From the Institute of Histology and Embryology,
Faculty of Medicine, University of Lisbon, 1649-028 Lisbon, Portugal
and ¶ Gene Expression Program, European Molecular Biology
Laboratory, 69117 Heidelberg, Germany
Received for publication, September 25, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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The U2 small nuclear ribonucleoprotein auxiliary
factor (U2AF) is a heterodimeric splicing factor composed of 65-kDa
(U2AF65) and 35-kDa (U2AF35) subunits.
The large subunit of U2AF recognizes the intronic polypyrimidine tract,
a sequence located adjacent to the 3' splice site that serves as an
important signal for both constitutive and regulated pre-mRNA
splicing. The small subunit U2AF35 interacts with the 3'
splice site dinucleotide AG and is essential for regulated splicing.
Like several other proteins involved in constitutive and regulated
splicing, both U2AF65 and U2AF35 contain an
arginine/serine-rich (RS) domain. In the present study we determined
the role of RS domains in the subcellular localization of U2AF. Both
U2AF65 and U2AF35 are shown to shuttle
continuously between the nucleus and the cytoplasm by a mechanism that
involves carrier receptors and is independent from binding to mRNA.
The RS domain on either U2AF65 or U2AF35 acts
as a nuclear localization signal and is sufficient to target a
heterologous protein to the nuclear speckles. Furthermore, the results suggest that the presence of an RS domain in either U2AF subunit is sufficient to trigger the nucleocytoplasmic import of the
heterodimeric complex. Shuttling of U2AF between nucleus and cytoplasm
possibly represents a means to control the availability of this factor
to initiate spliceosome assembly and therefore contribute to regulate splicing.
Pre-mRNA splicing is an essential step in eukaryotic gene
expression that is often regulated in a cell type-specific or
developmental manner. The splicing reaction takes place in the
spliceosome, a multicomponent RNA-protein complex containing five
uracil-rich small nuclear ribonucleoproteins
(snRNP)1 and 50-100
non-snRNP protein splicing factors (1, 2). To ensure the precise
excision of introns, components of the spliceosome recognize weakly
conserved sequences in the pre-mRNA (3). The decision to splice a
given pre-mRNA and the selection of splice sites takes place during
the first stages of spliceosome assembly. These early steps are
therefore crucial for splicing regulation (3-5). The stable
association of U2 snRNP with the 3' region of the intron (3, 6, 7)
requires an auxiliary factor, U2AF (8). U2AF consists of two subunits
that form a stable heterodimer (9). U2AF65 binds directly
to the intronic polypyrimidine tract and is essential for splicing
(10). U2AF35 recognizes the 3' splice site dinucleotide AG
(11-13) and is required for the regulated splicing of a subset of
pre-mRNAs (14).
Regulatory mechanisms underlying the selection of alternative exons
remain poorly defined (5, 15). A significant advance in understanding
splice site recognition was provided by the observation that exon
sequences also play a critical role in splicing. Constitutive as well
as regulated exon splicing enhancers have been identified (16, 17).
These sequences contain binding sites for SR proteins (18, 19),
a family of essential splicing factors with a modular structure
consisting of one or two RRMs and a C-terminal arginine/serine (RS)-rich domain, involved in protein-protein interactions (20, 21).
According to a current model, SR proteins may activate splice site
choice by binding to an enhancer sequence and recruiting the splicing
machinery to the adjacent intron (16, 17, 22), acting as bridging
factors between the 5' and 3' splice sites (23-25).
The subcellular distribution of splicing factors has been shown to be
complex and dynamic, and it is increasingly believed that it may play a
role in the regulation of splicing. Splicing factors accumulate in the
nucleus, concentrating in structures commonly designated as nuclear
speckles (26). Several lines of evidence suggest that nuclear speckles
represent sites where splicing factors accumulate when they are not
actively engaged in the splicing reaction. By an as yet unknown
mechanism, splicing factors are recruited from the speckles to the
sites of transcription, where splicing of most introns takes place
(27).
