From the Institut Jacques Monod, Unité Mixte de
Recherche 7592, CNRS, Universités Paris VI et VII, 2 Place
Jussieu, Tour 43, 75251 Paris Cedex 05, France, the § Center
for Cell Signaling, University of Virginia, Charlottesville, Virginia
22908, the ¶ U526-Laboratoire de Virologie, 06107 Nice Cedex
2, France, and the
Institut de Génétique
Moléculaire,
34297 Montpellier Cedex 5, France
Received for publication, December 3, 2002, and in revised form, December 20, 2002
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ABSTRACT |
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The adenovirus VA1 RNA (VA1), a 160-nucleotide
(nt)-long RNA transcribed by RNA polymerase III, is efficiently
exported from the nucleus to the cytoplasm of infected cells, where it
antagonizes the interferon-induced antiviral defense system. We
recently reported that nuclear export of VA1 is mediated by a
cis-acting RNA export motif, called minihelix, that comprises a
double-stranded stem (>14 nt) with a base-paired 5' end and a 3-8-nt
protruding 3' end. RNA export mediated by the minihelix motif is
Ran-dependent, which indicates the involvement of a
karyopherin-related factor (exportin) that remained to be determined.
Here we show using microinjection in Xenopus laevis oocytes
that VA1 is transported to the cytoplasm by exportin-5, a nuclear
transport factor for double-stranded RNA binding proteins. Gel
retardation assays revealed that exportin-5 directly interacts with VA1
RNA in a RanGTP-dependent manner. More generally, in
vivo and in vitro competition experiments using
various VA1-derived, but also artificial and cellular, RNAs lead to the
conclusion that exportin-5 preferentially recognizes and transports
minihelix motif-containing RNAs.
Nucleo-cytoplasmic transport of most RNAs and proteins is
dependent on soluble receptors called karyopherins that can dock at and
translocate through the nuclear pore complex. Interaction between cargo
and karyopherin Our understanding of the nuclear export of RNAs has been greatly
facilitated by the study of viral RNAs. For this reason, we focused our
attention on the adenovirus VA1 RNA (VA1), a
160-nt1-long RNA transcribed
by RNA polymerase III that massively accumulates in the cytoplasm of
infected cells. It serves to antagonize the interferon-induced cellular
antiviral defense system. Indeed, VA1 binds and inhibits the
double-stranded RNA-dependent protein kinase R (PKR), which
otherwise phosphorylates eIF2 RNA Mutants--
VA
t-RNA Electrophoretic Mobility Shift Assay--
EMSA was performed in
binding buffer (20 mM Hepes, pH 7.9, 50 mM KCl,
5 mM NaCl, 0.2 mM EDTA, 0.5 mM
dithiothreitol, and 10% glycerol) containing 250 nM
Mut10 to reduce unspecific binding, in a final sample volume of 20 µl. Recombinant proteins were preincubated with unlabeled competitors
for 10 min at room temperature and incubated with the radiolabeled
probe for 25 additional minutes. Then, 1 µl of loading
DTT-dithiothreitol buffer (0.6 mg ml Xenopus laevis Oocyte Microinjections--
Oocytes injections
and analysis of microinjected RNA by denaturing gel electrophoresis and
autoradiography were performed as described previously (7). Stability
of competitor VA RNA has been verified previously (7).
VA1 consists of three functional domains, an apical stem-loop
required for PKR binding, a central domain responsible for PKR inhibition, and a terminal stem, which brings together the 5' and 3'
ends of the RNA. The 3' end consists in an unpaired oligouridine stretch characteristic of polymerase III transcripts that not only acts
as a transcription termination signal but also as a primary binding
site for the La protein. To analyze the role of the oligouridine
stretch in the nuclear export of VA RNA, this sequence was replaced by
a UAG sequence (Fig. 1A).
Corresponding wild type (VA
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
is governed by the GTPase Ran. The asymmetric
distribution of the Ran regulatory proteins provides a steep gradient
of RanGDP (cytoplasmic)/RanGTP (nuclear) across the nuclear envelope
that ensures the directionality of nuclear transport (1, 2). Nuclear
import receptors unload their cargo upon binding to RanGTP in the
nucleus, whereas RanGTP is used to assemble export complexes which are
in turn destabilized by dissociation of RanGTP in the cytoplasm (3,
4).
and leads to the inhibition of protein
synthesis (5, 6). Adenovirus VA1 RNA contains a new cis-acting RNA
export motif that comprises a double-stranded stem (>14 nt) with a
base-paired 5' end and a 3-8-nt protruding 3' end and that can
tolerate some mismatches and bends (7). This export signal, called
minihelix, is present not only in VA1 but in a large family of small
viral and cellular RNAs transcribed by polymerase III. RNA export
mediated by the minihelix motif is Ran-dependent, which
indicates the involvement of a karyopherin-related factor (exportin).
