* Department of Biology, University of California at San Diego, La Jolla, California 92093-0347; and Department of
Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706
The 97-kD O-linked glycoprotein, Nup98, is a component of the Xenopus laevis nuclear pore complex and the only vertebrate GLFG nucleoporin identified (Powers, M.A., C. Macauley, F. Masiarz, and D.J. Forbes. 1995. J. Cell Biol. 128:721-736). We have investigated possible roles of xNup98 in the nucleocytoplasmic transport of proteins and RNAs by analyzing the consequences of injecting monospecific polyclonal antibodies to xNup98 into X. laevis oocytes. We show here that nuclear injection of anti-xNup98 inhibited the export of multiple classes of RNAs, including snRNAs, 5S RNA, large ribosomal RNAs, and mRNA. In contrast, the export of tRNA was unaffected. Injection of antixNup98 into the oocyte cytoplasm had no effect on export of any of the RNAs. Significantly, nuclear injection of anti-xNup98 antibodies did not inhibit import of either karyophilic proteins or snRNPs. This latter result is in agreement with our previous finding that Nup98 is not an essential element of the protein import pathway. Thus, Nup98 plays a role specifically in RNA export from the nucleus, and it appears to be an essential component of multiple RNA export pathways.
Trafficking across the nuclear envelope occurs exclusively through the nuclear pore complex, which
both imports proteins and small nuclear ribonucleoproteins (snRNPs)1 and exports RNAs and ribosomal subunits. In addition to the proteins of the pore, nucleocytoplasmic transport requires soluble factors such as the
importin The nuclear pore complex itself is a large and elaborate
structure of 120 MD in vertebrates, comprising ~100 different proteins, many of which are present in multiple copies (for review see Rout and Wente, 1994 Much progress has been made recently in our knowledge of the nuclear pore complex. In yeast, multiple nucleoporin genes have been identified, and mutational analysis
has linked functional or structural phenotypes with specific gene products (for review see Doye and Hurt, 1995 At present, Nup98 is the only vertebrate nucleoporin
identified that contains GLFG repeats. Interestingly, reconstituted nuclei that lack this GLFG nucleoporin are competent for nuclear protein import. However, xNup98-
depleted nuclei remain small and fail to replicate their
DNA, indicating an essential nuclear role for Nup98, but
not one in import (Powers et al., 1995 The GLFG family in the yeast Saccharomyces cerevisiae
includes five nucleoporins: Nup49, Nup54, Nup100,
Nup116, and Nup145 (Wente et al., 1992 Export of different classes of RNA, including snRNAs,
mRNA, tRNA, and ribosomal RNA, occurs via distinct pathways (for review see Izaurralde and Mattaj, 1995 Given the contributions of GLFG nucleoporins to nuclear export in yeast, we asked whether xNup98 might be
an essential component of the vertebrate RNA export machinery. For this, we have used Xenopus laevis oocytes,
which allow for microinjection of transport substrates and
potentially inhibitory antibodies into either the nuclear or
cytoplasmic compartment. We find that affinity purified antibodies to Xenopus Nup98, when injected into oocyte
nuclei, selectively inhibit the nuclear export of multiple,
but not all, classes of RNAs. However, xNup98 antibodies
do not significantly impair nuclear import of either snRNPs
or karyophilic proteins. These results argue strongly that
Xenopus Nup98 functions as a common element in multiple pathways of RNA export from the nucleus, but not as
an essential component of nuclear import pathways.
DNA Templates for In Vivo and In Vitro
RNA Synthesis
Templates for in vivo RNA synthesis in oocytes were plasmid DNAs containing genes for X. laevis U4 snRNA (Vankan et al., 1990 Antibodies for Microinjection or Immunoprecipitation
Affinity purified polyclonal rabbit antibodies to the Xenopus nucleoporins
xNup98 and xNup200 were prepared as previously described (Macaulay
et al., 1995 Oocyte Injection and Analysis of 32P-labeled RNAs
Made In Vivo
Stage V and VI oocytes were obtained from X. laevis frogs as described
(Lund and Dahlberg, 1989
Immunoprecipitation of snRNA Precursors
Deproteinized nuclear RNAs were immunoprecipitated with anti-m7Gantibodies bound to protein A-Sepharose (Sigma Chemical Co., St. Louis,
MO) as previously described (Neuman de Vegvar and Dahlberg, 1990;
Terns et al., 1993b In Vitro Synthesis of 32P-labeled RNAs and Analysis
of Transport
In vitro transcription with SP6 RNA polymerase (for AdML pre-mRNA
and U1Sm
In Vivo Synthesis of 35S-labeled Nuclear Proteins and
Analysis of Protein Import
To produce 35S-labeled Xenopus karyophilic proteins, 100-150 stage V and
VI oocytes were incubated for 24 h in 1 ml of MBS-H medium (Gurdon
and Wickens, 1983 Anti-xNup98 Antibodies Inhibit Nuclear Export of
snRNA and 5S RNA, but Not tRNA
Previously, we showed that the Xenopus nucleoporin
xNup98, which is located both in the nuclear interior and
at the nucleoplasmic side of the nuclear pore, is not essential for protein import (Powers et al., 1995 Anti-xNup98 antibodies, prepared and affinity purified,
as previously described, recognized a single WGA binding
protein from Xenopus egg extracts (Fig. 1 A, lane 3; Powers et al., 1995 To examine their effect on RNA export, xNup98 specific antibodies were injected into Xenopus oocyte nuclei
together with a mixture of plasmid DNAs encoding Xenopus U4 snRNA, 5S rRNA, and tRNATyr (Fig. 2, In sharp contrast to the control injections, injection of
antibodies to xNup98 strongly inhibited the export of U4
snRNA from the oocyte nuclei (Fig. 2, A and B, lanes 3 and 4). At both the 4 and 21 h timepoints, the level of U4
RNA in the cytoplasm of oocytes injected with antixNup98 was greatly reduced relative to the level in oocytes
injected with either buffer or the control antibody (Fig. 2,
A and B; lanes 2, 4, and 6). This inhibition of export was
apparent even at incubation times as short as 2 h (data not
shown).
