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Molecular and Cellular Biology, December 2000, p. 8996-9008, Vol. 20, No. 23
European Molecular Biology Laboratory,
D-69117 Heidelberg, Germany1; Institute
of Histology and Embryology, Faculty of Medicine, University of Lisbon,
1699 Lisbon Codex, Portugal2; and
Institute of Biochemistry, Swiss Federal Institute of
Technology, CH-8092 Zürich, Switzerland3
Received 12 July 2000/Returned for modification 15 August
2000/Accepted 6 September 2000
Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p,
have been implicated in the export of mRNAs from the nucleus. The TAP
protein includes a noncanonical RNP-type RNA binding domain, four
leucine-rich repeats, an NTF2-like domain that allows
heterodimerization with p15 (also called NXT1), and a
ubiquitin-associated domain that mediates the interaction with
nucleoporins. Here we show that TAP belongs to an evolutionarily
conserved family of proteins that has more than one member in higher
eukaryotes. Not only the overall domain organization but also residues
important for p15 and nucleoporin interaction are conserved in most
family members. We characterize two of four human TAP homologues and
show that one of them, NXF2, binds RNA, localizes to the nuclear
envelope, and exhibits RNA export activity. NXF3, which does not bind
RNA or localize to the nuclear rim, has no RNA export activity.
Database searches revealed that although only one p15
(nxt) gene is present in the Drosophila
melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind
TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15
mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.
mRNAs are exported from the nucleus
as large ribonucleoprotein complexes (mRNPs). To date, proteins
directly implicated in this process include several nucleoporins and
RNA binding proteins (hnRNPs), an RNA helicase of the DEAD-box family
(Dbp5), and the nuclear pore complex (NPC)-associated proteins Gle1p,
TAP and Mex67p, and RAE1 (also called Gle2p) (reviewed in references
22, 28, and 32). Mex67p is
essential for mRNA export in Saccharomyces cerevisiae, while RAE1 is essential for mRNA export in
Schizosaccharomyces pombe (9, 27, 36). Their
vertebrate homologues, TAP and RAE1, have also been implicated in the
export of cellular mRNAs (6, 8, 12, 15, 20, 31).
We identified TAP as the cellular factor which is recruited by the
constitutive transport element (CTE) of simian type D retroviruses to
promote nuclear export of their genomic RNAs (12). In
Xenopus oocytes, titration of TAP with an excess of CTE RNA
prevents cellular mRNAs from exiting the nucleus (12, 30,
33). This suggests a role for this protein in the export of
cellular mRNA.
Considerable progress has been made in defining TAP structural and
functional domains (see Fig. 1) and in identifying its binding
partners. TAP partners include various nucleoporins (4, 17);
p15 (also called NXT1), a protein related to nuclear transport factor 2 (NTF2) (7, 17); transportin, which mediates TAP nuclear
import (4); and several mRNP-associated proteins, such as
E1B-AP5, RAE1 (4), and members of the Yra1p/REF family of proteins (37, 39). Binding of TAP to these mRNP-associated proteins is mediated by its N-terminal domain (residues 1 to 372) (4, 39). This domain includes a noncanonical RNP-type RNA binding domain (RBD) and four leucine-rich repeats (LRRs) and exhibits
general RNA binding affinity and specific binding to the CTE RNA
(8, 12, 24).
TAP heterodimerizes with p15 via its NTF2-like domain (residues 371 to
551) (4, 40). Nucleoporin binding by TAP in vitro and in
vivo is mediated by a domain located at the very C-terminal end of the
protein (residues 508 to 619) (4, 5). The similarities of
this domain to the ubiquitin-associated (UBA) domain allowed the
prediction that residues located in a conserved loop were implicated in TAP-nucleoporin interaction (40).