This exportin is distinct from Crm1 and exportin-t (7, 8). Therefore,
we sought to identify cellular factors that bind to and mediate the
export of minihelix-containing RNAs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
IV, Mut10 mutants, and artificial
stems have been described previously (7). VARdm and Mut9 mutants are
derived from VA
IV. VARdm contains a 3'-terminal oligouridine
stretch replaced by a UAG sequence. Mut9 presents a 6-base mispairing
in the 5' end. These mutants have been generated by PCR and confirmed
by sequencing. In vitro transcription was performed as
described previously (7).
1 heparin, 1 mg
ml
1 bromphenol blue) was added, and incubation was
pursued for another 5 min. Half of each sample was loaded on a 5%
non-denaturing polyacrylamide gel, and electrophoresis was carried out
at a constant voltage of 13.3 V cm
1 at 4 °C in 0.5×
TBE (45 mM Tris borate, 1 mM EDTA).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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IV) and mutant (VARdm) radiolabeled RNAs
were transcribed in vitro and injected into
Xenopus oocyte nuclei. Radiolabeled U1 small nuclear
RNA with a mutated Sm-binding site (U1
Sm) and tRNAPhe
were co-injected as internal control of exported RNA and U6
ss RNA
that is not transported from the nucleus was used as a control of
injection. Nucleocytoplasmic distribution of these RNAs was analyzed
following a 3-h incubation (Fig. 1B). As described
previously, about 40-50% of VA
IV was detected in the cytoplasm
3 h after injection (7). In contrast, a complete nuclear export of
VARdm was observed in the same experimental conditions. This result indicates that the 3'-oligouridine stretch is not necessary for export
of VA1 and rather retains this RNA in the nucleus as it has been
previously shown for human Y1 RNA and U6 RNA (11, 12).
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Fig. 1.
Specific characteristic of the VA1 RNA export
pathway. A, structure of the VA1-derived or artificial
terminal stem RNA mutants. The ability of these RNAs to be exported
from the nucleus of Xenopus oocytes is indicated on the
right. B, a mixture of 32P-labeled
VADIV or VARdm, U1DSm, U6Dss, and tRNAPhe was injected into
oocyte nuclei in the absence or presence of 2.5 pmol of the indicated
competitor. After the indicated time at 19 °C, total (T),
nuclear (N), and cytoplasmic (C) RNAs were
extracted and analyzed by polyacrylamide gel electrophoresis in
denaturing conditions.
Study of the determinant responsible for the cytoplasmic localization
of VA1 led to the identification of a new cis-acting RNA export motif
within the terminal stem. This structural motif consists in a
double-stranded stem (>14 nt) with a base-paired 5' end and a 3-8-nt
protruding 3' end (7). We thus used either VA1-derived or artificial
RNA containing a functional or altered minihelix structure and tested
their ability to compete out the export of VARdm in Xenopus
oocyte nuclei. Both VAIV and Stem20, which form a terminal stem
mimicking that of VA1, efficiently prevented this process (Fig. 1,
A and B) but did not affect the nuclear export of
U1
Sm and tRNAPhe, which are exported by Crm1 and
exportin-t, respectively. It should be noted that cellular RNA Y1,
which also presents a minihelix motif required for its nuclear export,
also competes out the transport of VA1 but not tRNA or U1
Sm (7, 8).
In contrast, mutant RNAs that display mispairing of the 5' end (Mut9
and MM3), a shorter stem (Stem12), or no terminal stem (Mut10) poorly
interfere with nuclear export (Fig. 1, A and B).
The correspondence between the capacity of a given RNA to be exported
from the nucleus (7) and to inhibit VARdm nuclear export strongly
suggested that RNAs containing a minihelix motif utilize a unique,
saturable, and structure-dependent nuclear export pathway.
We previously reported that RNA export mediated by the minihelix motif
is controlled by the GTPase Ran, which indicates the involvement of a
karyopherin-related factor (exportin). In addition, we also found that
exportin-5, a karyopherin related to human Crm1 and the
Saccharomyces cerevisiae Msn5/Kap142p, acts as a nuclear
export receptor for proteins containing double-stranded RNA binding
domains (dsRBD) (13). Since the structural basis of the minihelix motif
corresponds to a double-stranded RNA stem, we tested the ability of
exportin-5 to interact with VARdm in a EMSA. No detectable binding of
recombinant exportin-5 or RanGTP could be observed on a VARdm probe
(Fig. 2A, lanes 2 and 3). In contrast, exportin-5 was able to directly bind
VARdm in a RanGTP-dependent manner (Fig. 2A,
compare lanes 4 and 5). The specificity of the exportin-5/minihelix interaction was further confirmed by the ability
of VARdm and Stem 20 to compete out the formation of this complex (Fig.
2A, lanes 6 and 9), whereas
minihelix-derived mutants that are not exported did not interfere with
this complex (Fig. 2A, lanes 7, 8,
10, and 11). In agreement with these results, EMSA experiments using either VARdm or Mut9 as probe indicate that
exportin-5 interacts better with VARdm than with Mut9 (data not shown).