Normally, m7G-capped precursor snRNA is rapidly exported to the cytoplasm. There, it becomes complexed
with cytoplasmic Sm proteins, and its cap is hypermethylated to the mature m2,2,7G form. The fully matured snRNP
can then be imported back into the nucleus (Mattaj, 1986
The export of both 5S RNA and tRNA occurs by different pathways than U4 snRNA, as determined by competition studies (Jarmolowski et al., 1994 Anti-xNup98 Antibodies Impair mRNA Export
Although both mRNA and pre-snRNAs contain 5 In control oocytes, the spliced AdML mRNA was exported in a time-dependent manner (Fig. 4, mRNA, lanes
3 and 4, 7 and 8). In contrast, in anti-xNup98 injected oocytes, the majority of spliced mRNA remained in the nucleus (Fig. 4, lanes 1 and 2, 5 and 6). The level of nuclear
mRNA at 4.5 h in anti-xNup98 injected oocytes (Fig. 4, lane
5) was reduced relative to the level at 1.5 h (Fig. 4, lane 1),
due to the RNA turnover that occurs in the nucleus when
export is inhibited; still, the majority of the mRNA observed was nuclear, not cytoplasmic (Fig. 4, lanes 5 and 6).
U1Sm In parallel experiments, we found that the simultaneous
injection of anti-xNup98 antibodies with snRNA and premRNA resulted in little or no inhibition of export (data
not shown). The necessity for preinjection to allow binding
of antibody to antigen supports our conclusion that inhibition of RNA export results from specific interaction of the
antibodies with xNup98. Taken together, the results of
Figs. 2, 3, and 4 indicate that both mRNA and snRNA pathways for export are effectively inhibited by the binding of antibodies to xNup98.
Anti-xNup98 Antibodies Decrease Ribosomal
RNA Export
We next asked whether xNup98 function was also required for export of the large 18 and 28S ribosomal RNAs,
which occurs by yet another distinct RNA export pathway
(Bataillé et al., 1990
Anti-xNup98 Antibodies Have No Effect on Nuclear
Protein Import
We have previously shown that xNup98 is not required for
nuclear protein import in reconstituted nuclei (Powers et al.,
1995
Anti-xNup98 Antibodies Do Not Prevent
snRNP Import
Certain small RNAs, like U6 snRNA (Vankan et al., 1990
By monitoring nuclear transport in Xenopus oocytes in the
presence of monospecific antibodies to xNup98, we have
demonstrated that this GLFG nucleoporin functions in the
export of multiple classes of RNA, but not in the export of
tRNA. Further, our results show that Nup98 does not have
an essential role in nuclear protein import, supporting our
previous conclusion from nuclear reconstitution studies.
Similarly, we find that Nup98 is not required for the import of spliceosomal snRNPs. Thus, Nup98 represents the
first vertebrate nuclear pore component for which a unidirectional role in multiple RNA export pathways has been
established.
The existence of distinct pathways of nuclear export for
different classes of RNA is suggested both by kinetic analyses (Zasloff, 1983 The next question is what then recognizes the different
class-specific export factors. One possibility is that a general export receptor recognizes NES signals present in the
individual class-specific factors and ferries the RNA-protein complexes to the pore. Alternatively, different RNAs,
via their class-specific factors, may bind directly to a protein or proteins of the nuclear pore. Nup98, localized both
on the nucleoplasmic side of the pore and within the nucleus, potentially on intranuclear filaments, is ideally positioned to mediate either the binding of a general export
receptor, or the binding of individual class-specific factors.
Antibodies to xNup98 impair snRNA, mRNA, 5S RNA, and
ribosomal RNA export, indicating involvement of this nucleoporin in all of these pathways (Figs. 2, 4, and 5). The
saturability of tRNA export indicates that this class of
RNA also has a specific export system (Zasloff, 1983 All classes of RNA export, including tRNA export, can
be blocked by certain inhibitors; however, because these
componds also block protein import, it is unclear if they
affect RNA export directly. For example, injection of the
lectin WGA inhibits export of all classes of RNA (Featherstone et al., 1988 If a general export receptor carries the different RNAs
to the pore, what might that export receptor be? It is presently not known whether either of the components of the
heterodimeric import receptor, importins If importin Several other regions of Nup98 present interesting
possibilities for a potential function in export. A distinct
sequence of 40 amino acids devoid of any repeat motifs is
present near the NH2 terminus of Nup98, within the
GLFG repeat domain (amino acids 170-192; Radu et al.,
1995b Alternatively, the COOH-terminal domain of Nup98
contains a sequence of ~150 amino acids; this domain has
no GLFG repeats but exhibits weak homology to certain
RNA binding proteins (amino acids 737-892; Radu et al.,
1995b In summary, we have demonstrated that the GLFG nucleoporin Nup98 plays a central role in the export of multiple classes of RNAs. The future identification of the nuclear export factors that specifically interact with Nup98
and Nup153 (Bastos et al., 1996/
heterodimer, which binds directly to nucleartargeted proteins, and the GTPase, Ran, with its associated stimulatory and recycling factors (for review see Moore
and Blobel, 1994
; Powers and Forbes, 1994
; Melchior and
Gerace, 1995
; Görlich and Mattaj, 1996
; Sazer, 1996
).
; Davis, 1995
).