In this study, we identify TAP homologues in Homo sapiens,
Caenorhabditis elegans, and Drosophila
melanogaster. The overall domain organization of these proteins,
including residues important for p15 and nucleoporin interaction, is
conserved. Two of four putative human TAP homologues, NXF2 and NXF3,
have been characterized in detail. These proteins show 57 and 44%
sequence identity to TAP. Neither binds specifically to the CTE RNA,
indicating that they may exhibit different substrate specificities.
Both proteins are localized within the nucleoplasm, but only NXF2
associates with the nuclear envelope and exhibits detectable RNA export
activity. In addition, a human homologue of p15 (p15-2 [also called
NXT2]) was characterized. p15-2 binds to TAP, NXF2, and NXF3 with
affinities similar to those of p15. Like p15-1, a fraction of p15-2
localizes to the nuclear envelope. Both human p15 homologues
participate in RNA nuclear export. Together, our results indicate that
the TAP-p15-mediated export pathway has diversified in higher
eukaryotes and in humans includes at least two p15 proteins and
multiple TAP-like putative mRNA export factors.
Homology searches and sequence analysis.
Homologues of TAP
and p15 were retrieved in expressed sequence tag (EST) databases, human
genomic DNA, and the Drosophila and C. elegans
genomes, as well as in various protein sequence databases, using the
BLAST suite of programs (3). Multiple sequence alignments
were constructed by using CLUSTAL W (42) and manually
refined on the SEAVIEW alignment editor (11). To predict the
genomic structure of TAP and p15 homologues in various organisms, the
Genewise program (http://www.sanger.ac.uk/Software/Wise2/) was
employed. If cDNA and/or EST sequences were available, the BLASTN
program for genomic sequences was also used to determine genomic structures.
Plasmids.
Full-length human NXF2, NXF3, p15-2a, and p15-2b
cDNAs were amplified by PCR using the human testis Marathon-Ready cDNA
library (Clontech) as a template and primers introducing the
appropriate restriction sites. The 3' primers were designed according
to the predicted cDNAs and included stop codons. The 5' primers were designed using sequence information obtained after performing 5' rapid
amplification of cDNA ends reactions with the same human testis cDNA
library and oligonucleotides lying in the p15-2a, p15-2b, NXF2, and
NXF3 coding region that is represented in the corresponding human
genomic sequences or I.M.A.G.E. Consortium cDNA clones. The NXF2
sequence is represented in the IMAGp998P054498Q2 and IMAGp998C234110Q2
clones. NXF3 is represented in the IMAGp998M08574Q2 clone (see
http://www.rzpd.de for further information). The p15-2a and p15-2b
cDNAs are represented in the genomic sequence AL031387. The complete
NXF2 and NXF3 cDNA was cloned into pGEMT-easy (Promega) and
sequenced. p15-2a cDNA was directly cloned in pGEXCS
(29) and sequenced. The cDNAs present in these plasmids were
used for all further subcloning steps.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
TAP (NXF1) Belongs to a Multigene Family of
Putative RNA Export Factors with a Conserved Modular
Architecture
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-globin 5' untranslated region
inserted between the HindIII and EcoRI sites.
For the coexpression assay (see Fig. 5F), p15-1 and p15-2a cDNAs were
cloned in the NcoI/NotI or
NcoI/BamHI sites of vector pET28c (Novagen), respectively.
598-626 was
made by digesting pGEX4T-1-NXF2 3xAla598 with PpuMI and
NotI and inserting the released fragment into
pGEX4T-1-NXF2 cut with the same enzymes. Constructs encoding GST
fusions of the NXF2 amino acids 1 to 377 and 102 to 203 (RBD) were
generated by inserting the corresponding PCR products into
pGEX4T-1 digested with BamHI and NotI. A
plasmid encoding a GST fusion of NXF3 amino acids 90 to 192 (RBD)
was generated by inserting the corresponding PCR product into pGEXCS
digested with NcoI-BamHI. All PCRs were performed with the Expand high-fidelity PCR system (Roche). The integrity of the
PCR products was confirmed by sequencing.
