50%
Xenopus extracts alone or supplemented with NXT1 (100 µg/ml) or in 15% Xenopus extracts alone or supplemented
with NXT1 (250 µg/ml). After 60 min of incubation at 20°C,
mRNAs were extracted from nuclear fractions (N) and from
incubation buffer (S) and analyzed by gel electrophoresis.
|
|
A subsaturating concentration of extracts (15%) induced only a low
level of tRNA and Rab11 mRNA export compared to the level
observed with 50% extracts (respectively 29% ± 7% and 25% ± 10%
of the export observed with 50% extracts). When recombinant NXT1
was
added this a subsaturating concentration of extracts, the
export level
of tRNA and Rab11 mRNA was strongly increased (respectively
110% ± 10% and 77% ± 15% of the export observed with 50%
extracts
[Fig.
8B,
8C, and
9C). These data show that NXT1 protein is
able
to stimulate the CRM1-independent export of tRNA and
mRNA. In
addition, we found that the NXT1 NN48,50KK mutant is
twofold less
effective than NXT1 wt in its ability to stimulate tRNA
export
(Fig.
8B and C). These results indicate that the ability of NXT1
to bind RanGTP is required to activate nuclear export of
tRNA.
| |
DISCUSSION |
We developed an assay that reconstitutes nuclear export of U
snRNAs, tRNA, and mRNAs in vitro. Selective permeabilization of the plasma membrane and nuclear envelope of human cells permitted nuclear loading of a homogenous population of in vitro-transcribed RNAs. The ability of Xenopus egg membranes to repair
permeabilized human nuclear envelope and restore selective protein
import (6) was used to reseal RNA-loaded nuclei and study
nuclear export. In agreement with the in vivo observations, not only
was U1, tRNA, and Rab11 mRNA nuclear export extract and
energy dependent but U1 transport was also strongly stimulated by the
5' monomethylated cap structure (7, 15, 22). In contrast, U3
was retained into the nucleus both in vitro and in vivo despite its 5'
cap structure, indicating that reconstituted nuclei conserved the selectivity of nuclear export (50). Other reconstitution
assays for RNA nuclear export have been previously described. Release of endogenous RNA from isolated mammalian nuclei has been studied previously and found to be not strictly dependent on energy, but in
these studies neither integrity of nuclear envelope nor intactness of
RNA was carefully controlled (2, 45). More recently,
synthetic nuclei assembled in Xenopus egg extracts on
paramagnetic DNA-coated beads containing a U1 template were shown to
transcribe U1 RNA and support U1 nuclear export in a temperature- and
energy-dependent manner. However, the precise nuclear export pathway in
this model, and in particular the cap dependence of U1 transport, was
not analyzed (3).
Nuclear export of U1 in vitro was inhibited upon addition of either the
importin -binding site of importin or RanQ69L in the extracts,
indicating that it likely requires ongoing NLS-mediated nuclear import
or at least nuclear accumulation of essential export factors present in
the extracts. U1 export depends on the integrity of its 5' cap
structure, a cis-acting export signal recognized by CBC
(20). CBC is a shuttling complex whose NLS-dependent import
is ensured by karyopherins and . One can assume that an
efficient nuclear import of CBC present in the extracts is essential
for the cap-dependent export of U1. As reported previously for studies
using microinjection in Xenopus oocyte nucleus, U1 was
exported by a CRM1-mediated pathway in vitro (11). However, whether CRM1 interacts directly with CBC or through an NES-containing bridging factor remains elusive. Although CRM1 binds
NES-containing proteins in a RanGTP-dependent manner, GTP hydrolysis is
not required for translocation but rather triggers dissociation of the
cargo-CRM1 complex after translocation (26, 43).
Surprisingly, data obtained both in vivo and in vitro indicate that
expression of RanQ69L in the nucleus inhibits U1 nuclear export
(19), suggesting that Ran could be required for an
intranuclear step prior export complex assembly. Alternatively, an
additional, essential and very limiting export factor could exist such
that its GTP hydrolysis-dependent cytoplasmic release from the export
complex and subsequent recycling is crucial for an efficient export of U1.
Using the in vitro assay, we show here that the NTF2-related protein
NXT1 stimulates the nuclear export of U1 snRNA. NXT1 was found
previously to activate the CRM1-dependent nuclear export of
leucine-rich NES-containing protein (5) and thus represents a factor involved in both protein and RNA nuclear export routes mediated by CRM1. In addition, our results provide the first
direct evidence that NXT1 is also involved in the CRM1-independent
export of both tRNA and mRNA. Indeed, the interaction between
NXT1 and the mRNA export factor TAP has been clearly
established, but the specific role of NXT1 in the TAP-mediated
mRNA export pathway remains elusive (25). The
ability of NXT1 to interact with RanGTP is clearly required to activate
nuclear export. NXT1 could stimulate the formation of or stabilize
cargo-containing export complexes either by increasing affinity of
export receptors for RanGTP or by binding to both RanGTP and export
receptor. This hypothesis is supported by the ability of NXT1 to bind
both RanGTP and TAP and by the involvement of Ran in mRNA
export (19), although the precise role of Ran in the
TAP-mediated mRNA export has not been established. Such
a hypothesis would also predict that NXT1 is able to interact with CRM1
and exportin t. Alternatively, it has been shown that NXT1 localizes
both in the nucleoplasm and to the NPC and is able to shuttle between
nucleus and cytoplasm (5). NXT1 could thus target
cargo-export receptor-RanGTP complexes to the NPC or mediate
interaction of these export complexes with specific nucleoporins
during translocation through the NPC. Although NXT1 has no effect
on classical NLS import in vitro (5), the possibility that
NXT1 could stimulate RanGTP import and play a role analogous to that of
NTF2 in RanGDP import cannot be formally excluded. Future
experiments should yield additional clues to the function of NXT1 in
nuclear export.
| |
ACKNOWLEDGMENTS |
B.O.N. and C.M. contributed equally to this work.
We thank B. Wolff (Novartis) for the generous gift of leptomycin B,
I. W. Mattaj, J. P. Bachellerie, B. Goud, and E. Lund for
U1Sm, U3, rab11, and tRNA constructs, respectively, G. Dreyfuss for
GST-M9, J. C. Courvalin for anti-gp210 antibodies, R. Bastos for
anti-Nup, and B. Burke for anti-Nup. We are grateful to D. Tenza and G. Raposo for their help in electron microscopy.
This work was supported by grants from the Association de Recherche
contre le Cancer and the Ligue contre le Cancer to C.D. and by funds
from the American Cancer Society grant RPG98-048-01-CSM to B.M.P.
B.O.N. is supported by Rhone Poulenc Rorer.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Transport Nucléocytoplasmique, Unité Mixte de Recherche 144 Institut Curie-CNRS, 26 rue d'Ulm, 75248 Paris Cedex 05, France.
Phone: 0033 1 42346366. Fax: 0033 1 42346367. E-mail:
dargemon{at}curie.fr.
Present address: Laboratoire de la Dynamique Nucléaire et de
la Plasticité du Genome, Unité Mixte de Recherche 218 Institut Curie-CNRS, 75248 Paris Cedex 05, France.
| |
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Molecular and Cellular Biology, July 2000, p. 4562-4571, Vol. 20, No. 13
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