A major determinant of the subcellular localization of SR proteins is
the RS domain. The RS domain can function as a NLS, and it is also
thought to be responsible for targeting the proteins to the nuclear
speckles (28-30). Changes in the phosphorylation of the RS domain are
sufficient to alter the protein-protein interactions established by SR
proteins (31, 32) and to modulate their subcellular distribution (33,
34). Two novel transport receptors, termed transportin-SR and
transportin-SR2, bind specifically to the phosphorylated RS domain of
SR proteins (35, 36). Although splicing factors are predominantly
detected in the nucleus, at least some members of the SR protein family
have the ability to shuttle between the nucleus and the cytoplasm (37).
The RS domain has been shown to be necessary, but not sufficient, for
export of the shuttling SR proteins to the cytoplasm (30). Based on the
observation that SR proteins can influence splice site selection in a
concentration-dependent manner (19, 38, 39), it has been
proposed that transport to the cytoplasm may provide a way to control
the nuclear availability of these proteins and, hence, contribute to
regulate splicing (37, 40).
Although they are not members of the SR protein family, both
U2AF65 and U2AF35 contain an RS-like domain
(10, 41). The RS domain of U2AF65 is located in the
N-terminal region of the protein and has never been shown to mediate
the establishment of protein-protein interactions. Rather, it
facilitates base pairing between the U2 small nuclear RNA and the
branch point (42) and plays a role in the targeting of this protein to
active splicing sites (43). In human U2AF35, the RS domain
is C-terminal and interacts with the RS domain of SR proteins (20).
In the present work we analyzed the role of the RS domain in the
subcellular distribution of the heterodimeric splicing factor U2AF.
Despite the fact that RS residues in U2AF are more dispersed and have
less periodicity than in SR proteins, the data presented here indicate
that they play a similar role in subcellular localization. We show that
the RS domain on either U2AF65 or U2AF35 acts
as a nuclear and subnuclear localization signal. We also show that both
U2AF65 and U2AF35 shuttle continuously between
the nucleus and the cytoplasm by a mechanism that involves carrier
receptors and is independent from binding to mRNA. Finally, we
provide evidence suggesting that the presence of an RS domain in either
U2AF subunit is sufficient to trigger the nucleocytoplasmic import of
the heterodimeric complex. Taken together these results suggest that
nucleocytoplasmic transport of RS-containing splicing factors may
represent a common pathway to control the availability of these
proteins to initiate spliceosome assembly and, therefore, contribute to
regulate splicing.
Cell Culture, Heterokaryon Assays, and Fluorescence
Microscopy--
Human HeLa and murine NIH 3T3 cells were cultured as
monolayers in modified Eagle's medium and Dulbecco's modified
Eagle's medium, respectively (Life Technologies, Inc.).
Drosophila SL2 cells were grown in suspension in
Schneider's medium at room temperature. All media were supplemented
with 10% fetal calf serum (Life Technologies, Inc.). Heterokaryons
were obtained as previously described (44). Briefly, 1.5 × 106 3T3 cells or 2 × 107 SL2 cells were
plated over subconfluent HeLa cells grown on coverslips on P35 Petri
dishes. 3T3 cells were allowed to adhere for 3 h at 37 °C, 5%
CO2 in the presence of 20 µg/ml emetine. Adhesion of SL2
cells to the coverslips was induced by incubation in serum-free medium
for 15 min at 29 °C, 5% CO2. The cells were then placed in complete modified Eagle's medium with 20 µg/ml emetine and incubated for an additional 3 h. To induce cell fusion, coverslips were rinsed in phosphate-buffered saline (PBS) and placed on a drop of
polyethylene glycol (PEG 1500; Roche Molecular Biochemicals) for 2 min.
The coverslips were then washed in PBS and further incubated in culture
medium at 37 °C for 4 h (for HeLa-3T3 heterokaryons) or at
29 °C for 7 h (for HeLa-SL2 heterokaryons)
Cells on coverslips were briefly rinsed in PBS, fixed in 3.7%
formaldehyde (freshly prepared from paraformaldehyde) diluted in PBS
for 10 min at room temperature, and washed in PBS. The cells were then
permeabilized with 0.5% Triton X-100 in PBS for 10 min at room
temperature and washed in PBS. Immunofluorescence and confocal
microscopy were performed as described (44). Endogenous U2AF65 was detected using monoclonal antibody MC3 (43), and
HA-tagged constructs were visualized using monoclonal antibody 12CA5-I
(Berkeley Antibody Co., Richmond, VA). Additionally, the following
antibodies were used: monoclonal antibody anti-hnRNP C 4F4, (45) and
monoclonal antibody anti-SC35 splicing factor (46).