For expression of GFP fusions in mammalian cells, NXF2 and NXF3 cDNAs
were excised from the pGEMT-easy clones with the restriction enzymes
BamHI/HindIII (NXF2) or
EcoRI/BamHI (NXF3) and inserted into the vector
pEGFP-C1 (Clontech) digested with
BglII/HindIII or
EcoRI/BamHI, respectively. pEGFP-N3 (Clontech)
derivatives encoding Staphylococcus aureus protein A (zz)
fusions of p15-1 and p15-2a were constructed in two steps: first, p15-1
and p15-2a cDNAs were cloned into the vector pRN3zz using the
NcoI/BamHI restriction sites. Then, fragments
encoding the zz tag fusions were released by digesting the
corresponding plasmids with HindIII/NotI and
inserted into pEGFP-N3 cut with the same enzymes. Additional plasmids
used in this study were previously described (4, 8, 24, 39).
In vitro translation and expression of recombinant proteins. For generation of 35S-labeled in vitro-translated proteins, the combined in vitro transcription-translation (TnT) kit from Promega was used following the instructions of the manufacturer. Translation was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent autoradiography. E. coli BL21(DE3) was used when proteins were expressed with the pGEX vectors, while E. coli M15[pREP4] was used for expressing proteins cloned into the pQE vectors. Recombinant proteins were purified as previously described (12).
GST pull-down and in vitro RNA binding assays. Gel retardation assays and GST pull-down assays were performed as previously described (4, 8, 39). For in vitro synthesis of a 43-nucleotide RNA probe, pBluescribe was linearized with BamHI and transcribed with T3 RNA polymerase. The amounts of unlabeled competitor RNAs used per binding reaction are indicated in the figure legends.
Ran binding assays.
The expression and purification of NTF2
and the expression of importin fragments have been described previously
(21). Wild-type Ran or zzRanQ69L was immobilized on
immunoglobulin G (IgG)-Sepharose in the presence of the Rna1 or RCC1
and energy-regenerating system, respectively. E. coli
lysates (250 µl) supplemented with equal amounts (5 µM) of purified
NTF2, p15-1, or p15-2a, and a lysate from E. coli expressing
importin (fragment 1-452) were subjected to binding to 15 µl of
IgG-Sepharose beads coated with RanGDP or RanQ69L-GTP. Binding was
performed for 4 h at 4°C in a final volume of 1.5 ml of binding
buffer (50 mM HEPES [pH 7.6], 200 mM NaCl, 2 mM magnesium acetate).
After being washed three times with binding buffer, the bound protein
was eluted with 1.5 M MgCl2 and precipitated with
isopropanol. The starting material and the bound fractions were
analyzed by SDS-PAGE and Coomassie blue staining.
Immunofluorescence. HeLa cells were transfected with FuGENE6 (Roche). Approximately 20 h after transfection, the cells were fixed with 3.7% formaldehyde for 10 min and subsequently permeabilized with 0.5% Triton X-100 for 15 min or were extracted first with 0.5% Triton X-100 for 1 min on ice and then fixed in formaldehyde. Indirect immunofluorescence assays were performed as previously described (2). The zz fusion proteins were visualized with a rabbit polyclonal anti-protein A antibody (Sigma) (dilution, 1:1,000 in phosphate-buffered saline supplemented with 5% fetal calf serum and 0.05% Tween 20) and a secondary Cy3-coupled anti-rabbit IgG antibody (diluted 1:4,000). Coverslips were mounted in VectaShield medium (Vector Labs).
DNA transfection and CAT assays.
Human 293 cells were
transfected by the calcium phosphate method. The cells were transfected
at 50% confluency in 6-cm-diameter dishes with a plasmid DNA mixture.
