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Molecular and Cellular Biology, May 1999, p. 3540-3550, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Carboxyl Terminus of RNA Helicase A Contains a
Bidirectional Nuclear Transport Domain
Hengli
Tang,1
David
McDonald,
Tamara
Middlesworth,1
Thomas J.
Hope, and
Flossie
Wong-Staal1,*
Department of Biology1
and Department of Medicine,3 University
of California, San Diego, and Infectious Disease Laboratory,
The Salk Institute for Biological Sciences,2 La
Jolla, California
Received 31 August 1998/Returned for modification 5 October
1998/Accepted 29 January 1999
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ABSTRACT |
Human RNA helicase A was recently identified to be a shuttle
protein which interacts with the constitutive transport element (CTE)
of type D retroviruses. Here we show that a domain of 110 amino acids
at the carboxyl terminus of helicase A is both necessary and sufficient
for nuclear localization as well as rapid nuclear export of glutathione
S-transferase fusion proteins. The import and export
activities of this domain overlap but are separable by point mutations.
This bidirectional nuclear transport domain (NTD) has no obvious
sequence homology to previously identified nuclear import or export
signals. However, the Ran-dependent nuclear import of NTD was
efficiently competed by excess amounts of the nuclear localization
signal (NLS) peptide from simian virus 40 large T antigen, suggesting
that import is mediated by the classical NLS pathway. The nuclear
export pathway accessed by NTD is insensitive to leptomycin B and thus
is distinct from the leucine-rich nuclear export signal pathway
mediated by CRM1.
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INTRODUCTION |
Macromolecular trafficking across
the eukaryotic nuclear envelope occurs through the nuclear pore
complexes and involves specific signal-receptor interactions. Nuclear
localization signals (NLS) are capable of conferring the nuclear import
and retention functions to heterologous nonnuclear proteins
(3). Both the classical NLS, first identified in simian
virus 40 (SV40) large T antigen, and the bipartite NLS, found in a
number of Xenopus nuclear proteins, contain stretches of
basic amino acid residues. NLS-bearing proteins interact with the NLS
receptor, namely, importin 
, in the cytoplasm. Subsequent binding to
importin 
targets the NLS-importin / complex to the nuclear
pore for translocation across the membrane (12, 31). Another
type of nuclear import signal, termed M9, was found in heterogeneous
nuclear ribonucleoprotein (hnRNP) A1 and several related hnRNPs
(26, 39). M9 specifies both nuclear import and export of
hnRNP A1 and bears no sequence resemblance to the classical or
bipartite NLS. The only known receptor protein for M9 is a protein
distantly related to importin , named transportin (1, 9,
36). Additional nuclear import sequences have been identified,
but their receptors are currently unknown (16, 19, 27). A
signal from hnRNP K is believed to bypass the requirement of a soluble
receptor and interact directly with the nuclear pore (27).
Nuclear export of RNA and protein is less well understood but also
appears to be signal mediated and energy dependent. The best-characterized, leucine-rich nuclear export signals (NES) use CRM1,
also a distant relative of importin , as their functional export
receptor (6, 10, 32, 40, 43). Leucine-rich NES complexes
with CRM1 and RanGTP in the nucleus prior to translocation through the
nuclear pore. This complex formation is sensitive to leptomycin B
(LMB). Different classes of cellular RNA were shown to use distinct
pathways for nuclear export (18). Recently, the receptor for
nuclear export of tRNA, named exportin-t, was shown to be another
member of the importin family (21).
The nuclear export of cellular mRNA is tightly coupled to splicing,
such that only completely spliced mRNA is exported into the cytoplasm.
However, export of unspliced viral RNA is necessary for the expression
and replication of retroviruses. For lentiviruses, a viral regulatory
protein named Rev promotes the nuclear export of unspliced and
incompletely spliced viral RNA by binding to its cognate RNA sequence,
the Rev response element (RRE) (4, 5, 24). Rev contains a
leucine-rich NES and uses CRM1 as the export receptor (4,
6). In simple retroviruses, the unspliced RNAs contain a
cis-acting, constitutive transport element (CTE) that
interacts directly with cellular factors to achieve nuclear export
(2, 34, 41, 48). We previously showed that human RNA
helicase A specifically binds functional but not mutant CTE in vitro
(42) and that antibodies to helicase A blocked CTE function
when microinjected into human cells (23). We also showed
that helicase A shuttles constantly between the nucleus and cytoplasm
despite its apparent steady-state nuclear localization (42).