Construction and Expression of Fusion Proteins--
The
HA-U2AF65
Green fluorescent protein (GFP) fusion constructs were obtained by
restriction digestion and subcloning into the appropriate pEGFP-C
vector (CLONTECH, Palo Alto, CA).
GFP-U2AF65 was obtained by digestion of
pBluescript-
GFP-U2AF35 was obtained by digestion of pGEX-3X
U2AF35 with EcoRI and cloning of the
U2AF35 insert into pEGFP-C1 linearized with
EcoRI. DNA restriction analysis was performed to isolate
constructs with oriented inserts. GFP-U2AF35
DNA for transfection assays was purified using the Qiagen plasmid DNA
midi-prep kit (Qiagen GmbH, Hilden, Germany). HeLa cells were
transiently transfected with FuGene6 Reagent (Roche Molecular Biochemicals) using 1 µg of DNA. For double transfections, 0.5 µg
of each DNA was used. Cells were analyzed at 16-24 h after transfection. Western blot analysis of transfected cells was performed for all constructs as described (43) using anti-HA (Berkeley Antibody
Co.) or anti-GFP antibodies (Roche Molecular Biochemicals).
RNA Binding Assays--
Wild type and mutant RRM1mut proteins
were expressed and purified from Escherichia coli as fusions
with glutathione S-transferase from amino acids 95 to 475 of
U2AF65. These proteins lack the arginine-serine (RS)-rich
domain, which was deleted to facilitate the analysis of RNA binding
exclusively through the RRM motifs of the protein and minimize
nonspecific interactions (50). The purified proteins were incubated
with 5 fmol (10,000 cpm) of a radioactively labeled RNA corresponding to a region of Drosophila sl-2 pre-mRNA containing a
stretch of 11 consecutive uridines, which constitute a high affinity
binding site for U2AF65 (10, 51-53). After incubation for
15 min on ice, the mixtures were fractionated by electrophoresis on
nondenaturing polyacrylamide gels as described (54).
The RS Domain on Either U2AF Subunit Functions as a NLS--
The
RS domain of SR splicing factors has been shown previously to function
as a NLS (28, 29) and to be necessary for export of the shuttling SR
proteins to the cytoplasm (30). To test whether the RS domain of the
U2AF polypeptides plays a similar role, we tagged the two U2AF subunits
with GFP and examined their subcellular localization (Fig.
1). GFP expression levels vary widely in
the transfected population, with very high expressors showing aberrant
protein distribution patterns (data not shown). Therefore, we selected
for analysis cells that expressed the GFP fusion proteins close to
endogenous levels. As depicted in Fig. 1, both GFP-U2AF65
(B) and GFP-U2AF35 (C) show a
distribution similar to that of endogenous U2AF65 detected
using a specific antibody (A). In all cases the staining is
diffuse throughout the nucleoplasm, excluding the nucleoli, with a
higher concentration in speckles, as confirmed by double-labeling with
SC35 monoclonal antibody (data not shown). Contrasting with the results
previously obtained using purified polyclonal antibodies raised against
synthetic peptides of U2AF65 and U2AF35 (41),
we observe no evidence for the localization of either U2AF subunit in
the coiled body. Considering that similar results were obtained with an
anti-U2AF65 monoclonal antibody (43) and epitope-tagged
U2AF65 (43) and U2AF35 (data not shown) as well
as GFP-U2AF65 and GFP-U2AF35 fusion proteins
(data not shown), it is most likely that U2AF does not associate with
coiled bodies.
As expected, expression of GFP alone produces a uniform staining in
both the nucleus and the cytoplasm (Fig. 1D). Fusion of GFP
to the RS domain of either U2AF65 or U2AF35 is
sufficient to target the chimeric protein to the nucleus and to nuclear
speckles (Fig. 1, E-H and F-I), demonstrating
that this domain functions as both a NLS and a speckle targeting
signal. Surprisingly, the GFP-RS chimeras also accumulate in the
nucleoli. Since these are small proteins, they probably enter the
nucleolus by diffusion and are retained by nonspecific binding of the
RS domain to rRNA (50).