Structurally, the pore consists of a core of eight spokes surrounding a central transporter which spans the nuclear envelope. This core structure is flanked by a cytoplasmic
ring, from which fibers project into the cytoplasm, as well
as a nuclear ring from which a basket-like structure extends into the nucleoplasm (for review see Pante and Aebi,
1993
; Rout and Wente, 1994
). Additional long fibers
project from the basket into the nucleus (Cordes et al.,
1993
). Both the cytoplasmic fibers and the nuclear basket
have been hypothesized to play roles in the initial binding
of transport substrates to the pore. Indeed, scanning electron microscopy of Balbiani ring transcripts shows movement through the basket (Kiseleva et al., 1996).
).
In vertebrates, 12 of the potential ~100 nucleoporins have
been identified and localized to specific substructures of
the pore (for review see Pante and Aebi, 1993
). Of these
12, approximately half contain repeated peptide motifs: FXFG in the majority (for review see Fabre and Hurt,
1994
; Davis, 1995
), and GLFG in a single protein, Nup98
(Powers et al., 1995
; Radu et al., 1995b
). Several lines of
evidence implicate the FXFG family of nucleoporins as
crucial to nuclear transport. In vivo, antibodies to the
FXFG family impair both nuclear protein import and RNA
export (Dabauvalle et al., 1988
; Featherstone et al., 1988
;
Terns and Dahlberg, 1994
; Lund and Dahlberg, manuscript in preparation). Recently, a member of this family
was reported to play a role in the export of polyA+ RNA
(Bastos et al., 1996
). FG repeat-containing nucleoporins of
the cytoplasmic fibers (Nup 358 and Nup214) and the nuclear basket (Nup153 and Nup98) have been shown to
bind soluble transport factors in vitro (Moroianu et al.,
1995
; Radu et al., 1995a
; Radu et al., 1995b
). Moreover,
binding experiments using isolated yeast nucleoporins suggest that interactions between such repeat domains and
import factors may be functionally important in transport (Rexach and Blobel, 1995
; Nehrbass and Blobel, 1996
). Finally, in vitro nuclear reconstitution has indicated the importance of FXFG nucleoporins in nuclear import (Dabauvalle et al., 1990
; Finlay and Forbes, 1990
; Finlay et al.,
1991
; Miller and Hanover, 1994
).
). Immunofluorescence shows that xNup98 is present both at the pore and
within the nuclear interior (Powers et al., 1995
), and rat
Nup98 has been localized within the pore to the nuclear
basket by immunoelectron microscopy (Radu et al.,
1995b
). In vitro binding assays indicate that rat Nup98 can
interact with soluble cytoplasmic import factors (Moroianu et al., 1995
; Radu et al., 1995b
). However, given the
localization of Nup98 at the nuclear basket and within the
nucleus, the functional significance of this latter interaction is not understood.
; Wimmer et al.,
1992
). Mutations in members of this family have pleiotropic effects on yeast nuclear function, including aberrant
nuclear envelope structure, nuclear accumulation of polyA+
RNA, and impaired nuclear import (for review see Doye
and Hurt, 1995
). Nup49 and Nup54 are essential proteins
present in a multiprotein complex that is primarily required for nuclear protein import (Schlenstedt et al., 1993
;
Grandi et al., 1995
). Deletion of the essential Nup145 gene
results in a defect not in protein import, but in poly A+
RNA export (Fabre et al., 1994
). Nup100, Nup116, and
Nup145 each contains a related domain that can bind homopolymeric RNA in vitro (Fabre et al., 1994
). A similar
domain is found in rat Nup98, which shows strong homology to this subset of the GLFG family (Radu et al., 1995b
);
peptide analysis of Xenopus Nup98 indicates that this domain is conserved in Xenopus (Powers et al., 1995
). In
yeast, the presence of a single gene containing this putative RNA-binding domain is sufficient for cell viability;
thus Nup145, Nup116, and Nup100 appear to serve a redundant function, most likely in the export of RNA.
). This
conclusion is based both on kinetic analyses and on experiments demonstrating that a given RNA is able to saturate
its own export but not that of the other classes of RNA
(Zasloff, 1983
; Bataillé et al., 1990
; Terns et al., 1993a
; Jarmolowski et al., 1994
; Boelens et al., 1995
; Pokrywka and
Goldfarb, 1995
; Simons et al., 1996
). These studies have
led to a model in which export of different RNAs is mediated by distinct and class-specific saturable factors. In support of this model, recent studies have identified several
soluble RNA binding factors in vertebrates, such as the
cap binding complex (CBC), TFIIIA, and the Rev protein
of HIV-1, which bind to snRNAs, 5S RNA, and unspliced
HIV RNA, respectively (Guddat et al., 1990
; Izaurralde and Mattaj, 1992
; Fischer et al., 1995
; Izaurralde et al.,
1995
). It is thought that each may specifically facilitate the
nuclear export of the bound RNA. In contrast, the components of the vertebrate nuclear pore that constitute the
export machinery have remained, for the most part, uncharacterized.
Materials and Methods
), 5S rRNA
(Wolffe et al., 1986
), and tRNATyr (Gouilloud and Clarkson, 1986
). Templates for in vitro RNA synthesis were DNA fragments generated by PCR
amplification, as described previously for U1Sm
, U1
, U6 snRNAs (Terns
et al., 1993a
), U5 snRNA (Pasquinelli et al., 1995
), and NL15 RNA
(Grimm et al., 1997
). The template for AdML pre-mRNA was Sma1-linearized pSP64-Ad1(+/
A) plasmid DNA (Lund and Dahlberg, manuscript in preparation). pSP64-Ad1(+/
A) contains the Pst1-Sau3A1 fragment of pBSAd1 (Konarska and Sharp, 1987
) cloned between the Pst1
and BamH1 sites of pSP64 Poly(A) (Promega Corp., Madison, WI).
