From the European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany
Received for publication, January 16, 2001, and in revised form, March 1, 2001
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ABSTRACT |
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Human TAP and its yeast orthologue Mex67p are
members of the multigene family of NXF proteins. A conserved feature of
NXFs is a leucine-rich repeat domain (LRR) followed by a region related to the nuclear transport factor 2 (the NTF2-like domain). The NTF2-like
domain of metazoan NXFs heterodimerizes with a protein known as p15 or
NXT. A C-terminal region related to ubiquitin-associated domains (the
UBA-like domain) is present in most, but not all NXF proteins.
Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans NXF1 are essential for the export of messenger RNA from the nucleus. Human TAP mediates the export of simian type D retroviral RNAs bearing the constitutive transport element, but the precise role of TAP and p15 in mRNA nuclear export has not yet been
established. Here we show that overexpression of TAP/p15 heterodimers
bypasses nuclear retention and stimulates the export of mRNAs that
are otherwise exported inefficiently. This stimulation of mRNA
export is strongly reduced by removing the UBA-like domain of TAP and abolished by deleting the LRR domain or the NTF2-like domain. Similar
results are obtained when TAP/p15 heterodimers are directly tethered to
the RNA export cargo. Our data indicate that formation of TAP/p15
heterodimers is required for TAP-mediated export of mRNA and show
that the LRR domain of TAP plays an essential role in this process.
Metazoan TAP and its yeast orthologue Mex67p are members of an
evolutionarily conserved protein family, the NXF family, implicated in
the export of messenger RNA from the nucleus (1). Mex67p, the
Saccharomyces cerevisiae NXF homologue, and the
Caenorhabditis elegans protein NXF1 are essential for the
export of bulk polyadenylated RNAs to the cytoplasm (2, 3), whereas
human TAP (also called Hs NXF1) has been directly implicated in the
export of simian type D retroviral RNAs bearing the constitutive
transport element (CTE)1 (4).
In Xenopus laevis oocytes, titration of TAP with an excess of CTE RNA prevents cellular mRNAs from exiting the nucleus (4-6), strongly suggesting a role for TAP in mRNA nuclear export, but direct evidence has so far remained elusive.
Members of the NXF family of proteins have a conserved modular domain
organization consisting of a non-canonical RNP-type RNA-binding domain
(RBD), a leucine-rich repeat (LRR) domain, a middle region showing
significant sequence similarity to nuclear transport factor 2 (the
NTF2-like domain) and a C-terminal ubiquitin-associated (UBA)-like
domain (Fig. 1 and Refs. 1, 7, and 8). The LRR and the NTF2-like
domains are the most conserved features of NXF proteins, whereas the
RBD and the C-terminal UBA-like domain are not always present in NXF
proteins (1, 3).
The N-terminal half of TAP includes the LRR domain, the RBD, and a less
conserved region upstream of the RBD (fragment 1-372, Fig. 1). This
protein fragment exhibits general RNA binding affinity and mediates
binding to several mRNA-associated proteins such as E1B-AP5 (9) and
members of the Yra1p/REF protein family (10, 11). Furthermore, the RBD
of TAP is required in cis to the LRR domain for specific
binding to the CTE RNA (7). Hence, the RBD and the LRR domain are
essential for TAP-mediated export of CTE-containing cargoes (1, 7, 12,
13). Mutations within the LRR domains of TAP and Mex67p have been
reported to affect cellular mRNA export (11, 12), but these
mutations involve residues that have important structural roles and
their substitution probably results in nonspecific structural aberrations.
The NTF2-like domain of metazoan NXFs mediates binding to a protein
known as p15 or NXT. p15 is also related to NTF2 (8, 15, 16) but unlike
NTF2, which forms homodimers, p15 heterodimerizes with the NTF2-like
domain of NXF proteins (1, 8). The human genome encodes at least two
p15 homologues, p15-1 and p15-2, and both interact with TAP (1). The
NTF2-like domain also occurs in Schizosaccharomyces pombe
and S. cerevisiae Mex67p, although there is no obvious p15
homologue encoded by the yeast genome (8, 15, 16). In S. cerevisiae Mex67p, this domain is implicated in the interaction
with a protein known as Mtr2p (14, 17). Prediction of Mtr2p secondary
structure and the observation that co-expression of human TAP and p15
in S. cerevisiae partially restores growth of a strain
carrying the otherwise lethal mex67/mtr2 double
knockout, suggest that Mtr2p may be a p15 functional analogue (8, 16).
A C-terminal fragment of TAP, the NPC-binding domain (Fig. 1, fragment
508-619), mediates direct interactions with nucleoporins and is
necessary and sufficient for the localization of TAP to the nuclear rim
(1, 9, 18). This fragment comprises the entire UBA-like domain and part
of the NTF2-like domain, but p15 binding by TAP is not required for its
interaction with nucleoporins (9). The UBA-like domain on its own
(fragment 567-619) is not sufficient to localize TAP at the nuclear
rim in vivo, but single amino acid changes in a conserved
loop of this domain (NWD at positions 593-595 in human TAP)
severely impair binding of TAP, Hs NXF2, and Ce NXF1 to
nucleoporins in vitro and in vivo (1, 3, 8, 18).
This suggests that high affinity binding to nucleoporins requires both
the UBA-like domain and at least part of the NTF2-like domain. The
UBA-like domain is conserved in yeast Mex67p, but only the S. cerevisiae protein has been shown to interact directly with
nucleoporins (20). As with the metazoan proteins (1, 9), Mex67p lacking
the UBA-like domain no longer localizes to the nuclear envelope (14),
suggesting that the mode of interaction of yeast and metazoan NXF
proteins with nucleoporins is conserved.