In this paper, we report the identification and characterization of a
nuclear transport domain (NTD) in helicase A which directs the
bidirectional trafficking of fusion proteins. This domain presumably
plays an important role in the posttranscriptional regulation of
retroviruses by helicase A.
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MATERIALS AND METHODS |
Plasmid constructions.
The cDNA clone of human RNA helicase
A was a kind gift from J. Hurwitz (22). Plasmid pSK-helicase
A was first digested with BglII, blunt ended with Klenow
polymerase, and then digested with EcoRI. A 3.4-kb fragment
that encodes amino acids 1 to 1136 of helicase A was recovered. pEGFP-1
(Clontech) was digested with BamHI, blunt ended, and then
digested with NotI. A 0.7-kb fragment encoding the green
fluorescent protein (GFP) was recovered. The 3.4- and 0.7-kb fragments
were then used in a three-way ligation with the
EcoRI-NotI-digested pcDNA3. The appropriate
construct encodes amino acids 1 to 1136 of helicase A with GFP fused to the carboxyl terminus and is called GFP/HelA-
CTD. The GFP coding sequence from pEGFP-1 was PCR amplified and cloned into the
BamHI and EcoRI site of pcDNA3 (Invitrogen) such
that the EcoRI site at the 3' end of the coding sequence can
be used for in-frame cloning of GFP fusion proteins. This plasmid was
designated pc-GFP. pSK-helicase A was digested with ApaI and
EcoRI and then blunt ended with Klenow polymerase, and a
3.9-kb blunt fragment (including the 3' untranslated region) encoding
amino acids 137 to 1269 of helicase A was recovered and cloned into the
EcoRI-digested but blunt-ended pc-GFP vector to make
GFP/HelA-ApaI. GFP/ApaI-CTD was made in a similar way, but the
ApaI-BglII fragment of helicase A was cloned into
pc-GFP. To make the GFP-helicase carboxyl-terminal domain fusion
proteins, different carboxyl-terminal domain fragments of helicase A
were PCR amplified and cloned into the EcoRI site of pc-GFP.
Site-directed mutagenesis was carried out by a method similar to that
used with the Transformer kit of Clontech. Mutated sequences were used
for testing subcellular distributions of the protein domain they
encode. Myc-PK plasmid was described by Siomi and Dreyfuss
(39). Wild-type and mutant NTDs were PCR amplified as
KpnI-NotI fragments and cloned into the
KpnI and NotI sites of pcDNA3/Myc-PK to make
Myc-PK-NTD and Myc-PK-NTD mutant plasmids. Myc-NPc-TNLS has been
described by Michael et al. (26). Wild-type and mutant NTDs
were PCR amplified as XhoI-XbaI fragments and cloned into the corresponding sites of pcDNA3/Myc-NPc-TNLS to make
NPc-TNLS-NTD, NPc-TNLS-K62, and NPc-TNLS-R65. GFP-TNLS-NTD and
mutant constructs were made by cloning the
EcoRI-XbaI fragments of the corresponding
NPc-TNLS plasmids into EcoRI-XbaI-digested pc-GFP. pGST-NTD was made by cloning the EcoRI fragment of
GFP-NTD into yeast vector pGAD10, resulting in pGAD10-NTD. The
BamHI-BglII insert of this plasmid was then
cloned into the BamHI site of pGEX-2T (Pharmacia) to yield
pGST-NTD. pGST-K62 was made in a similar fashion.
Cell cultures, transfections, and Western blotting.
HeLa and
Cos-1 cells were maintained in Dulbecco's modified Eagle's medium
with 10% fetal calf serum. Transfections were done by calcium
phosphate precipitation. In experiments where transcription and
translation inhibitors were included, they were added 45 h after
transfection, 3 h prior to fixation of the cell for
immunofluorescence assay. For the Western blot, Myc-PK-NTD plasmids
were transfected into Cos-1 cells, and cells were harvested 48 h
later and lysed in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis buffer. DNA was sheared by passage through a syringe
and boiling. Total proteins were either treated with alkaline
phosphatase for 1 h at 37°C or directly loaded onto a 12.5%
polyacrylamide gel. The separated proteins were transferred onto a
nitrocellulose membrane and Myc-tagged proteins were detected with
monoclonal antibody 9E10 (Babco).