; Powers et al., 1995
). To generate preparations suitable for oocyte injection, affinity purified antibodies were further concentrated to
0.5-1 mg immunoglobulin/ml using a microconcentrator (Microcon 50;
Amicon Corp., Danvers, MA). Immunoblots were performed as described
previously, using peroxidase-conjugated secondary antibodies and a chemiluminescent substrate (Powers et al., 1995
). Crude sera containing the
polyclonal rabbit anti-m7G antibodies (Munns et al., 1982
) used for immunoprecipitation of precursor snRNAs were a generous gift of T. Munns
(Washington University School of Medicine, St. Louis, MO).
). For in vivo synthesis of RNAs, 12-15 nl of a
solution containing a mixture of plasmid DNAs (Fig. 1, legend) plus blue
dextran (as a marker) was injected into the oocyte nucleus. To monitor synthesis of the RNAs, ~1.0 µCi of
-[32P] GTP (NEN-Dupont, Boston,
MA) was injected into the cytoplasm of each oocyte, and the intracellular
distribution of the newly made transcripts was assayed with time. When
used, control IgG and anti-nucleoporin antibodies were either coinjected
with the DNA into the nucleus (6-15 ng/oocyte) or injected separately
into the cytoplasm (15-30 ng/oocyte) prior to the cytoplasmic injection of
labeled GTP. Nuclei and cytoplasms were isolated from successfully injected oocytes (those with blue nuclei) by manual dissection under mineral oil (Lund and Paine, 1990
), using at least three oocytes per time point.
Isolation of RNAs from the nuclear and cytoplasmic fractions using proteinase K and phenol and analysis in denaturing 8% (30:0.8) polyacrylamide gels were done as detailed elsewhere (Pasquinelli et al., 1995
). Autoradiograms were exposed for 18-72 h, and transport was quantitated by
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analysis.
Fig. 1.
Specificity of affinity-purified anti-xNup98 antibodies.
(A) Affinity purified anti-nucleoporin antibodies (-98 and
-200) or a nonspecific rabbit IgG was used to probe immunoblots containing Xenopus WGA binding proteins (Xenopus eluate, XE)
and a nuclear pore fraction from Xenopus annulate lamellae
(AL). (B) Affinity purified anti-xNup98 was used to probe blots
containing either E. coli lysate (control), lysate from strains expressing regions of rat Nup98, or Xenopus cytosol (XC). rNup98
corresponds to the nearly full length protein, amino acids 43-824.
The GLFG repeat domain (GLFG) corresponds to amino acids
43-490. The RNA binding domain (RBD) corresponds to amino
acids 618-824.
[View Larger Versions of these Images (43 + 26K GIF file)]
). The RNAs in both precipitate and supernatant fractions were analyzed by PAGE, and the levels of m7G-capped snRNA precursors were quantitated by PhosphorImager analysis.
, U1
, and U5 snRNAs) or T7 RNA polymerase (for U6 snRNA and NL15 RNA) was done as described previously (Melton et al.,
1984
; Pasquinelli et al., 1995
) using
-[32P]GTP as the label. AdML premRNA, U1Sm
, U1
, and U5 snRNA transcripts were synthesized with
m7GpppG caps, and U6 snRNA was synthesized with a
-mpppG cap
(Terns et al., 1993a
). All RNA products were purified by electrophoresis in 8% denaturing polyacrylamide gels. For use in transport assays, 12-15
nl of a mixture of RNAs (1-10 fmol each, see Figs. 2 and 4, legends) was
injected into the nucleus or cytoplasm of oocytes. For nuclear injections,
U6 snRNA, which is not exported to the cytoplasm (Vankan et al., 1990
;
Terns et al., 1993a
), and blue dextran (Jarmolowski et al., 1994
) was included as controls for the accuracy of injections and dissections. After different times of incubation, the intracellular distributions of the injected RNAs were analyzed as described above for in vivo transcribed RNAs.
Fig. 2.
Differential effects
of anti-xNup98 antibodies on
transport of in vivo synthesized RNA polymerase II
and III transcripts. A mixture
of DNA templates encoding
U4 snRNA, 5S rRNA, and
tRNATyr was injected into oocyte nuclei together with
buffer (Control) or monospecific antibodies against nucleoporins xNup98 (-98) or
xNup200 (
200). 2 h later,
[32P]GTP (~1.0 µCi/oocyte) was injected into the
cytoplasms of all oocytes. After 4 (A) or 21 h of labeling
(B), oocytes (3-6/timepoint) were manually dissected, and
the intracellular distributions
of the newly made RNAs
were determined by analyses
of 0.5 oocyte equivalents of
the nuclear (N) and cytoplasmic (C) RNAs in 8% denaturing polyacrylamide gels.
The RNAs made from the injected genes are indicated.
Endogenous oocyte RNAs
made in the absence of injected genes (No DNA) are
shown in B, lanes 7 and 8. The level of 5S DNA templates was ~10-fold higher in
A than in B.
[View Larger Version of this Image (73K GIF file)]
Fig. 4.
Inhibition of mRNA export by anti-xNup98 antibodies.
Anti-xNup98 antibodies (-98) or buffer (Cont.) was injected
into oocyte nuclei and 2 h later the same oocytes received a second nuclear injection with a mixture of 32P-labeled AdML premRNA, U1Sm
RNA, and U6 RNA that had been synthesized in
vitro using SP6 or T7 RNA polymerases. At 1.5 (lanes 1-4) and
4.5 h (lanes 5-8) after RNA injection, splicing of the AdML premRNA and export of the spliced mRNA and U1Sm
RNA were
monitored by analyses of nuclear (N) and cytoplasmic (C) RNAs
isolated from individual oocytes. The injected mixture of RNAs
(Inj.) is shown in lane 9. U1Sm
RNA, which lacks a functional
Sm binding site, is exported to the cytoplasm but cannot be imported back into the nucleus. U6 RNA is retained in the nucleus and serves as a marker for the accuracy of the second nuclear injection and oocyte dissections.