Previous studies have focused on the role of the individual domains of
TAP in the export of CTE-bearing RNAs (1, 7, 9, 12, 13, 19). In this
study we investigated the role of TAP domains in the export of cellular
mRNAs. To this end, we developed assays to test mRNA export
stimulation by TAP/p15 heterodimers in cultured cells and in
Xenopus oocytes. These assays are based on the observation
that overexpression of TAP with p15 bypasses nuclear retention and
stimulates export of mRNAs that are normally not exported
efficiently. Using these assays, we show that in the presence of p15,
only full-length TAP efficiently stimulates export of a variety of
mRNA export cargoes. The RBD is dispensable for the stimulation of
mRNA export by TAP whereas the LRR and the NTF2-like domains are
essential for this function. The first 60 amino acids of TAP and the
UBA-like domain contribute substantially, but are not strictly required
for TAP-mediated export of cellular mRNA. These results were
confirmed by directly tethering TAP/p15 heterodimers to the RNA export cargo.
Plasmids--
Most plasmids used in this study have been
described before (1, 4, 7-9, 12). TAP
Plasmid pCMV128 has been described (22, 23); plasmid pCH110 encoding
Expression of Recombinant Proteins--
Glutathione
S-transferase protein fusions were expressed in E. coli BL21(DE3) strains. E. coli M15[pREP4] strain was
used for expressing proteins cloned into the pQE60zz vector.
Recombinant proteins were purified as previously described (4). For
oocyte injections recombinant proteins were dialyzed against 1.5 × phosphate-buffered saline supplemented with 10% glycerol.
DNA Transfections and CAT Assays--
DNA transfections and CAT
assays were performed essentially as described before (1), with the
following modifications. Human 293 cells were transfected using
Polyfect transfection reagent (Qiagen) according to the manufacturer's
instructions. The transfected DNA mixture consisted of 0.25 µg of the
CAT reporter plasmid pCMV128, 0.5 µg of pEGFP-C1 plasmid encoding TAP
or TAP mutants, and/or 0.5 µg of pEGFP-N3 plasmid encoding zzp15.
Transfection efficiency was determined by including 0.5 µg of pCH110
plasmid (Amersham Pharmacia Biotech), as Xenopus Oocyte Microinjections--
All DNA templates for
in vitro synthesis of labeled RNAs have been described.
These were dihydrofolate reductase mRNA, U5 Overexpression of TAP/p15 Heterodimers Promotes the Nuclear Exit of
Inefficiently Spliced pre-mRNAs--
Previously, we reported an
assay that allows quantifying TAP-mediated stimulation of RNA nuclear
export in cultured cells (1). In this assay, a TAP protein expression
vector is co-transfected with the chloramphenicol acetyltransferase
(CAT) gene encoded by the reporter plasmid pDM138 (22). This plasmid
harbors the CAT coding sequence inserted into an intron, which is not
efficiently spliced (22). Cells transfected with this plasmid retain
the unspliced pre-mRNA in the nucleus, yielding only trace levels of CAT enzyme activity (22). Expression of TAP/p15 heterodimers bypass
nuclear retention and promotes the export of the inefficiently spliced
pre-mRNA, resulting in a 14-16-fold increase in CAT activity (1).
In this study, we improved the sensitivity and expanded the dynamic
range of the assay by using the reporter plasmid pCMV128 (Fig.
2A). This plasmid is related to pDM138 but has a CMV
promoter instead of an SV40 promoter (23). Consequently, the basal
level of CAT enzyme activity in cells transfected with pCMV128 is
10-fold higher than in cells transfected with pDM138 (not shown).
Furthermore, co-expression of TAP/p15 heterodimers with the reporter
pCMV128 caused a 44-fold increase in CAT activity (Fig. 2B).
RNase protection analysis confirmed that TAP/p15 heterodimers enhanced
cat gene expression by allowing the unspliced transcripts to
enter the cytoplasm (not shown). As reported (1), overexpression of TAP in the absence of exogenous p15 results in a significant but modest increase of CAT activity, although, the levels of expression of TAP
were also reduced (Fig. 2, B and C). Moreover,
overexpression of p15 in the absence of exogenous TAP or in the
presence of deletion mutants of TAP that cannot bind p15 (TAP The LRR and NTF2-like Domains of TAP Are Essential for Its Export
Activity--
Using pCMV128 as a reporter we have investigated the
role of TAP domains in RNA export. Tested TAP mutants include a
deletion of the first 60 amino acids (TAP-(61-619)) and
deletions of the RBD, the LRR, the NTF2-like and the UBA-like
domains (TAP
In the absence of exogenous p15, none of the TAP mutants stimulated
significantly cat gene expression (Fig.
2B, white bars), but their
expression levels were also reduced in comparison with that of TAP
(Fig. 2C). In the presence of p15, TAP mutants that bind p15
were expressed at a steady-state level comparable with that of
wild-type TAP (Fig. 2C). Nevertheless, when assayed for the
ability to induce CAT expression from pCMV128, TAP
Since deletions of entire domains may affect multiple interactions or
TAP folding, we tested the effect of introducing point mutations in the
LRR (see below) and the UBA-like domains (Fig. 2B). The
mutations targeted conserved residues exposed on the surface of the
protein and were designed on the known three-dimensional structure of
the LRR domain and the predicted structure of the UBA-like domain (7,
8). TAP mutants W594A and D595R have a single amino acid change in the
conserved loop of the UBA-like domain and have impaired nucleoporin
binding (1, 8). These mutants have an effect which is similar to
removing the entire UBA-like domain (Fig. 2B), so we
conclude that this domain is critical for TAP-dependent RNA
nuclear export.