These data clearly indicate that exportin-5 specifically recognizes the
minihelix motif, and this interaction is prevented by a 5' protruding
end or limited stem length.
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We then compared the competition efficiency of VARdm and various
cellular RNAs on the exportin-5-VARdm complex. Addition of 1 pmol of unlabelled VARdm already affected the formation of the radiolabeled probe-exportin-5-RanGTP complex that was completely prevented with 50 pmol of VARdm (Fig. 2, B and
C). No significant competition could be observed using
U1sm that do not contain the minihelix motif, and a poor competition
was observed using Mut10. hY1 RNA presents a 18-nt-long double-stranded
stem with mismaches, a base-paired 5' end, and a protruding 3' end,
thus corresponding to a minihelix structure less optimal than the VA1. Consistently, 5-fold more hY1 RNA than VARdm were required to compete
out the formation of the radiolabeled probe-exportin-5-RanGTP complex.
Finally tRNAPhe that contains a highly degenerated
minihelix motif displayed a very weak competition effect (Fig. 2,
B and C). Similar results were obtained with
purified Escherichia coli tRNA than in vitro transcribed human tRNAPhe. Together, these data indicate
that exportin-5 preferentially recognizes minihelix-containing RNAs
with affinity varying as a function of the minihelix structure.
To determine whether the ability of exportin-5 to interact with
minihelix structure in a RanGTP-dependent manner correlates with its ability to mediate the nuclear export of minihelix-containing RNAs, we examined the effect of microinjection of recombinant exportin-5 on the transport of various RNA in Xenopus
oocytes (Fig. 3). For this purpose, a
mixture of labeled VARdm, U3, U1Sm, Mut10, and tRNAMet
RNAs were injected into oocyte nuclei following preinjection of control
buffer or recombinant exportin-5, and their nucleocytoplasmic distribution was analyzed after short incubation periods (12-30 min).
It should be noted that t-RNAMet used in this experiment
contained an additional GGG at the 5' end allowing the formation of a
pseudo-minihelix structure 2 nt longer than mature tRNA. This tRNA is
normally exported to the cytoplasm only after the processing of the GGG
5' end (9, 10). As shown on Fig. 3, A and B,
preinjection of exportin-5 dramatically accelerated VARdm export and
weakly stimulates the export of unprocessed tRNA. Importantly, the
exportin-5 did not significantly affect the export of other RNAs.
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Altogether, our results demonstrate that exportin-5 promotes the
nuclear export of the viral VA1 RNA. More generally, this exportin
appears to specifically interact with the minihelix RNA motif in a
RanGTP-dependent manner and is likely responsible for the
nuclear transport of viral or cellular RNAs containing such a motif.
Based on our results, an optimal exportin-5-interacting minihelix
presents a 20-nt-long double-stranded stem with a base-paired 5' end
and a protruding 3' end. It has been reported recently that exportin-5
is able to stimulate nuclear export of tRNA in microinjected
Xenopus oocytes nuclei when transport of endogenous tRNA was
artificially saturated (14). However, in normal experimental conditions, we found that exportin-5 does not affect tRNA transport. In
addition, although exportin-5 can bind tRNA, our results show that its
affinity for the optimal minihelix structure is much higher. This
strongly suggest that tRNAs, which contain a degenerate minihelix, do
not represent a preferential cargo for exportin-5 but can eventually
use this transport receptor when their own transport pathway using
exportin-t is deficient. Interestingly, the predicted secondary
structures of intermediates of the recently discovered class of
micro-RNAs and siRNAs resemble the minihelix motif and might mediate
their nuclear export by the exportin-5 pathway. In addition, exportin-5
acts as a nuclear export receptor for proteins containing dsRBD. This
could occur through an indirect interaction mediated by dsRNA similarly
to exportin-5/eEF1A interaction via tRNA (15), but one can certainly
not exclude that certain dsRBD-containing proteins might participate in
the nuclear export of minihelix RNAs.
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ACKNOWLEDGEMENTS |
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We thank members of the laboratory for helpful and stimulatory discussions and M. Rodriguez, U. Nehrbass, and J. Salamero for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from Sidaction, the Association de Recherche contre le Cancer, and the Agence Nationale de Recherche contre le SIDA.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.
** To whom correspondence should be addressed: Inst. Jacques Monod, Unité Mixte de Recherche 7592, CNRS, Universités Paris VI et VII, 2 Place Jussieu, Tour 43, 75251 Paris Cedex 05, France. Tel.:/Fax: 33-1-44276956; E-mail: dargemont@ijm.jussieu.fr.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.C200668200
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ABBREVIATIONS |
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The abbreviations used are: nt, nucleotide; PKR, protein kinase R; EMSA, electrophoretic mobility shift assay; dsRBD, double-stranded RNA binding domains.
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