[View Larger Version of this Image (64K GIF file)]
) containing 0.5-1.0 mCi of [35S]methionine (TRAN35SLABELTM; ICN Radiochemicals, Irvine, CA). Labeled nuclei were isolated by manual dissection of oocytes under mineral oil (Lund and Paine, 1990
) and collected in a 0.5-ml microfuge tube on ice. After removal of excess mineral oil, 50-100 nuclei were homogenized in 10-20 µl of protein
buffer (10 mM MOPS, pH 7.2, 75 mM KCl, 25 mM NaCl containing 1 mM
DTT, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin) by repeated
pipetting. The homogenate was centrifuged at 14,000 rpm for 4 min in a
microfuge; the clarified supernatant was collected and either used immediately or stored at
70°C for later injection. For protein import assays,
35-50 nl of 35S-labeled nuclear extract (~0.25 oocyte equivalents) was injected into each oocyte cytoplasm. Where used, control IgG and either anti-nucleoporin antibodies or wheat germ agglutinin (WGA; Vector Labs, Inc., Burlingame, CA) was injected separately into the nuclei or
cytoplasms before cytoplasmic injection of the import substrate. For
WGA, 8-10 nl or 15-30 nl of a saturated solution (~20 mg/ml in 88 mM
NaCl, 10 mM TrisHCl, pH 8.0) was injected per nucleus or cytoplasm, respectively. After overnight incubation, oocytes (at least 5/sample) were
manually dissected, and the intracellular distribution of labeled proteins
was determined by analysis of 0.5-1.0 oocyte equivalents of the nuclear
and cytoplasmic fractions in 10% polyacrylamide gels containing SDS
(Adolph et al., 1977
). Autoradiograms of the dried gels were exposed for
2-14 d at room temperature.
Results
). To determine
if Nup98 is required for transport out of the nucleus, we
examined the effect of anti-xNup98 antibodies on the nuclear export of different classes of RNA.
). This antibody also specifically recognized
a single protein of identical size in the enriched nuclear
pores of Xenopus annulate lamellae, indicating that the
antibody does not cross-react with any other component
of the pore (Fig. 1 A, lane 4; Meier et al., 1995
). In addition, the antibody was seen to cross-react with both the
GLFG repeat and RNA binding domains of the homologous rat Nup98 (Fig. 1 B, lanes 2-4). A nonspecific rabbit
IgG as well as antibodies to the Xenopus nucleoporin
p200, which is the homolog of the mammalian nucleoporin
Nup214/CAN and is localized to the cytoplasmic face of
the pore (Miller and Hanover, 1994
; Kraemer et al., 1994
;
Macaulay et al., 1995
), were used for control antibody injections.
-98). The
DNA templates were also injected with either buffer alone
(Fig. 2, Control) or with affinity purified antibodies against
the Xenopus nuclear pore protein, Nup200 (
-200). AntixNup200 antibodies injected into the nucleus have no access to their cytoplasmic antigen and serve as controls for
nonspecific effects of antibody injection. After a short incubation to allow for the binding of the antibodies and formation of transcription complexes, [32P]GTP was injected
into the oocyte cytoplasm. At various time points, oocytes
were manually dissected into nuclear and cytoplasmic fractions, and the newly synthesized, labeled RNAs in
each fraction were isolated and resolved by PAGE.
).
It was thus important to determine whether the U4 RNA
in the nuclei of anti-xNup98 injected oocytes had been exported to the cytoplasm and reimported, or had remained in the nucleus due to a block in export. To determine this,
we immunoprecipitated nuclear RNAs with an antibody
that specifically recognizes the m7G-cap common to RNA
polymerase II transcripts (Munns et al., 1982
). In control
oocytes at both 4 and 21 h, the vast majority of U4 RNA in
the nucleus consisted of the mature, nonprecipitable
m2,2,7G-capped form, indicating it had been reimported
(Fig. 3, lanes 1-3 and 7-9; Neuman de Vegvar and Dahlberg, 1990). In contrast, the U4 RNA present in nuclei
of anti-xNup98 treated oocytes at 4 h was almost exclusively the m7G-capped, precursor forms (Fig. 3, lanes 4-6).
Thus, export of newly synthesized snRNAs had been effectively blocked by microinjection of anti-xNup98 antibodies into the nucleus. Even at 21 h, only a small amount
of mature U4 RNA had accumulated in the nuclei of antixNup98 treated oocytes (Fig. 3, lane 11), showing that the
inhibitory effect on snRNA export was persistent. When
anti-xNup98 antibodies were injected into the cytoplasm
of oocytes, no deficiency in RNA transport was observed,
consistent with the nuclear localization of the target Nup98
nucleoporin (data not shown). The reduced level of U4 snRNA in anti-xNup98 injected oocytes was not due to a defect in U4 transcription (data not shown) but to the inherent instability of m7G-capped precursor U4 snRNA, an
instability also seen in other cases where pre-U4 is retained in the nucleus (Lund, E., and J.E. Dahlberg, manuscript in preparation; Her, L.-S., and E. Lund, unpublished
results).
Fig. 3.
Failure of snRNA precursors to leave the nucleus of
anti-xNup98 treated oocytes. Total nuclear RNAs (T), corresponding to those in lanes 1 and 3 of Fig. 2, A and B, were immunoprecipitated with anti-m7G antibodies, which are specific for
the cap structure of the nonexported, nuclear precursor snRNA.
The RNAs in the precipitate (P) and supernatant (S) fractions
were analyzed as in Fig. 2. Dots by lane 12 indicate m7G-capped
degradation products of pre-U4 RNA that also are observed when nuclear export is inhibited by other reagents.