TAP/p15 Heterodimers Trigger Nuclear Export When Tethered to the
RNA Export Cargo--
Next, we tested the effect of tethering TAP/p15
heterodimers directly to the pCMV128 pre-mRNA. In this context,
stimulation of RNA export by the various TAP mutants should be
independent of their ability to bind RNA or RNA-associated proteins.
TAP wild-type and mutants were fused to the C terminus of an HIV Rev
protein defective in export (RevM10). RevM10 carries two point
mutations in the nuclear export signal and, unlike wild-type Rev,
cannot promote export of RNAs bearing the HIV Rev response element
(RRE) (Ref. 27, reviewed in Ref. 28). However, RevM10 has an intact RRE-binding domain and can target the fusion protein to RRE-bearing RNAs (27-29). Vectors expressing RevM10 fusions were co-transfected into 293 cells with the CAT reporter plasmid pCMV128, which carries the
RRE inserted in the intron (23). Expression of RevM10-TAP fusion
moderately stimulated CAT activity, in contrast its co-expression with
p15 increased CAT activity 250-fold (Fig.
3A). Similar results were
recently reported by Guzik et al. (29). Conversely,
tethering of p15 via the RevM10 protein had no significant effect on
CAT activity, but its co-expression with TAP resulted in a 150-fold stimulation of cat gene expression (Fig. 3A).
When neither TAP nor p15 were fused to RevM10 a 40-fold
stimulation of CAT activity was measured, in agreement with data shown
in Fig. 2A. Thus, the higher CAT activity measured when
either subunit of the TAP/p15 heterodimer was fused to RevM10 is likely
to be due to its direct binding to the RRE.
Using this assay we then tested the effect of deleting individual TAP
domains. Western blot analysis indicate that in the presence of p15,
the expression levels of TAP mutants fused to RevM10 were comparable to
that of the wild type control (not shown). When TAP was tethered to the
RNA, removing the first 60 amino acids reduced its export activity by
2.5-3-fold (Fig. 3B and Table I). Unexpectedly, we found
that deletion of the RBD increased the ability of RevM10-TAP fusion to
promote cat gene expression. A possible explanation for this
observation is that removing the RBD reduces the nonspecific binding of
TAP to other RNAs, thereby increasing the pool of protein able to bind
to the RRE-containing RNA. The export activity of RevM10-TAP was
reduced by removing the LRR domain and completely abolished by deleting
the NTF2-like or the UBA-like domains (Fig. 3B and Table I).
In summary, when TAP is directly tethered to its cargo the RBD becomes
dispensable for its export activity while the LRR, the NTF2-like
domain, and the UBA-like domain still play a critical role. However, a
protein fragment comprising these domains (TAP-(200-619)) exhibited
14% of the activity of wild type TAP, suggesting that residues
upstream of the RBD are important for TAP function. This result is
consistent with the observation that the first 60 amino acids of TAP,
although not strictly necessary, contribute to its export activity
(Figs. 2B and 3B).
Mutations in the LRR Domain Impair TAP-mediated RNA
Export--
The mechanism by which deletion of the LRR domain
abolishes TAP function is unclear because TAP TAP Stimulates the Export of mRNAs That Are Otherwise
Inefficiently Exported--
To investigate whether TAP can directly
stimulate export of cellular mRNA, Xenopus oocyte nuclei
were coinjected with purified recombinant TAP and a mixture of labeled
RNAs. This mixture consisted of U5
We also found that TAP directly stimulated export of other mRNAs.
First, we decreased the export rate of Role of TAP Domains in mRNA Export Stimulation in Xenopus
Oocytes--
Next, we investigated the role of individual TAP domains
in mRNA export stimulation in Xenopus oocytes. The TAP
mutants described above were expressed in E. coli and
injected into Xenopus oocyte nuclei together with p15 and
the mixture of labeled RNAs described in Fig. 4A. After a
90-min incubation period, 34% of dihydrofolate reductase and Ftz
mRNAs and 48% of
The effect of introducing point mutations in the LRR domain and the
UBA-like domain of TAP was also tested. In the LRR domain only the
reverse-charge mutation of residues Glu318 and
Glu319 to Arg impaired stimulation of Ad-mRNA export by
TAP (TAP E318R,E319R; Fig. 5B, lanes 10-12). In contrast,
this mutant protein stimulated export of an U6-CTE chimeric RNA (Fig.
5B, lanes 10-12). TAP mutant D595R had the same effect as
deleting the entire UBA-like domain (Fig. 5A, lanes 25-27
versus 22-24).
Because all TAP mutants that were impaired in mRNA export
stimulation exhibited general RNA binding affinity in vitro
(not shown), their absence of export activity cannot be attributed to a
failure to bind RNA, but is likely to reflect impaired binding to p15
(for TAP This study provides direct evidence for a role of TAP/p15
heterodimers in nuclear mRNA export. TAP/p15 heterodimers directly stimulate the export of mRNAs that are otherwise exported
inefficiently. TAP/p15 heterodimers have no significant effect on the
export of mRNAs that are efficiently exported or are produced by
in vivo splicing of the corresponding pre-mRNA,
suggesting that TAP/p15 heterodimers are not limiting for these
cargoes. The observation that in Xenopus oocytes titration
of TAP by an excess of CTE RNA inhibits mRNA nuclear export
irrespective of whether or not the mRNA has been generated by
splicing (4-6), however, suggests that TAP/p15 heterodimers
participate in the export of both spliced and intronless mRNAs.