This mixture consisted of 0.5 µg of pDM138 (13, 14), 1 µg of pEGFP-C1 plasmid encoding green fluorescent protein
(GFP)-tagged versions of TAP or TAP homologues, and 1 µg of
pEGFP-N3 derivatives encoding zz-tagged versions of p15-1 or
p15-2a. The total amount of plasmid DNA transfected in each sample was
held constant by adding the appropriate amount of the corresponding
parental plasmids without an insert. The transfection efficiency was
determined by including 0.5 µg of pCH110 plasmid (Pharmacia) encoding
-galactosidase (-Gal), as -Gal expression from this vector is
not affected by increasing levels of TAP expression. Quail QT6 cells
were transfected using Lipofectamine Plus (Life Technologies). The
cells were transfected at 50% confluency in six-well dishes with a
plasmid DNA mixture. This mixture consisted of 0.5 µg of pCH110, 0.1 µg of pDM138 or pDM138-CTE, 0.5 µg of pEGFP-C1 plasmids encoding
GFP-tagged versions of TAP or TAP homologues, 0.5 µg of
pEGFP-N3zzp15-1, and 0.8 µg of pBSIISK. The cells were harvested
40 h posttransfection, and chloramphenicol acetyltransferase (CAT)
activity was measured as described previously (26). Protein
expression levels were analyzed by Western blotting with anti-GFP
antibodies kindly provided by Patrick Keller.
Nucleotide sequence accession numbers. The NXF2, NXF3, p15-2a, and p15-2b sequence data have been submitted to the EMBL database under accession numbers AJ277526, AJ277527, AJ277591, and AJ278323.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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TAP belongs to a multigene family of evolutionarily conserved
proteins.
We performed an extensive search for TAP homologues.
Using BLAST searches (3), we identified two homologues
in the C. elegans genome, four in the Drosophila
genome, and, in addition to TAP itself, four putative homologues in the
human genome, including the two previously identified (5)
(Fig. 1). In agreement
with the Human Genome Nomenclature Committee, genes encoding TAP-like proteins in higher eukaryotes were named nxf (for nuclear
export factor). To avoid confusion, we will call human NXF1 TAP.
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Multiplication of nxf genes has occurred independently
in different lineages.
Phylogenetic analysis of the NXF protein
family indicates that separate gene duplication events have occurred in
several eukaryotic lineages (Fig. 2).
Thus, although higher eukaryotes have several TAP homologues, they have
evolved independently, so there is no clear one-to-one relationship of
any isoforms.
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Characterization of the substrate binding domain of NXF2 and NXF3. cDNAs encoding human NXF2 and NXF3 were cloned by reverse transcription-PCR and sequenced (Fig. 3). To investigate whether these proteins interact with E1B-AP5 or the REF proteins and exhibit RNA binding activity, we performed in vitro binding assays.
[35S]methionine-labeled NXF2 and NXF3 were synthesized in vitro in rabbit reticulocyte lysates and assayed for binding to glutathione agarose beads coated with either GST-E1B-AP5, GST-REF1-II, or GST. Binding of TAP to the recombinant proteins was tested in parallel. Figure 4A shows that TAP, NXF2, and NXF3 could be selected on glutathione agarose beads coated with E1B-AP5 (lane 3) but not on beads coated with GST (lane 2). TAP and NXF2 bound to full-length REF1-II (fragment 1-163 [lane 6]) and to its C-terminal domain (fragment 103-163 [lane 5]) but not to its RBD (fragment 14-102 [lane 4]). In contrast, NXF3 did not interact with REF1-II. None of these interactions was affected by the presence of RNase A, indicating that they were not RNA mediated (data not shown; see reference 39 for TAP).
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NPC) or by deleting the RBD required for CTE binding (24). In contrast to TAP, neither NXF2 nor NXF3 could
promote specific CTE-dependent export (Fig. 4F), although these
proteins were expressed at comparable levels (data not shown).