[View Larger Version of this Image (81K GIF file)]
). When the export of
5S rRNA was examined, it was found to be substantially
reduced in the presence of nuclear xNup98 antibodies
(Fig. 2, lanes 2, 4, and 6). A reduced amount of 5S DNA
was injected for the 21-h timepoint to avoid overwhelming the signals from other transcripts (Fig. 2, legend). Strikingly, the export of tRNA was completely unaffected by
anti-xNup98 antibodies at all times. The persistance of
tRNA export in the presence of anti-xNup98 antibodies
makes it unlikely that the inhibition of U4 and 5S RNA
export resulted from physical obstruction of the nuclear
pore. Instead, we conclude that the binding of antibody to
xNup98 prevents a specific function of this protein, a function required for multiple pathways of RNA export.
m7Gcaps which serve as signals for export from the nucleus
(Hamm and Mattaj, 1990
; Izaurralde et al., 1992
; Terns et
al., 1993a
; Izaurralde et al., 1995
), transport of these RNAs
apparently occurs via different pathways (Jarmolowski et
al., 1994
; Fischer et al., 1995
). To determine whether the
export of mRNA is also dependent on Nup98 function, we
monitored the export of an mRNA that was generated by
splicing of injected 32P-labeled adenovirus major late
(AdML) pre-mRNA in oocyte nuclei (Hamm and Mattaj,
1990
; Lund and Dahlberg, manuscript in preparation). Oocytes were injected in the nucleus twice, first with either xNup98-specific antibodies or buffer, and then, after a
short incubation to allow for antibody binding, with a mixture of the 32P-labeled pre-mRNA and control RNAs. As
a control for the accuracy of the second nuclear injection,
we included labeled U6 snRNA, which is not exported
from the nucleus. As a further test for inhibition of snRNA export by anti-xNup98, we also included U1Sm
RNA, a mutant U1RNA which would normally be exported, but which lacks the Sm binding site required for
import back into the nucleus (Hamm and Mattaj, 1990
).
RNA also was exported to the cytoplasm in control oocytes, but was largely retained in the nucleus in antixNup98 injected oocytes. Control U6 snRNA and the lariat of the pre-mRNA, RNAs normally not exported,
stayed localized in the nucleus in both control and antixNup98 injected oocytes. A certain amount of unspliced
pre-mRNA export was observed in control injected oocytes, most likely due to initial saturation of the splicing machinery (Lund and Dahlberg, unpublished results).
Strikingly, however, this export was also sensitive to inhibition by anti-xNup98 antibodies.
; Fischer et al., 1995
; Pokrywka and Goldfarb, 1995
). In Xenopus oocytes, the rRNA genes are
highly amplified, so it is possible to monitor expression of the
endogenous rRNA genes simply by injection of [32P]GTP
(Peculis and Steitz, 1993
). We found that anti-xNup98 antibodies significantly retarded export of the large 18 and
28S ribosomal RNAs, as shown by the reduction in levels
of these newly made rRNAs in the cytoplasm (Fig. 5, lanes
4 and 5). The concomitant accumulation of excess mature
18 and 28S rRNAs in the nucleus (Fig. 5, lane 2) showed
that anti-xNup98 antibodies inhibited export but not the
synthesis or maturation of ribosomal RNAs. We also note
that export of rRNAs, in the form of large preribosomal subunits, is inhibited to a lesser extent than is the export of the much smaller 5S RNA particles. This is a further indication that inhibition does not result simply from nonspecific obstruction of the nuclear pore. Additionally, maturation of the nascent labeled rRNA transcripts requires the
assembly of the ribosomal subunits in the nucleus which,
in turn, depends on the import of ribosomal proteins (for
review see Hadjiolov, 1985
). Thus, the results in Fig. 5 suggested that protein import is not influenced by the antixNup98 antibodies.
Fig. 5.
Reduced export of
ribosomal RNAs in the presence of anti-xNup98 antibodies. Buffer (Control) or antibodies specific for xNup98 (-98) or xNup200 (
-200)
were injected into oocyte nuclei. 2 h later
[32P]GTP
(~1.0 µCi/oocyte) was injected into the cytoplasm of
all oocytes. After 20 h of labeling, the intracellular distribution of newly made endogenous ribosomal RNAs
was determined by analyses of 0.5 oocyte equivalents of nuclear (lanes 1-3) and cytoplasmic RNAs (lanes 4-6) in a 1.2% formaldehyde-agarose gel. The
electrophoretic mobilities of precursor (45, 36, 32, and 20S) and
mature (18 and 28S) ribosomal RNAs are indicated.
[View Larger Version of this Image (72K GIF file)]
). To test whether Nup98 is required for import in
vivo, we compared the nuclear import of karyophilic proteins in oocytes pre-injected with anti-xNup98 antibodies,
control IgG antibodies, or WGA, a potent inhibitor of protein import (Forbes, 1992
). After antibody injection, total
35S-labeled nuclear proteins (Dabauvalle et al., 1988
) were
injected into the cytoplasm of the oocytes. 24 h later, the
oocytes were dissected into nuclear and cytoplasmic fractions for determination of the intracellular distribution of
labeled proteins. Injection of anti-xNup98 antibodies into
the nucleus had no detectable effect on nuclear import, as
shown by the efficient uptake of the major labeled nuclear
proteins, N1/N2, as well as other nuclear proteins (Fig. 6,
lanes 2 and 4. Cytoplasmic injection of anti-xNup98 also had no effect on nuclear protein import (data not shown).
As expected, the import of proteins containing nuclear localization signals (NLS) was strongly inhibited in oocytes
that were injected with WGA (Fig. 6, lanes 5 and 6; Finlay
et al., 1987
; Dabauvalle et al., 1988
; Fischer et al., 1991
).
Thus, antibodies to xNup98 do not act as a physical block
to protein transit through the nuclear pore, but rather, define a specific and essential role for this nucleoporin in
RNA export.
Fig. 6.