The role of TAP domains in RNA export was analyzed in cultured cells
and in Xenopus oocytes. Despite the differences between these cell types and the export cargoes analyzed, the results obtained
in these two systems are in surprisingly good agreement (Table I).
These results indicate that TAP-mediated export of mRNA strictly
requires the LRR and NTF2-like domains, whereas deletion of the first
60 residues of TAP or of the UBA-like domain strongly impairs TAP
function. The functional importance of the LRR and NTF2-like domains is
underlined by the observation that these domains are the most conserved
among NXF proteins (1). Only the RBD is dispensable for mRNA export
stimulation by TAP. Similar results were obtained by tethering TAP to
the RNA via the RevM10 protein, although in this case TAP mutants
lacking the first 60 amino acids or the LRRs exhibited 39 and 27% of
the activity of wild type TAP, respectively. This suggests that these domains are crucial for cargo binding by TAP but can be deleted when
TAP is directly tethered to its cargo (Fig. 3).
Interaction of TAP with mRNP Export Cargoes--
The association
of TAP with cellular mRNA may be direct or mediated by
protein/protein interactions. Recently, several TAP partners that might
facilitate TAP binding to cellular mRNA have been identified. These
include E1B-AP5 (9), RAE1/Gle2 (9), and REF proteins (also called Yra
in yeast and Aly in mice) (10, 11, 34). Apart from these, other
RNA-binding proteins may act as adaptors between TAP and cellular
mRNPs. In particular, the splicing coactivator SRm160, the acute
myeloid leukemia-associated protein DEK, RNPS1 and Y14, together with
REFs, are components of a 335-kDa protein complex deposited by the
spliceosome 20-24 nucleotides upstream of a splice junction (25, 26).
These proteins, either individually or as a complex, bind mRNA in a splicing-dependent, but sequence-independent way or may
facilitate the recruitment of TAP to mRNA following splicing (26,
34-37). Therefore, TAP may not be limiting for spliced mRNAs
because splicing guarantees the recruitment of TAP partners, and hence
of TAP, in a sequence-independent manner. In contrast, the pCMV128
pre-mRNA and some intronless mRNAs, depending on their primary
sequence and/or their length, may not be able to recruit TAP partners
or TAP efficiently. Thus, TAP may be limiting for these particular cargoes so their export can be stimulated by TAP overexpression. Consistent with this, nuclear exit of inefficiently exported, intronless mRNAs can also be stimulated by microinjection of
recombinant REFs in Xenopus oocytes (34).
An Essential Role for the LRR Domain of TAP in mRNA Nuclear
Export--
In this article, we have presented evidence that the LRR
domain is essential for TAP function. Furthermore, we show that
residues located at the C-terminal edge of the concave face of the LRR domain are critical for TAP-mediated export of cellular mRNA but not of CTE-bearing RNAs. The concave Formation of p15/TAP Heterodimers Is Required for TAP-mediated RNA
Nuclear Export--
The essential role of p15 in TAP-mediated RNA
export was clearly demonstrated in cultured cells. Although
co-expression of p15 increased the steady-state expression levels of
TAP (Fig. 2C and Ref. 1), this effect was less dramatic than
the stimulation of cat gene expression. For instance, the
expression levels of GFP-TAP and RevM10-TAP were increased by a factor
of 2-3-fold in the presence of p15, however, CAT activity was
stimulated 40- and 250-fold, respectively, indicating that p15 not only
stabilizes TAP but it is absolutely required for its export activity.
The critical role of p15 in TAP-mediated export of intron-containing RNAs was also recently reported by Guzik et al. (29),
although in this study a truncated form of TAP, (TAP-(61-619)) was used.
In Xenopus oocytes, injection of TAP-(
p15 has been implicated in export of tRNAs but also in CRM1-mediated
export of U snRNAs and leucine-rich NESs (38, 39). This export function
of p15 was proposed to be dependent on its ability to interact with
RanGTP. Binding of p15 to RanGTP is controversial, as this interaction
could not be reproduced in other laboratories (1, 16). In
Xenopus oocytes microinjection of p15 did not stimulate
export of any of the RNA species tested (Figs. 4 and 5). Similarly, in
cultured cells and in the absence of exogenous TAP, overexpression of
p15 did not stimulate CAT expression even when it was tethered to the
RRE-bearing pre-mRNA via RevM10 (Fig. 3). Moreover, co-expression
of p15 with wild-type Rev did not significantly increase Rev-mediated
export of RRE-containing pre-mRNAs (29).3 Together, these
results suggest that p15 participates in mRNA export through its
heterodimerization with TAP, or other members of the NXF family, and
has no intrinsic export activity.
The UBA-like Domain of TAP Is Critical for Its Export
Activity--
The experiments described here indicate that the
UBA-like domain contributes substantially to the export function of
TAP, although a low but significant export activity was measured when this domain was deleted. Indeed, in the presence of p15, TAP Distinct Requirements for TAP-mediated Export of Cellular and Viral
mRNA--
The results obtained in this study reveal different
requirements for TAP-mediated export of cellular mRNA or of
CTE-bearing RNAs. The RBD is dispensable for TAP-mediated export of
mRNA but is essential for specific binding to the CTE RNA and
therefore, for TAP-mediated export of CTE-containing cargoes (1, 7, 12,
13). Conversely, the first 60 amino acids of TAP have an important role
in the stimulation of mRNA export, but are not required for
TAP-mediated export of CTE-bearing RNAs (1, 9, 12, 13). Due to the lack
of structural information, however, the role of these residues in
mRNA nuclear export cannot currently be analyzed further.