Heterokaryon assay.
Interspecies heterokaryons of HeLa and
NIH 3T3 cells were formed as described previously (27). For
GFP-CTD and GFP-NTD, cells were fixed 1 h after fusion. For the
GFP-TNLS-NTD series, NPc-TNLS-NTD series, and Rev, cells were fixed
3 h after fusion. To confirm the inhibitory effect of
cycloheximide on new protein synthesis, cells were metabolically
labeled with [35S]methionine and
[35S]cysteine 30 min after the addition of the
translation inhibitor, labeled for 1 h at 37°C, lysed, and
assayed for radiation incorporation.
Immunofluorescence analysis.
For GFP-transfected cells,
cells were fixed in 4% paraformaldhyde and viewed under a Nikon
fluorescence microscope. For Myc-PK or Myc-NPc-TNLS fusion
construct-transfected cells, the cells were fixed and stained with
monoclonal antibody 9E10 as described previously (26). In
all the heterokaryon experiments, 5 µg of Hoechst 33258 (Sigma) per
ml was added either in 3% bovine serum albumin (BSA) after
permeabilizing the cells with 0.2% Triton X-100 (for GFP fusion
proteins) or during the secondary-antibody staining (for NPc-TNLS
constructs). MEK-1 was stained with a polyclonal antibody purchased
from Santa Cruz Biotechnology Inc. GST-NTD was stained with a
monoclonal antibody against GST (Santa Cruz Biotechnology Inc.).
Recombinant protein purification, peptide conjugation, and
microinjection.
pGST-NTD was transformed into bacterial strain
BL21. Recombinant protein purification was carried out with a
commercial GST purification module (Pharmacia). The final protein
sample was concentrated to 3 mg/ml for microinjection. NIH 3T3 cells
growing on coverslips were injected by using an Eppendorf
microinjector. For the LMB experiments, cells were treated with 20 nM
LMB 6 h before injection. One group of coverslips was inverted
onto 50% polyethylene glycol to form polykaryons; 1 h after cell
fusion, GST-NTD was mixed with 1.5 mg of rhodamine-dextran per ml
before being injected into selected nuclei of the polykaryon. The cells were fixed for GST staining 30 min after injection. Another group of
coverslips was fixed for immunostaining with a polyclonal antibody against MEK-1. BSA conjugates were generated as previously described with either wild-type (CYTPPKKKRKLY) or mutant (CYTPPKTKLV) NLS peptide.
RNA gel shift assays.
Gel mobility shift experiments were
carried out as described previously (42). RNA helicase A
protein and helicase A protein with the carboxyl terminus deleted were
kind gifts from C. Lee and J. Hurwitz. GST-CTD was produced and
purified similarly to GST-NTD.
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RESULTS |
The carboxyl terminus of helicase A harbors an NLS.
N-terminal
and C-terminal deletions of RNA helicase A were generated and fused to
GFP (Fig. 1a). The subcellular
localization of fusion proteins was compared with that of endogenous
helicase A and of GFP alone. We observed that deletion of the
N-terminal amino acids (amino acids 1 to 135) did not affect the
nuclear localization of the fusion protein (GFP/ApaI). In contrast,
deletion of the carboxyl-terminal domain (CTD) (amino acids 1137 to
1269) both in the full-length context (GFP/HelA-C) and together with the N-terminal deletion (GFP/ApaI-C) rendered the fusion protein completely cytoplasmic (Fig. 1b), indicating that the C terminus of
helicase A is needed for its nuclear localization. To find out whether
the CTD of helicase A is sufficient for nuclear import, we fused the
CTD to GFP and examined the distribution of the fusion protein. GFP-CTD
also had a nuclear localization similar to that of endogenous helicase
A and GFP/ApaI. We therefore conclude that the CTD of helicase A is
both necessary and sufficient for nuclear localization.
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FIG. 1.
The carboxyl terminus of helicase A is both necessary
and sufficient for nuclear localization. (a) Schematic illustration of
various domains of RNA helicase A that were fused to GFP to map the NLS
of helicase A. Black bar, GFP; dashed line, deleted domain; open bar,
domains of helicase A. (b) Subcellular localization of different
helicase A domains. The minimal NLS was mapped to amino acids 1150 to
1259.