Thus, NXF2 displays general affinity for RNA as reported previously for
TAP and Mex67p (12, 17, 35, 39) and interacts with E1B-AP5
and Ref1-II, whereas NXF3 only interacts with E1B-AP5 and does not
exhibit detectable RNA binding activity. Neither protein specifically
interacts with the CTE RNA or mediates CTE-dependent export.
p15-2, a human p15 homologue that interacts with TAP and localizes
to the nuclear rim.
The NTF2-like domain of TAP heterodimerizes
with p15 and is conserved in most members of the NXF family. Searches
for p15 homologues revealed one nxt-like gene in
Drosophila and C. elegans and two in available
human genomic sequences (Fig. 5A). The
additional human nxt gene was named nxt2. The
proteins encoded by these human genes will be referred to as p15-1 and
p15-2, respectively. nxt2 is located on the X chromosome,
and its genomic structure more closely resembles those of the
homologues in other species than does the intronless nxt1
gene on chromosome 20 (Fig. 5A). Human EST sequences include two
alternative splice variants of nxt2 (p15-2a and p15-2b)
which differ in their 5' exons (Fig. 5A). The predicted p15-2 forms
were confirmed by cloning and sequencing the corresponding cDNAs.
Alignment of the deduced amino acid sequence of p15-2a with known
homologues and phylogenetic analysis suggest that the two human
nxt genes are the result of a recent duplication in the
nxt lineage (Fig. 5B and C).
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(fragment 1-452) bound
RanQ69L-GTP (Fig. 5E, lane 13). Thus, both p15-1 and p15-2a directly
interact with TAP but not with Ran.
Next, we tested whether the p15 proteins could also interact with NXF2
or NXF3. Untagged p15-1 and p15-2a were coexpressed in E. coli along with TAP, NXF2, or NXF3 fused to GST. The bacterial lysates were incubated with glutathione agarose beads, and after extensive washes, the bound proteins were eluted with SDS-sample buffer. Both p15-1 and p15-2a were copurified with TAP, NXF2, and NXF3
but not with GST (Fig. 5F). Moreover, coexpression of the NXF2 and NXF3
proteins with p15-1 or p15-2a significantly increased their stability,
as full-length NXF3 could not be expressed in E. coli in the
absence of p15 (data not shown).
NXF2, but not NXF3, binds to nucleoporins and localizes to the
nuclear rim.
To test nucleoporin binding of NXF2 and NXF3, we
immobilized bacterially expressed GST-CAN (fragment 1690-2090) or GST
on gluthatione agarose beads. The beads were then incubated with in vitro-translated TAP, NXF2, or NXF3. Both TAP and NXF2 bound CAN, while no significant binding was observed for NXF3 (Fig. 6A). In order to determine whether NXF2
could interact with other FG-repeat containing nucleoporins, pull-down
assays were performed with recombinant NXF2 or TAP fused to GST and in
vitro-translated CAN (fragment 1690-1894), Nup153 (fragment
895-1475), or full-length Nup98 and p62. The TAP mutants
W594A and D595R were used as negative controls (40). With
the exception of Nup98, the nucleoporins tested bound to NXF2 with
efficiencies similar to those with TAP (Fig. 6B, lane 6 versus lane 3).
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598-626) (Fig. 6C, lanes 7 and
8). As is the case for TAP, a single substitution of alanine for W599 was sufficient to disrupt nucleoporin binding (Fig. 6C, lane
5), while substitution of alanine for N600 or of arginine for
E598 had no significant effect (Fig. 6C, lanes 4 and 6).
To analyze nucleoporin binding in vivo, the subcellular localization of
NXF2 and NXF3 fused to GFP was investigated in transfected HeLa cells
and compared to that of GFP-tagged TAP. GFP-tagged NXF2 and NXF3 were
evenly detected in the nucleoplasm and were excluded from the
nucleolus. Moreover, a fraction of NXF3 could be detected in the
cytoplasm (Fig. 6D, 
Tx). To visualize a potential nuclear envelope
association, transfected cells were extracted with Triton X-100 prior
to fixation (Fig. 6D, +Tx). As previously observed for TAP, a fraction
of NXF2 was resistant to detergent extraction and remained associated
with the nuclear envelope while NXF3 was not detected at the nuclear
rim following Triton extraction. Furthermore, NXF2 and TAP mutants that
do not interact with nucleoporins in vitro were not detected at the
nuclear envelope upon Triton extraction (Fig. 6E).