Interestingly, this domain is the least conserved among the NXF
proteins (1). Our data suggest that the poor conservation of this
domain does not reflect a non-essential function but may confer
specific properties to the NXF proteins (i.e. substrate specificity or binding to specific partners).
The requirement of the NTF2-like and the UBA-like domains for CTE
export are cargo-dependent (9). In Xenopus
oocytes TAP-mediated export of CTE-bearing intron lariats is
independent of these domains (9, 12) while export of U6-CTE requires
the UBA-like domain but not the NTF2-like domain (9). In quail cells,
TAP-mediated export of an inefficiently spliced pre-mRNA carrying
the CTE in the intron is abolished by mutations or deletions of the
UBA-like domain (1, 19) but is only reduced by mutations preventing p15
binding (19).
Only the LRR domain is essential for export of both cellular mRNA
and CTE-bearing RNAs, but its role in these processes is different.
Indeed, reverse-charge mutations of Arg318 and
Arg319 impaired mRNA export stimulation by TAP but
supported CTE-dependent export (Ref. 7 and this study).
Thus, it is likely that the mRNA export defect of this mutant is
attributable to its inability to interact with some components of the
nuclear export machinery that is required for mRNA nuclear export
but bypassed by the CTE. This provides further support for the
hypothesis that the mode of interaction of TAP with cellular mRNA
is different from that with the CTE RNA and thus, that the CTE subverts
TAP from its normal cellular function (7, 9, 12). Moreover, these
results suggest that the assays described in this article are likely to reflect the genuine mRNA export activity of TAP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RBD corresponds to a deletion
of residues 119-198 in the TAP protein sequence (7), TAP
LRR has
residues 203-362 deleted (7), TAP
NTF2 and TAP
UBA correspond to
the deletions TAP
437-507 and TAP
567-613 described before (9).
TAP point mutants were generated using an oligonucleotide-directed in vitro mutagenesis system from Stratagene (Quick Change
Site-directed Mutagenesis). For expression of TAP and TAP mutants as
glutathione S-transferase fusions in
Escherichia coli, the corresponding cDNAs were cloned
between the NcoI and BamHI sites of vector pGEXCS (21). TAP and TAP mutants were expressed in mammalian cells as fusions
with green fluorescent protein (GFP). To this end, cDNA fragments
encoding TAP or TAP mutants were excised from the corresponding pGEXCS
constructs as NarI-BamHI fragments and cloned into the vector pEGFP-C1 (CLONTECH) between the
AccI and BamHI restriction sites. p15-1 was
expressed with an N-terminal tag consisting of two
immunoglobulin-binding domains from protein A of Staphylococcus
aureus (zz tag). For expression in E. coli, p15-1
cDNA was cloned into pQE70zz vector (1). Subsequently, the cDNA
fragment encoding zzp15-1 was excised from pQE60zzp15-1 plasmid using
the restriction sites HindIII-NotI and inserted into pEGFP-N3 vector (CLONTECH) cut with the
same enzymes. This deletes the GFP coding sequence. For expression of
TAP/p15 heterodimers in E. coli, a bicistronic plasmid was
constructed by inserting a ribosome-binding site followed by the p15-2a
cDNA downstream of the TAP coding sequence in plasmid
pGEXCS-TAP.
-galactosidase (
-gal) is from Amersham Pharmacia Biotech.
cDNAs encoding HIV-I Rev protein and the export-deficient mutant
RevM10 were kindly provided by Françoise Stutz (University of
Lausanne, Lausanne, Switzerland). These cDNAs were amplified by
polymerase chain reaction and cloned between the AgeI and
BsrGI sites of plasmid pEGFP-C1, thereby deleting the GFP
coding sequence. The 5' polymerase chain reaction oligo introduced a
HA-tag N-terminal so that Rev and RevM10 fusions can be detected by
Western blot using anti-HA antibodies. The resulting plasmids, pCMV-Rev
and pCMV-RevM10, were sequenced and used in subsequent cloning steps. Plasmids expressing RevM10 fusions of TAP mutants and p15-1 were generated by replacing the GFP coding sequence from the corresponding pEGFP-C1 plasmids by the HA-RevM10 coding sequence using the
AgeI and EcoRI restriction sites. TAP full-length
and TAP
567-613 were excised from the corresponding pGEXCS plasmids
as NarI-BamHI fragments. The BamHI
site was blunted by T4 DNA polymerase. These cDNAs were cloned into
the AccI-SmaI sites of vector pCMV-RevM10.
-galactosidase expression
from this vector is not affected by TAP overexpression. The total
amount of plasmid DNA transfected in each sample was held constant by
adding the appropriate amount of the corresponding parental plasmids
without insert, and was brought to a total of 2 µg by adding pBSSKII
plasmid when necessary. When Rev-M10 fusions were tested, the
transfected DNA mixture consisted of 0.25 µg of the CAT reporter
plasmid pCMV128, 0.5 µg of plasmids pCMV-RevM10-TAP or
pCMV-RevM10-p15, and/or 0.5 µg of plasmids pEGFP-C1-TAP or
pEGFP-N3zzp15. Transfection efficiency was determined by including 0.5 µg of pCH110 plasmid. Plasmids pCMV-Rev and pCMV-RevM10 were used as
positive and negative controls, respectively. Cells were harvested
48 h after transfection and CAT activity was measured as described
(24). Protein expression levels were analyzed by Western blot using
anti-GFP or anti-HA antibodies.