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To map the minimal determinants for nuclear import, we fused a series
of 5' and 3' deletions of the CTD to GFP and studied
their subcellular
localization. The minimal functional NLS was
mapped between amino acids
1150 and 1259 (Table
1; Fig.
1b).
The
inability of further CTD deletions to localize in the nucleus
was not
due to their having insufficient sizes, because M9 of
hnRNP A1, which
is less than 40 amino acids long, was able to
localize GFP in the
nucleus in the same experiments (data not
shown). Examination of this
minimal NLS sequence revealed no homology
to either the classical NLS
(SV40 T antigen or bipartite NLS),
M9 from hnRNP, or the nuclear
import-export signal of hnRNP K
(
27,
39).
The CTD also contains a functional NES.
We recently
demonstrated that helicase A is a shuttle protein (42) and
thus probably contains a nuclear export signal. We then tested the CTD
for its ability to direct the nuclear export of GFP-CTD. Inhibition of
transcription blocks the nuclear import of shuttling proteins, making
it possible to detect these steady-state nuclear proteins in the
cytoplasm (25, 35). In our previous experiments, actinomycin
D treatment caused endogenous helicase A to accumulate in the cytoplasm
of HeLa cells. Cytoplasmic accumulation of GFP-CTD was also observed in
transfected cells similarly treated with actinomycin D (data not
shown), suggesting that CTD is capable of mediating nuclear export.
To obtain more definitive evidence for the nuclear export function of
CTD, interspecies heterokaryon experiments were carried
out with
GFP-CTD or the minimal NLS fused to GFP. HeLa cells that
were
transfected with these constructs were fused to NIH 3T3 cells
to form
heterokaryons. Cycloheximide was added to inhibit de novo
protein
synthesis. The appearance of GFP in the mouse nuclei within
the
heterokaryon indicated that the fusion protein had moved out
of the
human nuclei and into the mouse nuclei. The mouse nuclei
were
distinguished from the human nuclei by punctate staining
with Hoechst
33258. A Myc-tagged form of NPc-TNLS (
26), which
contains
the core domain of
Xenopus nucleoplasmin and an NLS from
SV40 T antigen, was cotransfected as a nonshuttling nuclear protein
control. As shown in Fig.
2, GFP-CTD was
exported from the nucleus
of the transfected human cell while
Myc-NPc-TNLS stayed in the
same nucleus. Similar results were obtained
for the minimal NLS
domain (data not shown). Therefore, both CTD and
the minimal NLS
sequence also contain an NES. For this reason, we named
the domain
between residues 1150 and 1259 the NTD (nuclear transport
domain).
These results indicate that although GFP-NTD has a
steady-state
nuclear localization, it actually shuttles constantly
between
the nucleus and the cytoplasm.
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FIG. 2.
The carboxyl terminus of helicase A harbors an NES. An
interspecies heterokaryon assay confirmed the shuttling ability of NTD.
Mouse nuclei were identified by the punctate staining by Hoechst 33258. Anti-Myc antibody detected the cotransfected Myc-NPc-TNLS. GFP was
found only in transfected HeLa cell nuclei and mouse nuclei within the
heterokaryons that contained transfected human cells.
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The nuclear import and export activities of NTD are separable by
point mutations.
NTD, which contains part of the RGG box of
helicase A (22, 46), is rich in glycine and serine residues.
The N terminus of the domain contains several closely spaced basic
residues (Fig. 3). Site-directed
mutagenesis was used to identify specific amino acid residues that are
critical for the import and export functions of NTD. Point mutations
were introduced across the domain, and the mutants were tested for
their ability to direct the GFP fusion protein in and out of the
nucleus (Table 2). All the point mutants that retained the nuclear import function also maintained their nuclear
export activity. Mutations in two basic residues, lysine 1162 and
arginine 1165 (K62, R65, or R65L) abolished the nuclear localization of GFP-NTD, but deletion of arginine 1158 (R58) or a
neighboring tyrosine 1166 (Y66) did not have any effect. The loss of
import function of the K62 and R65 mutants was confirmed in another
experiment. NTD, R58, K62, R65, and Y66 were fused to the
carboxyl terminus of a Myc-tagged pyruvate kinase (39) and
transiently expressed in HeLa cells. The subcellular localization of
the fusion proteins was determined by immunostaining with a monoclonal
antibody against Myc. Myc-PK localized exclusively in the cytoplasm, as
previously reported (39). PK-NTD, PK-R58, and PK-Y66
were all localized in the nucleus, while the PK-K62 and PK-R65
derivatives were localized in the cytoplasm (Fig. 4a).