NXF2, but not NXF3, can stimulate RNA export.
We have
developed an assay that tests mRNA export stimulation of TAP and
its homologues. In this assay, TAP and TAP-like proteins are
cotransfected with the reporter plasmid pDM138 (14) in human 293 cells. As mentioned above, transfection with this plasmid yields
only trace levels of CAT enzyme activity (13, 14). However,
cotransfection with vectors expressing TAP and p15 nuclear retention to
be bypassed and export of the unspliced transcripts to be promoted,
increasing CAT activity (Fig. 7A). To test the effect of TAP-like
proteins on cat gene expression, human 293 cells were
cotransfected with a constant amount of pDM138 reporter plasmid,
plasmids encoding TAP, NXF2, or NXF3 together with plasmids encoding
p15-1 or p15-2a (Fig. 7A and D). Protein
expression levels were analyzed by Western blotting (Fig. 7B and C).
Overexpression of the TAP protein together with p15-1 increased
cat expression by 15- to 17-fold compared to the expression
levels obtained when only pDM138 vector was transfected, whereas
coexpression of TAP with p15-2a resulted in 13-fold activation of
cat expression (Fig. 7A). Coexpression of NXF2 with p15-1 or
p15-2a activated cat expression up to 10- or 6-fold,
respectively. This level of induction may reflect the lower expression
levels of NXF2 compared to TAP (Fig. 7B and C). In contrast, when
coexpressed with p15-1 or p15-2a, NXF3 had no effect on cat
activity, even though its expression levels in the presence of p15-1
were similar to that of TAP (Fig. 7A and B). Overexpression of p15-1 or
p15-2a, in the absence of exogenous TAP or in the presence of a
deletion mutant of TAP (TAP437-507) that no longer interacts with
p15, had no effect on CAT activity (Fig. 7A and D). In contrast,
overexpression of TAP or NXF2 in the absence of exogenous p15 resulted
in a significant but modest increase in CAT activity (Fig. 7A);
however, the levels of expression of TAP and NXF2 in the absence of p15
were also reduced (Fig. 7B). Therefore, p15-1 and p15-2a appear to
increase the stability of TAP-like proteins. In summary, TAP and NXF2,
when coexpressed with p15-1 or p15-2a, stimulate the export of the
unspliced pre-mRNA, while NXF3 did not stimulate CAT gene
expression. The ability of TAP and NXF2 to export pDM138 pre-mRNA
is likely to reflect the genuine export activity of these proteins, as
this activity could be abolished with various mutations in the TAP
sequence, implying the need for a functional protein (Fig. 7D). In
particular, this activity requires the LRRs and the NTF2-like domain.
In contrast, the RBD and the NPC binding domain of TAP were not
strictly required (Fig. 7D) (see Discussion). Thus, the lack of RNA
export activity of NXF3 may not be due to the truncation of the UBA
domain but to the deletion of part of the LRRs.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we present a combined evolutionary and functional characterization of the family of TAP-like proteins in higher eukaryotes. Members of this protein family were called NXF. We have further characterized two of four putative human NXF proteins, namely, NXF2 and NXF3, and have shown that NXF2 exhibits RNA export activity.
NXF proteins heterodimerize with p15. Previously, we have shown that TAP heterodimerizes with p15-1 via its NTF2-like domain (40). In this study, we show that the human genome encodes at least two p15 homologues and that both interact with TAP, NXF2, and NXF3 and participate in RNA nuclear export. However, the precise role of p15 in TAP-mediated export is not well defined. p15 binding is not strictly required for TAP-mediated export of CTE-bearing substrates (4, 8, 16) but appears to play a role in the general export activity of the NXF proteins (Fig. 7A and D). Whether these effects are due to a direct role of p15 in export or to an increased stability of NXF proteins when coexpressed with p15 is unclear.