Sm and U6
ss
snRNAs, U6-CTE, and human initiator methionyl tRNA (6, 9). AdHML81,
Fushi tarazu (Ftz), and
-globin cDNAs have been described (25,
26). Ftz-218 and
-globin-247 cDNAs were kindly provided by
Hervé Le Hir (Brandeis University). Oocyte injections and
analysis of microinjected RNA by denaturing gel electrophoresis and
autoradiography were performed as described (6). Quantitation was done
by FluorImager (Fuji FLA-2000). The concentration of recombinant
proteins in the injected samples is indicated in the figure legends.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NTF2
and TAP 1-372) had no significant effect on CAT expression (Fig.
2B). These results indicate that formation of TAP/p15
heterodimers is required for TAP-dependent stimulation of
cat gene expression. These results also indicate that in
cells overexpressing TAP, p15 becomes limiting, and vice versa, and
thus no large pools of free TAP or p15 exist in vivo.
RBD, TAP
LRR, TAP
NTF2, and TAP
UBA) (Fig. 1 and Table
I). TAP fragments lacking the entire
C-terminal half (TAP-(1-372)) or the N-terminal half (TAP-(371-619))
were included as negative controls. All tested TAP mutants localize
within the nucleus when expressed in HeLa cells, with the exception of
TAP fragment 371-619, which distributes between the nucleus and
cytoplasm (Ref. 9 and data not shown).
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Fig. 1.
Domain organization of human TAP
protein. The N-terminal domain of TAP (residues 1-372)
includes the minimal CTE-binding fragment (residues 102-372) and
exhibits general RNA binding affinity. This domain also binds to
several mRNA-associated proteins such as E1B-AP5 and REF/Aly, and
carries an NLS recognized by transportin (1, 7, 9, 10). The N-terminal
domain consists of an RNP-type RNA-binding domain (yellow),
a leucine-rich repeat domain (green), and a less conserved
region upstream of the RBD (purple). The domain boundaries
of the RBD and the LRRs are as defined in the crystal structure of
these domains (7). The C-terminal half of TAP consists of an NTF2-like
domain (red) and a UBA domain (cyan). The minimal
TAP fragments sufficient for p15 or nucleoporin binding (9) are
indicated. Numbers indicate the position in the amino acid
sequence.
Role of TAP domains in RNA export
LRR was completely defective, whereas TAP-(61-619) and TAP
UBA exhibited low, but significant residual activity. In contrast, TAP
RBD retained 38% of the activity of wild-type TAP (Fig. 2B,
Table I). TAP mutant
NTF2, which does not bind p15 (8, 9), failed to
stimulate CAT activity (Fig. 2B), but the expression of this mutant protein was reduced in comparison with that of the wild-type control (Fig. 2C, lane 13 versus 5). We therefore generated
a second mutant in the NTF2-like domain of TAP by deleting residues 381-503. Despite that TAP-(
381-503) does not bind p15, its
expression at a steady-state level was comparable with that of TAP (not
shown). However, this protein does not stimulate cat gene
expression (Table I). These results provide strong support for the
conclusion that formation of TAP/p15 heterodimers is required for
TAP-mediated RNA export stimulation.
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Fig. 2.
The LRR and NTF2-like domains of TAP are
essential for RNA export stimulation in 293 cells. A,
schematical representation of the reporter gene encoded by the plasmid
pCMV128 (23). SD and SA indicate the splice donor
and acceptor sites of the intron. B, human 293 cells were
transfected with a mixture of plasmids encoding -Gal, CAT (pCMV128),
and either GFP alone or fused N-terminal to TAP or various TAP mutants
as indicated on the left. When indicated, a pEGFP-N3
derivative encoding zzp15 was co-transfected (+p15, black
bars). Cells were collected 48 h after transfection and
-Gal and CAT activity were determined. Data from three separate
experiments are shown as fold activation of CAT activity relative to
the activity measured when pCMV128 was co-transfected with parental
plasmids without insert (
). The numbers are mean ± S.D. C, protein expression levels were analyzed by Western
blot using anti-GFP antibodies. Lane 1 shows the
untransfected control. The position of TAP mutants fused to GFP, of GFP
itself, or of zzp15 is shown on the right.
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Fig. 3.
TAP/p15 heterodimers promote export when
tethered to RNA. A, human 293 cells were transfected
with pCMV128, pCH110, and plasmids encoding RevM10 fusions of TAP or
p15. When indicated, plasmids encoding GFP-TAP (+TAP) or zzp15 (+p15)
were co-transfected (black bars). As controls, the reporter
plasmids were co-transfected with empty vectors ( ) or vectors
expressing Rev or RevM10. Data from three separate experiments are
shown as fold activation of CAT activity relative to the activity
measured when GFP was coexpressed with pCMV128. The numbers
are mean ± S.D. B, 293 cells were transfected with
pCMV128, pCH110, zzp15, and plasmids encoding TAP or TAP mutants fused
to RevM10. The stimulation of cat gene expression by TAP
mutants in the presence of p15-1 measured in three independent
experiments is expressed as percentage of the activity of
RevM10-TAP.
LRR exhibits general
RNA binding affinity, binds to REFs, E1BAP5, p15, and nucleoporins in vitro, and localizes to the nuclear rim in
vivo (not shown). LRR domains have a crescent shape with the
convex surface formed by
-helices and the concave surface lined by
-strands (30). The concave
-sheet surface of LRR domains has been
proposed to mediate protein-protein interactions (30). At the concave
face of the LRR domain of TAP, there is a conserved electronegative area defined by residues Asp228, Glu318,
Glu319, Asp323, and Asp352 (7).