Lack of effect of
anti-xNup98 antibodies on
import of karyophilic proteins. Normal IgG (IgG),
anti-xNup98 (-98), or WGA
was injected into oocyte nuclei (IgG,
-98) or cytoplasms (WGA). 2 h later,
0.25 oocyte equivalents of
35S-labeled total soluble nuclear proteins were injected
into the cytoplasm of all oocytes. After 24 h incubation,
oocytes were manually dissected, and total nuclear (N) and cytoplasmic (C) extracts were
prepared from pools of 3-5 oocytes. 0.5 oocyte equivalents of the
extracts were analyzed on a 10% polyacrylamide gel containing SDS and 35S-labeled proteins were detected by autoradiography.
Arrows indicate the strongly labeled karyophilic proteins N1/N2
(Dabauvalle and Francke, 1982). Lines indicate the positions of
molecular weight markers of 200, 97, and 66 kD.
[View Larger Version of this Image (103K GIF file)]
;
Terns et al., 1993a
; Boelens et al., 1995
) and the synthetic
NL-15 RNA (Grimm et al., 1997
), are not normally exported from the nucleus. However, if injected into the cytoplasm, these RNAs can be imported into the nucleus via
the same WGA-sensitive pathway as NLS-containing proteins (Fischer et al., 1991
; Grimm et al., 1997
). Other
RNAs, like U1 and U5 snRNAs, which normally undergo
maturation in the cytoplasm, are imported via a separate,
snRNP-specific pathway (Mattaj, 1986
) that is less sensitive to WGA inhibition (Fischer et al., 1991
; Michaud and
Goldfarb, 1991
; Lund and Dahlberg, manuscript in preparation). We found that nuclear import of cytoplasmically injected NL-15 RNA was completely unaffected by the
presence of anti-xNup98 antibodies (Fig. 7), in agreement
with the results for protein import (Fig. 6). Likewise, antixNup98 antibodies had little if any effect on the import of
U5 or U1 snRNAs via the snRNP pathway (Fig. 7, lanes 2 and 3). In contrast, WGA completely blocked NL15 import and impaired U1 and U5 import (Fig. 7, lane 4).
Taken together, these results strongly argue that Nup98 is not essential for nuclear import of RNA.
Fig. 7.
Unimpaired import of
snRNPs and small RNAs in the
presence of anti-xNup98 antibodies. Oocytes that had been
pre-injected with antibodies or
WGA, as in Fig. 6, were injected
in the cytoplasm with a mixture
of 32P-labeled U1Sm, U1
, U5,
and NL15 RNAs that were made by in vitro transcription.
After 7 h of incubation, import of the RNAs was monitored by
polyacrylamide gel analysis of
0.5 oocyte equivalents of nuclear
RNAs isolated from pools of 3-5 oocyte nuclei. The mixture of injected RNAs (Inj.) is shown in lane 1; the slightly faster gel mobilities of U1
(a mutant U1 RNA lacking the U1A protein binding site) and U5 RNAs in lanes 2-4 are due to 3
end trimming
which occurs upon nuclear import of snRNPs (Neuman de Vegvar and Dahlberg, 1990). U1Sm
RNA lacks the Sm binding site
required for import and serves as a control for cytoplasmic contamination of the nuclear samples. Cytoplasmic fractions are not
shown, as RNA levels were in excess, and differences among
them were not significant.
[View Larger Version of this Image (53K GIF file)]
Discussion
; Jarmolowski et al., 1994
; Lund and
Dahlberg, manuscript in preparation) and by competition
experiments, which demonstrate that various RNAs (e.g.,
snRNA, mRNA, and tRNA) are unable to compete with
one another for export (Jarmolowski et al., 1994
; Boelens
et al., 1995
; Pokrywka and Goldfarb, 1995
; Simons et al., 1996
). These latter results indicate the existence of at least one specific limiting factor in the export of each class of
RNA. Since it is believed that RNA exits the nucleus as an
RNA-protein complex, it is likely that specific RNA binding proteins represent these limiting factors, and indeed
candidate proteins have been identified. TFIIIA, which binds
to 5S RNA, contains a nuclear export signal (NES) that
may mediate interaction with the export machinery (Guddat et al., 1990
; Fridell et al., 1996
). CBC, which binds to
m7G-capped RNAs, is thought to promote export of snRNA precursors (Izaurralde et al., 1992
). Antibodies to the
CBP20 protein, a CBC subunit, are capable of reducing the
export of snRNA (Izaurralde et al., 1995
). The CBC complex may also be involved in mRNA export (Visa et al.,
1996
), although antibodies to CBP20 have no effect on
mRNA export (Izaurralde et al., 1995
). The hnRNP A1
protein may fulfill the role of a class-specific export factor
for mRNA, since this protein binds to mRNA, shuttles between nucleus and cytoplasm, and contains a unique NES
sequence (Michael et al., 1995
). The retrovirus HIV-1 encodes its own specific export factor, the Rev protein,
which recognizes a sequence present only in incompletely spliced viral RNAs (for review see Hope and Pomerantz,
1995
) and, via an NES sequence, mediates the export of
these RNAs to the cytoplasm (Fischer et al., 1995
). No
class-specific factor has yet been identified for tRNA.
), but
the lack of inhibition by anti-xNup98 antibodies (Fig. 2) implies that such export bypasses the need for Nup98.
Thus, the export of tRNA might involve binding to a different nucleoporin, or tRNA may enter the export machinery at a point downstream of Nup98. Interestingly, we
have previously found that tRNA export, unlike that of
most other RNAs, occurs independently of the Ran GTPase cycle (Cheng et al., 1995
).