The interaction between the NTF2-like domains of TAP and p15-1 is mediated by multiple rather than a few critical residues (40). Indeed, mutation of residues located at the heterodimer interface reduced but did not completely abolish heterodimerization in vitro (40). This provides an explanation for the TAP, NXF2, and NXF3 interactions with p15-1 and p15-2a studied here, as these interactions occur even though not all interface residues are conserved. The NTF2-like domain also occurs in Mex67p, the S. pombe and S. cerevisiae, NXF homologue (40). In S. cerevisiae, this domain is implicated in interaction with a protein known as Mtr2p (35, 38). Mtr2p does not exhibit obvious sequence similarities with p15, but secondary-structure predictions suggest that it could be a p15 analogue (40).Functional conservation of the UBA-like domain. The nucleoporin binding domain of TAP includes a UBA domain (40). The known structure of UBA domains suggests a conserved loop (NWD at positions 593 to 595 in human TAP) is involved in the interaction with nucleoporins. Replacement of these three residues by alanines was previously shown to block the ability of TAP to promote CTE-dependent export of a precursor mRNA in quail cells (15). Moreover, single-amino-acid changes in this loop are sufficient to impair binding of TAP and C. elegans NXF1 to nucleoporins in vitro and in vivo (40, 41) (Fig. 6). The mutational analysis of the UBA-like domain of NXF2 presented here further supports the prediction that the conserved loop residues of the UBA-like domain have a critical role in the interaction of NXF proteins with nucleoporins. Conversely, based on these observations, it was possible to predict that NXF proteins having a truncated UBA domain are unlikely to directly interact with nucleoporins. This prediction was confirmed for NXF3.
Surprisingly, in spite of its conservation, the UBA domain of Mex67p is not sufficient for nucleoporin binding, and Mex67p lacking the UBA domain can bind to nucleoporins in the presence of Mtr2p (38). The possibility that vertebrate NXF proteins lacking the NPC binding domain can still interact with nucleoporins in the presence of p15 requires further investigation; however, NXF3 did not associate with nucleoporins either in vivo or in vitro when coexpressed with p15 (data not shown). TAP binds Nup98 in vitro, but this interaction is not required for its localization at the nuclear rim, as in Nup98
/ cells, TAP remains at the nuclear
envelope (43). The observation that NXF2 localizes at the
nuclear rim and can promote export of the pDM138 pre-mRNA in the
absence of Nup98 binding is consistent with the observation that
Nup98/ cells do not display an overt export
inhibition phenotype (43). Thus, although TAP and NXF2
interact with multiple nucleoporins in vitro, the nucleoporins that are
responsible for their localization at the nuclear rim remain to be
identified. Moreover, the mechanism by which NXF proteins regulate
their binding to nucleoporins is unknown.
Role of NXF proteins in RNA nuclear export. In yeast, Mex67p is involved in the export of bulk polyadenylated RNAs (36). Evidence for an essential role in mRNP export has been recently obtained for the C. elegans protein NXF1 (41). Moreover, TAP is directly implicated in the export of simian type D retroviral RNAs bearing the CTE (8, 12). Although we cannot exclude the possibility that some TAP homologues may have a function other than export, the conservation of their structural organization and the observation that at least three members of the NXF family are implicated in RNA export suggest that these proteins are likely to participate, directly or indirectly, in the export of cellular mRNAs to the cytoplasm. The diversification of NXF proteins in higher eukaryotes compared to yeast may reflect a greater substrate complexity or tissue-specific requirements. Consistent with this possibility is the observation that human NXF5 is expressed in brain (Lin et al., submitted).