Reverse-charge mutations of Asp228 (TAP D228K), which is
conserved within the NXF family, and of Asp323, did not
affect TAP-mediated expression of the cat gene (Table I).
Asp352 plays a structural role and was not mutated (7). In
contrast, reverse-charge mutations of residues Glu318 and
Glu319 (TAP E318R,E319R) dramatically reduced stimulation
of cat gene expression by TAP in the two assays described
above (Table I). This mutant protein, however, stimulated export of an
excised intron lariat bearing the CTE (7) and of an U6-CTE chimeric RNA
(see below), indicating that it is properly folded. Thus, residues
Glu318 and Glu319, which are located at the
C-terminal edge of the concave surface of the LRR domain (opposite to
Asp228), appear to be engaged in interactions that are
critical for TAP-mediated export of cellular mRNA but not of
CTE-bearing RNAs.
Sm and U6
ss snRNAs, the human
initiator methionyl tRNA (tRNAMet), and various mRNAs
that differ in their export efficiencies. These were dihydrofolate
reductase,
-globin, and fushi tarazu (Ftz) mRNAs and a mRNA
derived from the adenovirus major late region (Ad-mRNA). U6
ss
RNA is not exported from the nucleus and serves as an internal control
for nuclear injection (31). U5
Sm RNA is exported via the CRM1 export
pathway but, unlike wild type U5, is not subsequently reimported into
the nucleus (32). Immediately after injection, all RNAs were nuclear
(Fig. 4, lanes 1-3). After a
90-min incubation period, in control oocytes 67% of the dihydrofolate reductase and
-globin mRNA and 42% of the Ftz mRNA were
cytoplasmic (Fig. 4A, lanes 4-6). As reported (33, 34),
Ad-mRNA was less efficiently exported (28% export, Fig. 4A,
lanes 4-6). Coinjection of recombinant TAP, however, resulted in
a 2.6-fold stimulation of Ad-mRNA export, as 74.5% of this
mRNA was detected in the cytoplasm (Fig. 4A, lanes
10-12). When recombinant p15 was coinjected with TAP, export of
Ftz and Ad-mRNA was stimulated up to 1.9- and 3-fold, respectively
(Fig. 4A, lanes 13-15). Nuclear exit of the efficiently exported mRNAs (e.g. dihydrofolate reductase and
-globin) was only slightly or not further stimulated (Fig. 4A,
lanes 13-15). Recombinant p15, in the absence of TAP, had no
effect on the export of any of the RNA species tested (Fig. 4A,
lanes 7-9). Stimulation of mRNA export by TAP was specific,
since export of tRNA and U5
Sm RNA was not affected (Figs. 4 and
5). Furthermore, the stimulatory effect
of TAP could be obtained reproducibly using different preparations of
recombinant protein and in several independent experiments (e.g. Figs. 4, A-C, and 5, A and
B).
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Fig. 4.
TAP stimulates export of mRNAs that are
normally inefficiently exported. A-C, Xenopus
oocyte nuclei were injected with mixtures of 32P-labeled
RNAs and purified recombinant proteins as indicated. RNA samples from
total oocytes (T), nuclear (N), and cytoplasmic
(C) fractions were collected immediately after injection
(t0: lanes 1-3) or 90 min after injection in
panels A and B. In lanes 4-15 of
panel C, samples were collected 40 min after injection. RNA
samples were analyzed on 8% acrylamide, 7 M urea
denaturing gels. One oocyte equivalent of RNA, from a pool of 10 oocytes, was loaded per lane. The concentration of recombinant
glutathione S-transferase-TAP in the injected samples was
0.8 mg/ml. The concentration of zzp15 in the injected samples was 2 mg/ml except for lanes 13-15 of panel C, in
which p15 was injected at 10 mg/ml. On the left of
panels B and C, the numbers in
brackets indicate the size of the transcripts. The
stimulation of export for each of the mRNAs tested was quantified
and expressed as fractional stimulation relative to the export measured
in control oocytes. The numbers on the right of
the panels represent export stimulation by TAP/p15 heterodimers
relative to control oocytes (fold stimulation of export).
View larger version (69K):
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Fig. 5.
Role of TAP domains in mRNA nuclear
export stimulation. A, wild-type TAP and mutants were
expressed in E. coli as glutathione S-transferase
fusions. The recombinant proteins indicated above the lanes
were injected into Xenopus oocyte nuclei together with
recombinant zzp15 and a mixture of radiolabeled RNAs. This mixture
consisted of dihydrofolate reductase, -globin, Ftz, and
Ad-mRNAs, U5
Sm, and U6
ss snRNAs, and human initiator
methionyl tRNA. The concentration of recombinant TAP and TAP mutants in
the injected samples was 0.8 mg/ml and that of zzp15 was 2 mg/ml.
B, purified recombinant TAP and TAP E318R,E319R were
injected into Xenopus oocyte nuclei together with the
mixture of radiolabeled RNAs described in panel A
supplemented, with U6-CTE RNA. zzp15 was not included as it interferes
with the export of U6-CTE RNA. In both panels, RNA samples from total
oocytes (T), nuclear (N), and cytoplasmic
(C) fractions were collected immediately after injection
(t0: lanes 1-3, in both panels) or 90 min after
injection, and analyzed on 8% acrylamide, 7 M urea
denaturing gels. One oocyte equivalent of RNA, from a pool of 10 oocytes, was loaded per lane.