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FIG. 3.
Sequence of the RNA helicase A NTD. (a) Protein sequence
of helicase A NTD. Residues that were mutated in this study are
underlined. Positions of residues are those of Lee and Hurwitz
(22) but adjusted to the carboxyl-terminal sequence of
nuclear DNA helicase II as given by Zhang and Grosse (46).
We independently sequenced the carboxyl terminus of RNA helicase A
clone 1 (22), from which we identified NTD; we found that it
is identical to that of nuclear DNA helicase II. (b) Positive residues
are important for NTD and other nuclear import signals. Arginines and
lysines are underlined; residues in boldface type are essential for the
NLS activity of the protein. Only part of the NTD sequence is shown in
panel b.
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FIG. 4.
Subcellular localization and expression of NTD mutants
in HeLa cells. (a) Subcellular localization of NLS mutants. Myc-PK
localized in the cytoplasm (39); NTD and R58 targeted
this protein into the nucleus, while K62 and R65 did not. (b)
Immunoblotting analysis of PK-NTD and mutant fusion proteins. PK was
truncated by a KpnI site at amino acid 443 during cloning so
that PK-NTD is approximately the same size as PK. PK-K62 and
PK-R65 had a lower gel mobility than expected, possibly because NTD
is modified in the cytoplasm. Phosphatase treatment did not alter the
gel mobility of PK-K62 and PK-R65. M.W., molecular weight.
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The efficient expression of the PK-NTD fusion proteins in mammalian
cells was verified by Western blotting with the antibody
against the
Myc tag (Fig.
4b). PK was truncated at the carboxyl
terminus when fused
to the NTD series, and so Myc-PK-NTD is about
the same size as Myc-PK.
This analysis revealed that the PK-
K62
and PK-
R65 derivatives had
a lower mobility than PK-NTD and PK-
R58.
This reduced
electrophoretic mobility was probably due to posttranslational
modifications of NTD in the cytoplasm of eukaryotic cells, since
GST
fusion proteins of NTD and
K62 expressed in
Escherichia
coli exhibited similar electrophoretic mobility (data not shown).
Treatment
of the transfected cell lysates with alkaline phosphatase did
not alter the mobility of PK-
K62 and PK-
R65 (Fig.
4b), suggesting
that the shift is not due to
phosphorylation.
K62 and
R65 were further studied for their ability to mediate
export. Wild-type NTD,
K62, and
R65 were fused to the carboxyl
terminus of Myc-NPc-TNLS, and the resultant hybrid proteins were
tested
for their abilities to shuttle between the cytoplasm and
nucleus by
interspecies heterokaryon assays. As shown in Fig.
5a, both mutants still shuttled. The
K62 and
R65 NTD mutations
were also able to facilitate the
shuttling of GFP-TNLS in heterokaryon
assays (data not shown).
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FIG. 5.
NTD import mutants retain nuclear export function. (a)
NPc-TNLS had a nuclear localization and did not shuttle in heterokaryon
assays; both import mutants (K62 and R65) shuttled as well as the
wild-type NTD when fused to carboxyl terminus of NPc-TNLS. Arrows
indicate the mouse nuclei in the heterokaryons. (b) K62 directed the
rapid export of GST fusion protein when injected in to nucleus.
GST-K62 was purified from E. coli as a recombinant
protein and injected into either the nuclei or the cytoplasm of NIH 3T3
cells. The cells were then fixed for an immunofluorescence assay with a
GST monoclonal antibody after a 30-min incubation at 37°C. The
injection site was identified by coinjecting 1.5 mg of
rhodamine-dextran per ml with GST-K62. Hoechst staining identifies
the nuclei.
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The effects of the
K62 NTD mutation on import and export function
were further studied by microinjection analysis of proteins.
A fragment
encompassing
K62 NTD was fused to GST (GST-
K62).
The expressed
recombinant protein was purified and microinjected
into either the
cytoplasm or the nuclei of NIH 3T3 cells. When
injected into the
cytoplasm, the fusion protein stayed in the
cytoplasm (Fig.