NXF proteins may associate with cellular mRNPs directly, by binding to the mRNA, or indirectly, through interaction with hnRNP-like proteins. Recently, several TAP partners that could facilitate its interaction with cellular mRNPs have been identified. These include E1B-AP5, RAE1, and members of an evolutionarily conserved family of hnRNP-like proteins, the Yra1p/REF proteins (4, 37, 39). Aside from these, other RNA binding proteins may also facilitate TAP binding to the mRNPs, as Y14, an hnRNP-like protein that preferentially associates with spliced mRNAs, has recently been shown to be present in a complex containing TAP (18). In vitro, Y14 interacts with TAP and NXF2 (data not shown); therefore, it is possible that Y14, directly or indirectly, recruits NXF proteins to mRNPs in a post-splicing-dependent manner (18). Besides Y14, several other proteins form splicing-dependent interactions with mRNA. These include the splicing coactivator SRm160, the acute myeloid leukemia-associated protein DEK, and several unidentified proteins (23, 25). These proteins may also link NXFs with mRNAs. NXF proteins may then be more efficiently loaded on mRNAs that have been produced by splicing. Therefore, it is possible that the pDM138 precursor mRNA cannot efficiently compete with other mRNAs for binding to NXFs, i.e., NXFs may be limiting for this substrate; hence, its export can be stimulated by overexpression of NXF-p15 heterodimers. Consistent with this is the observation that overexpression of TAP-p15 heterodimers does not stimulate export of reporter mRNAs that are efficiently expressed (I. C. Braun and E. Izaurralde, unpublished data). TAP-mediated export of pDM138 pre-mRNA requires the LRRs and the NTF2-like domain, while deletion of the RBD or of the NPC binding domain impaired but did not abolish export (Fig. 7D). This is in sharp contrast to the situation observed when TAP-mediated export of pDM138-CTE is monitored in quail cells. In this case, both the RBD and the NPC binding domain are absolutely required (Fig. 4F). The RBD of TAP is required in cis to the LRRs for specific binding to the CTE RNA (24). Hence, this domain is essential for TAP-mediated export of CTE-containing substrates, whereas binding of TAP to pDM138 pre-mRNA may be mediated by protein-protein interactions. We have previously reported that the requirements for the NTF2-like and UBA domains of TAP are substrate dependent (4, 8). Indeed, in Xenopus oocytes, TAP-mediated export of CTE-bearing intron lariats is independent of these domains (8), while export of U6-CTE requires the UBA domain but not the NTF2-like domain (4). In quail cells, TAP-mediated export of pDM138-CTE pre-mRNA is abolished by mutations or deletions of the UBA domain (15) (Fig. 4) but is only reduced by mutations preventing p15 binding (16). Finally, the C-terminal domain of Mex67p is important but not essential for mRNA export (38). Thus, the functionality of NXF protein domains may be influenced by the specific set of proteins associated with the RNP export substrate. Discovery of the precise roles of NXF protein domains and the mechanism by which these proteins mediate directional transport of RNP substrates across the NPC remains an important goal for the future.| |
ACKNOWLEDGMENTS |
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The technical support of Michaela Rode is gratefully acknowledged. We thank Tom Hope and Matthias Dobbelstein for the kind gift of plasmids pDM138 and pDM138-CTE. We thank Birthe Fahrenkrog, Maarten Fornerod, Iain W. Mattaj, and Christel Schmitt for critical reading of the manuscript. We are grateful to Barbara Felber, Guy Froyen, and Peter Marynen for communicating their results prior to publication.
This study was supported by the German Ministry of Research and Technology (BMBF), the Swiss National Science Foundation, the European Molecular Biology Organization (EMBO), and the Human Frontier Science Program Organization (HFSPO).
A.H. and M.S. contributed equally to this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Phone: 0049 6221 387 389. Fax: 0049 6221 387 518. E-mail: izaurralde{at}embl-heidelberg.de.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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