; Bataillé et al., 1990
; Neuman de Vegvar and Dahlberg, 1990), but it binds to N-acetylglucosamine residues present on multiple glycoproteins of the
nuclear pore (Nup200, Nup98, and p62 in Xenopus; Forbes, 1992
), making conclusions about the role of any given nucleoporin difficult to interpret. Similarly, nuclear injection
of the monoclonal antibody, 414, which binds to the FXFG
repeat sequences found in some nucleoporins (Nup200,
Nup153, and p62), blocks numerous classes of RNA export but not that of tRNA (Terns and Dahlberg, 1994
;
Lund and Dahlberg, manuscript in preparation). However, because this antibody recognizes both Nup153 on the
basket and p62 in or near the central transporter (Pante
and Aebi, 1993
), its inhibition of export can not be attributed to any specific nucleoporin. Both these reagents,
WGA and mAb 414, also block the import of proteins into
the nucleus (Finlay et al., 1987
; Featherstone et al., 1988
;
Lund and Dahlberg, manuscript in preparation). Thus,
only now in the case of Nup98 has a monospecific reagent implicated a single vertebrate nucleoporin in a specific
transport process, RNA export.
and
, might
play a role in export through the pore. In nuclear import,
importin
binds to the NLS of a nuclear-targeted protein;
importin
then acts to dock the import receptor complex
to proteins of the nuclear pore (for review see Melchior
and Gerace, 1995
; Görlich and Mattaj, 1996
). The observations that importin
can bind to numerous repeat-containing nucleoporins on blots, including Nup98, led to a
proposal that Nup98 has a role in nuclear protein import
(Moroianu et al., 1995
; Radu et al., 1995b
). However, in
our earlier studies, we found that nuclei reconstituted in
the absence of Nup98 were competent for protein import,
indicating that binding of importin
to Nup98 is not essential for this process (Powers et al., 1995
). Consistent
with this, we find here that anti-xNup98 antibodies inhibit
export but have no significant effect on import of either
proteins or snRNPs. Thus, while there may be an interaction between Nup98 and importin
, we have found no
functional evidence supporting a role for Nup98 in nuclear
import.
has a function in RNA export, it might be
mediated through the interaction of this factor with the
GLFG domain of Nup98 (Radu et al., 1995b
). Indeed, in
yeast, importin
interacts with the GLFG domain of
Nup116, a homolog of Nup98, in a two hybrid assay. Moreover, the GLFG domain of Nup116 is the only GLFG domain of any yeast nucleoporin essential for cell viability,
and overexpression of this domain in yeast results in loss
of poly A+ RNA export (Iovine et al., 1995
). However, in
a separate study using recombinant subdomains of nucleoporins, yeast importin
was found to interact with the
yeast Nup1 FXFG domain but not with the GLFG domains of either Nup145 or Nup54 (Nup116 was not tested
in this assay; Rexach and Blobel, 1995
). These conflicting results raise questions about the specificity of interactions between purified, recombinant proteins in vitro and emphasize the importance of conducting parallel functional
assays, as was done with Nup116 (Iovine et al., 1995
). During protein import, vertebrate importin
traverses the
pore but remains bound to the nuclear side of the pore and
is not released into the nucleoplasm (Görlich et al., 1995
).
One possibility is that the GLFG domain of Nup98 may
provide a final docking site for this import factor before it
is recycled to the cytoplasm, thus explaining the interaction of Nup98 with importin
.
). A segment of this region, rich in charged residues,
is identical in Xenopus and rat Nup98, highly conserved in
yeast Nup116, and absent from other members of the GLFG family (Nup49, Nup54, Nup100, and Nup145; Wente
et al., 1992
; Wimmer et al., 1992
; Fabre et al., 1994
; Wente
and Blobel, 1994
). In Nup116, a somewhat larger region
containing this sequence is essential for function, as indicated by the loss of cell viability when it is deleted (Iovine
et al., 1995
). Because of its strong evolutionary conservation, this small domain may well be important for the
RNA export function of Nup98.
). A homologous region is present in the yeast nucleoporins Nup100, Nup116, and Nup145, but presumably serves a redundant function in the three yeast nucleoporins, since the presence of any one copy of the sequence is
sufficient for cell viability (Fabre et al., 1994
). This sequence may be involved in RNA export, but in no case has
the defect in yeast poly A+ RNA export been linked directly to deletion of this putative RNA-binding sequence.
In vitro, the region was found to bind preferentially to
poly rG homopolymers (Fabre et al., 1994
). Under similar
conditions, we find that Nup98 from Xenopus egg extracts
also exhibits preferential binding to poly rG over other homopolymers (Powers and Forbes, unpublished results).
However, as was the case for the yeast GLFG proteins
(Fabre et al., 1994
), only a very small fraction of the input
xNup98 bound to the homopolymer. Since many other
proteins of the egg extract also bind preferentially to poly
rG, the significance of such in vitro interactions should be
interpreted with caution. When poly rG and poly rI are injected into oocyte nuclei, inhibition of RNA export has
been observed (Jarmolowski et al., 1994
); but it remains to
be determined whether such inhibition of export is due to
binding and inhibition of Nup98 or to inhibition of another
component of the RNA export machinery. Because our polyclonal antibody strongly interacts with both the GLFG and
RNA-binding domains of rNup98 (Fig. 1 B), we can not
precisely distinguish whether both or only one domain is
involved in RNA export. It will be interesting to test even
more specific antibody tools as they are developed.
), either alone or complexed with an RNA export substrate, will be important
for understanding the role of this and other nucleoporins
in RNA export.
Received for publication 4 September 1996 and in revised form 21 November 1996.
This research was funded by grants from the National Institutes of Health to D.J. Forbes (GM 33279) and to J.E. Dahlberg and E. Lund (GM 30220).The authors wish to thank Drs. Katharine Ullman, Christian Grimm, and Amy Pasquinelli for helpful discussions, Dr. Sanjay Vasu for rNup98 peptide fragments, Dr. Brian Miller for annulate lamellae preparations, Dr. Ted Munns for anti-m7G-antibodies, and Dr. Arianne Grandjean and Ms. Michelle Barr for preparation of oocytes for injection.
AdML, adenovirus major late; CBC, cap binding complex; NES, nuclear export signal; NLS, nuclear localization signal; snRNPs, small nuclear ribonucleoproteins; WGA, wheat germ agglutinin.