-globin and Ftz mRNAs by
reducing the length of these transcripts from 360 and 343 nucleotides
to 247 and 218 nucleotides, respectively (34). Fig. 4B shows
that TAP stimulated the export of the shortened mRNAs (lanes
7-9). The effect of TAP was more dramatic on the export of
Ad-mRNA and Ftz-218 mRNA, which are the least efficiently exported mRNAs. Second, we reduced the incubation time from 90 to
40 min so that less than 42% export was observed for all mRNAs tested (Fig. 4C, lanes 4-6). Under these conditions, p15
alone had no effect on export even though a 5-fold higher molar
concentration was injected compared with Fig. 4A (Fig.
4C, lanes 13-15). Coinjection of TAP, with or without p15,
stimulated the export of all mRNAs, except dihydrofolate reductase
mRNA (Fig. 4C, lanes 7-12). Again, the stimulatory
effect of TAP was more dramatic on the export of mRNAs that were
less efficiently exported, suggesting that TAP is limiting for these
cargoes. TAP/p15 heterodimers had no significant effect on the export
of
-globin, Ftz and Ad-mRNA produced by in vivo
splicing of the corresponding pre-mRNAs (not shown).
-globin mRNA moved to the cytoplasm, while
only 13% of Ad-mRNA was exported (Fig. 5A, lanes 4-6).
Coinjection of full-length TAP resulted in a 4.2-fold stimulation of
Ad-mRNA export (Fig. 5A, lanes 7-9, and Table I). TAP
RBD stimulated Ad-mRNA export by 3.4-fold (Fig. 5A, lanes
13-15). In contrast, deletion of either the N-terminal 60 amino
acids (TAP-(61-619)), the LRR, the NTF2-like domain, or the UBA-like domain strongly reduced or abolished the export activity of TAP (Fig.
5A, lanes 10-12 and 16-24, Table I).
NTF2 and TAP
381-503) or to nucleoporins (for TAP
UBA and TAP D595R). It is currently unclear which interactions are
affected by deleting the LRR domain or the first 60 amino acids of TAP.
TAP-(61-619) has a reduced in vitro affinity for E1B-AP5
(9), but the significance of this interaction for TAP function in
vivo has not yet been established.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet surface of LRR domains has been proposed to mediate protein-protein interactions (30). Deletion of the entire LRR domain of TAP does not affect binding to its
known partners (E1BAP5, REFs, p15, and
nucleoporins).2 This suggests
that the LRR domain of TAP binds one or more unidentified cellular
ligands that are bypassed by the CTE, but is essential for export of
cellular mRNA.
381-503) and
TAP-(
NTF2), which have no affinity for p15 (Refs. 8 and 9, this study), resulted in no export activity (Table I). Because in oocytes
the recombinant proteins were stable (not shown), this suggests that
TAP/p15 heterodimer formation is required for mRNA nuclear export.
On the other hand, coinjection of p15 with TAP only slightly increased
the mRNA export stimulation observed when TAP alone was injected,
suggesting that p15 may not be limiting in the oocytes.
UBA and
TAP W594A exhibited between 9 and 13% of the export activity of
wild-type TAP (Table I). In S. cerevisiae, deletion of the UBA-like domain of Mex67p resulted in a thermosensitive growth phenotype and accumulation of polyadenylated RNAs within the nucleus, indicating that the UBA-like domain of Mex67p is required, but not
essential for efficient mRNA nuclear export (14). In addition, Mex67p mutants lacking the UBA-like domain no longer localized to the
nuclear rim (14). Overexpression of Mtr2p compensated for the lack of
the UBA domain and restored growth, but not the nuclear envelope
localization of the protein. Similarly, NXF proteins lacking the
UBA-like domain do not localize at the nuclear rim when coexpressed
with p15 (1). Thus, in vivo Mex67p and metazoan NXF proteins
lacking the UBA-like domain have a residual export activity in the
presence of Mtr2p or p15 and may still be able to interact transiently
with nucleoporins, although at equilibrium they are no longer localized
at the nuclear rim. The NTF2-like domain might, therefore, mediate
transient binding to nucleoporins when the UBA domain is not present
thereby sustaining a residual export activity. Consistent with this
hypothesis, it has recently been shown that high affinity
interactions between nucleoporins and transport receptors are
dispensable for NPC passage (40). For instance, NTF2 homodimers
localize to the nuclear rim and facilitate the nuclear import of
RanGDP, however, a point mutation that abolishes nuclear rim
localization (i.e. high affinity binding to the NPC) reduces
but does not abolish the import function of NTF2 (40).
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ACKNOWLEDGEMENTS |
---|
We thank Tom Hope and Karen L. Beemon for the kind gift of plasmid pCMV128 and Francoise Stutz for the plasmids encoding HIV-1 Rev and Rev-M10 mutant. We are grateful to Brian Guzik and Marie-Louise Hammarskjöld for communicating results prior to publication and Kevin J. Chalmers and Scott Kuersten for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the European Molecular Biology Organization (EMBO) and the Human Frontier Science Program Organization (HFSPO).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: EMBL,
Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel.: 49-6221-387-389; Fax: 49-6221-387-518; E-mail: izaurralde@embl-heidelberg.de.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M100400200
2 I. C. Braun, A. Herold, M. Rode, E. Conti, and E. Izaurralde, unpublished results.
3 I. C. Braun, A. Herold, and E. Izaurralde, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CTE, constutive transport element; RBD, RNA-binding domain; LRR, leucine-rich repeat; NTF2, nuclear transport factor 2; UBA, ubiquitin associated-like domain; GFP, green fluorescent protein; HA, hemagglutinin; CAT, chloramphenicol acetyltransferase; snRNA, small nuclear RNA; CMV, cytomegalovirus; RRE, Rev response element; Ad-mRNA, adenovirus mRNA.
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