5b), but
when injected into the nuclei, it became
cytoplasmic after 30 min of
incubation at 37°C (Fig.
5b). These
results confirm the observations
obtained in the transfection
studies of the
K62 NTD mutation. Again,
the
K62 mutation disrupted
the nuclear import function of NTD while
having no effect on its
nuclear export. These results demonstrate that
the import and
export functions of the NTD are separable activities of
the
NTD.
Since the NTD contains part of the RGG box that is implicated in RNA
binding, it was possible that the export of NTD was bridged
by RNA
(e.g., CTE) which was being exported by other NES-containing
proteins.
To test this possibility, the ability of GST-CTD to
bind to the CTE was
analyzed in a gel mobility shift assay. As
shown in Fig.
6a, although helicase A with CTD deleted
had a weaker
binding to CTE than did native helicase A, CTD itself was
not
sufficient for CTE binding in vitro. To further address the
importance
of cellular RNA in NTD export, RNase A was coinjected into
the
nucleus of Cos-1 cells with GST-
K62. The presence of RNase in
the injected cells disrupted the integrity of the nucleus, as
evidenced
by the accumulation of the injection marker, rhodamine-dextran,
in the nucleoli of these cells. However, GST-
K62 was exported
efficiently in the same cells (Fig.
6b). Taken together, these
results
suggest that the export function of NTD is not mediated
by binding to
RNA.
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FIG. 6.
RNA binding does not play a significant role in NTD
export. (a) GST-CTD, helicase A protein, and helicase A with the C
terminus deleted were tested for CTE binding in RNA gel shift assays.
Full-length helicase A and the C-terminally deleted helicase A formed
complexes with CTE probe, while GST-CTD was not able to interact with
CTE in vitro. (b) RNase (5 mg/ml) was coinjected into Cos-1 nuclei with
GST-K62 and rhodamine-dextran. Cells were incubated for 30 min after
injection before being fixed and stained for GST and DNA (Hoechst).
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The nuclear import of NTD can be competed with NLS peptides.
Although the NTD sequence bears no resemblance to a classical or
bipartite NLS, the fact that closely spaced basic residues are critical
for import raised the possibility that NTD uses the importin /
pathway. To address this question, excess unlabeled TNLS peptides
conjugated to BSA were injected into the cytoplasm of NIH 3T3 cells
along with both labeled BSA-NLS and GST-NTD. BSA conjugates containing
mutant NLS peptides were used as a control for the specificity of the
competition. Injection of fluorescein isothiocyanate-labeled BSA-NLS
into the cytoplasm resulted in the nuclear import of the conjugates
after 30 min of incubation at 37°C (Fig.
7a), indicating that the BSA conjugates
are functional as nuclear import substrates. In the presence of the
BSA-mutant NLS conjugates, both the BSA-TNLS conjugates and GST-NTD
were efficiently imported into the nuclei after 30 min (Fig. 7b, top panel). However, the nuclear import of both BSA-NLS and GST-NTD was
competed by excess unlabeled BSA-NLS peptides (Fig. 7b, bottom panel).
These results indicate that the nuclear import of NTD has the same
sensitivity to excess NLS peptides as to conjugates containing TNLS.
Microinjection experiments with T24N Ran also showed that the nuclear
import of NTD is Ran dependent (data not shown), as expected for the
classical NLS pathway. The glycine-rich feature of NTD also prompted us
to determine if it interacts with transportin. NTD did not bind to
transportin in the yeast two-hybrid assay, whereas the hnRNP A1 M9
region readily bound to transportin (data not shown).
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FIG. 7.
Classical NLS peptides compete for NTD import. Peptides
representing the wild-type or mutant NLS of SV40 T antigen were
synthesized, purified, and conjugated to BSA. (a) Labeled BSA-NLS was
imported into the nucleus after being injected (inj) into the cytoplasm
of NIH 3T3 cells. The injection site was identified by the coinjected
rhodamine-dextran. (b) Unlabeled BSA-mutant NLS (top) or BSA-NLS
(bottom) was coinjected into the cytoplasm of NIH 3T3 cells with
GST-NTD or fluorescein isothiocyanate-labeled BSA-NLS. The GST-NTD was
stained with a monoclonal antibody against GST and detected by indirect
fluorescence microscopy. The nuclei were identified by Hoechst
staining.
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The nuclear export of NTD is not sensitive to LMB treatment.
We tested if the nuclear export of helicase A NTD is sensitive to LMB,
which disrupts the function of the leucine-rich NES. GST-NTD was
injected into selected nuclei of NIH 3T3 cell polykaryons in the
presence of 20 nM LMB, a concentration which is 10 times that used to
inhibit Rev export (45). Normal shuttling of GST-NTD would
be evidenced by its appearance in the uninjected nuclei within the
polykaryon (Fig. 8a). The efficacy of LMB
treatment on nuclear export in this study was controlled by analysis of the mitogen-activated protein kinase kinase (MEK-1) in parallel. MEK-1
is a shuttle protein with a steady-state cytoplasmic localization and
contains a leucine-rich NES at the amino terminus (11). While LMB treatment relocalized MEK-1 to the nucleus by blocking the
NES-mediated export as previously reported, it had no effect on GST-NTD
export (Fig. 8b). This result indicates that the export mediated by NTD
functions in a CRM1-independent manner.
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FIG. 8.
NTD-mediated export is insensitive to LMB. (a) NIH 3T3
cells were grown on coverslips and treated with polyethylene glycol to
form polykaryons. GST-NTD was then injected (inj) into selected nuclei
of polykaryons along with the injection marker rhodamine-dextran. The
cells were incubated at 37°C for 30 min before being fixed for
immunofluorescence assay. (b) LMB (20 nM) was included to inhibit NES
export. Cells stained for MEK-1 were similarly grown and treated with
LMB, but no injections were performed.
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DISCUSSION |
RNA helicase A contains a sequence that specifies bidirectional
nuclear-cytoplasmic trafficking.
ATP-dependent RNA helicase A was
first purified from HeLa nuclear extracts as an in vitro RNA helicase
activity (22). A subsequently described nuclear DNA helicase
II (46) is the same protein. Helicase A contains two
RNA-binding domains, a helicase core and an RGG box. Recent studies
suggest that this protein may be involved in gene regulation at both
the transcriptional and posttranscriptional levels (23, 29,
42). In spite of its predominantly nuclear localization
(47), helicase A apparently shuttles between the nucleus and
the cytoplasm (42). Here, we showed that a domain of 110 amino acids at the carboxyl terminus of helicase A is a bidirectional
NTD. Helicase A with a deletion of this domain accumulated in the
cytoplasm, in contrast to endogenous helicase A or GFP-helicase A
fusion proteins that contain the NTD. NTD also targeted a completely
cytoplasmic protein, pyruvate kinase, to the nucleus upon fusion to its
carboxyl terminus. These observations suggest that NTD is both
necessary and sufficient to mediate the nuclear localization of RNA
helicase A. In addition, the NTD contains a nuclear export signal,
since it was able to direct the rapid export of GST, GFP, a nuclear
form of GFP (GFP-TNLS), and NPc-TNLS in a variety of assay systems. The
import and export activities of NTD are separable by point mutations in
two basic residues (K62 and R65), which abolished the ability of the
NTD to direct nuclear import while sparing the export activity.
Nuclear import activity of the NTD.
Although no appreciable
sequence homology was found between NTD and other NLSs, a short stretch
of amino acids relatively rich in basic residues is found in NTD, the
U1A signal, KNS, and the NLS of Sam68. An arginine-to-alanine
substitution was shown to abolish the NLS function of Sam68
(16). While both K1162 and R1165 are crucial for the nuclear
import function of NTD, R1158 is dispensable for this function.
Interestingly, PK-K62 and PK-R65 mutants had a lower gel mobility
than the nuclear counterparts (PK-NTD, PK-R58), which had the
expected mobility. It is possible that NTD is modified at the
posttranslational level in the cytoplasm and that this putative
modification is related to its NLS activity. We showed that this
modification was not at the level of phosphorylation. Despite the lack
of sequence resemblance to classical NLS, NTD apparently uses the same
import pathway, since its nuclear import can be efficiently competed with an excess of BSA-conjugated NLS peptides.
Nuclear export activity of NTD.
Nuclear export activity of NTD
was demonstrated in several different systems. First, NTD was able to
direct the export of three different heterologous proteins in
interspecies heterokaryon assays. GFP-NTD, GFP-TNLS-NTD, and
NPc-TNLS-NTD all shuttled when tested, indicating the NTD can direct
nuclear export even in the presence of a strong NLS. Second, export and
reimport of GST-NTD were detected within 30 min after being injected
into selected nuclei of NIH 3T3 polykaryons, demonstrating the rapid
shuttling ability of NTD. Finally, a mutant form of NTD that lost its
NLS activity directed nuclear export upon injection into the nucleus. This export function of NTD is not mediated by RNA binding. The NTD
showed no ability to bind to the CTE in vitro, and coinjection of RNase
had no effect on export function.
Helicase A and Rev use distinct nuclear export pathways.
Leucine-rich NES have been found in a variety of cellular and viral
proteins (4, 7, 8, 15, 28, 37, 44). The human
immunodeficiency virus Rev activation domain is a prototypic NES that
is involved in the nuclear export of intron-containing human
immunodeficiency virus mRNAs. It has been shown recently that CRM1, a
cellular protein distantly related to importin , serves as the
functional export receptor for Rev NES. Competition experiments with
Xenopus oocytes suggest that CTE and Rev-RRE utilize
different cellular factors for RNA export (34, 38). Therefore, if helicase A plays a role in CTE-mediated export, it should
function in a CRM1-independent manner. LMB disrupts the complex
formation of NES, CRM1, and RanGTP by binding to CRM1, thus blocking
Rev export. In functional studies, LMB blocked Rev-RRE-mediated but not
CTE-mediated gene expression (33). We found that
NTD-mediated export was resistant to 20 nM LMB, a concentration 10-fold
higher than that previously observed to block Rev-dependent mRNA export (45), indicating that NTD does not use CRM1 as its export
receptor. This observation is consistent with a possible role for
helicase A in CTE-mediated export. Our recent observation that the
injection of antisera specific for helicase A perturbs CTE function in
human somatic cells further supports an important role of this protein in CTE function. The human protein TAP was recently also identified as
a cellular CTE-binding protein that is involved in CTE-mediated export
in Xenopus oocytes (13). The importance of TAP
and that of helicase A in CTE function are not mutually exclusive.
Rather, both proteins may be required to facilitate efficient RNA
export. Alternatively, the two proteins may function at different steps in the CTE-mediated processing and export of unspliced RNA expressed by
type D retroviruses. Further functional studies will help address the
biological significance of helicase A in CTE-mediated RNA export.
Why simple and complex retroviruses have evolved different pathways to
achieve the same function is not clear. By requiring
the accumulation
of an early viral protein, Rev, to direct the
nuclear export of
partially spliced and unspliced RNA, complex
retroviruses are
temporally regulated and may thus have an advantage
in delaying immune
system surveillance by the host and building
up a larger burst size for
viral production. Simple retroviruses
are often less cytopathic and
express lower levels of virus. For
this type of chronic infection, a
constitutive cellular export
pathway seems to be sufficient. Although
simple and complex retrovirus
mRNAs apparently use distinct cellular
receptors for nuclear export
of unspliced mRNA, they may still have
some common step(s) in
their posttranscriptional regulation, e.g., in
their interfacing
with the splicing machinery to make unspliced RNA
available for
export. Our recent data (
23) that helicase A
may be involved
in the Rev transactivation pathway at a step prior to
nuclear
export are consistent with such a
hypothesis.
| |
ACKNOWLEDGMENTS |
We thank C. G. Lee and J. Hurwitz for the helicase A cDNA,
Gideon Dreyfuss for Myc-PK and Myc-NPc-TNLS plasmids, B. Wolff for LMB,
M. Vodika and M. Emerman for NLS peptides, T. R. Reddy for help
with the constructions of pGST-NTD, W. D. Xu for help with
site-directed mutagenesis, K. Kuhen for critical reading of the
manuscript, and F. Gage and the James B. Pendleton Trust for the use of
their microscopic facilities. We also thank C. Goodwin for his
excellent technical assistance throughout the study.
This study was supported in part by NIH grant AI35477 to T. Hope and
NIH grant GM56089 to F. Wong-Staal.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Stein Clinical
Research Building, University of California, San Diego, La Jolla, CA 92093-0665. Phone: (619) 534-7957. Fax: (619) 534-7743. E-mail: fwongstaal{at}ucsd.edu.
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Abstract
Introduction