Immunoblotting analysis of transfected cells using an anti-GFP antibody
(Fig. 1J) shows that a single protein band is observed for
each GFP construct used in this work. This observation excludes the
possibility that truncated GFP chimeras are contributing to the
observed localization patterns. The GFP-RS35 construct migrates with an
apparent weight that is slightly heavier than expected, possibly due to
phosphorylation of this domain.
Both U2AF Subunits Shuttle Continuously between Nucleus and
Cytoplasm--
Having established that the RS domain of
U2AF65 and U2AF35 plays a role in nuclear
import similar to the RS domain of shuttling SR proteins, we next asked
whether U2AF is also exported from the nucleus to the cytoplasm. To
address this question, we have made use of an interspecies heterokaryon
assay (55, 56). The migration of human U2AF65 was monitored
in human/Drosophila heterokaryons produced by polyethylene glycol-induced fusion of HeLa and SL2 cells (Fig.
2). Heterokaryons were kept in culture
for 4-7 h in the presence or absence of the protein synthesis
inhibitor emetine. Specific identification of human U2AF65
was possible because the monoclonal antibody MC3 does not cross-react with Drosophila proteins (Fig. 2A; Ref. 43). As a
control, heterokaryons were double-labeled with a monoclonal antibody
specific for human hnRNP C, a protein that is always restricted to the
nucleus (56). In the absence of emetine, both human U2AF65
and hnRNP C proteins synthesized in the cytoplasm of heterokaryons progressively accumulated in the Drosophila nuclei (data not
shown). In contrast, when protein synthesis was inhibited, no human
hnRNP C protein could be detected in Drosophila nuclei,
whereas human U2AF65 was readily visible (Fig. 2,
B and C). From this we conclude that during the
course of the experiment human U2AF65 molecules migrated
from the HeLa nucleus to the cytoplasm of the heterokaryon and from
there they were imported into the Drosophila nucleus. Thus,
U2AF65 shuttles between nucleus and cytoplasm.
To determine whether the small subunit of U2AF also shuttles, we
performed a HeLa/3T3 cell heterokaryon assay. HeLa cells expressing
GFP-U2AF35 were fused with mouse 3T3 cells in the presence
of the protein synthesis inhibitor emetine. As depicted in Fig. 2,
D-F, GFP-U2AF35 migrates to the 3T3 nuclei,
whereas the nonshuttling protein hnRNP C remains exclusively detected
in the human nuclei. Thus, GFP-U2AF35 is exported from the
nucleus to the cytoplasm.
Given that nuclear pore complexes allow the diffusion of molecules up
to 60-70 kDa (57), the results obtained with interspecies heterokaryons could be due to passive leakage through the pores. To
investigate whether U2AF is exported by diffusion or requires a
receptor-mediated energy-driven mechanism, we performed a
temperature-shift assay, as described by Michael et al.
(58). The rational for the assay is that at 4 °C both
receptor-mediated nuclear import and export are blocked, whereas
diffusion is unaffected. As a positive control we fused GFP to a
classical NLS (the SV40 large T antigen NLS) and expressed it in HeLa
cells (Fig. 3). Because GFP is a small
protein (~27 kDa), it may diffuse across the pores, but due to the
presence of an NLS, it is actively imported. At physiological
temperature in the presence of a protein synthesis inhibitor, GFP-NLS
diffuses out of the nucleus. However, because it is rapidly
re-imported, the protein is exclusively detected in the nucleus (Fig.
3A). In contrast, at low temperature in the absence of
protein synthesis, GFP-NLS continues to leak out of the nucleus, and
because NLS-mediated import is blocked, the protein progressively
accumulates in the cytoplasm (Fig. 3B). Contrasting with the
results obtained with GFP-NLS, both U2AF65 and
GFP-U2AF35 are exclusively localized in the nucleus at
either 37 or 4 °C (Fig. 3, C-F). This implies that
transport of U2AF to the cytoplasm involves an
energy-dependent carrier-mediated pathway.
RNA Binding Is Dispensable for U2AF Shuttling--
Two
different mechanisms may account for the shuttling of an RNA-binding
protein. Either it is a passive cargo of the mature mRNAs in
transit to the cytoplasm, or it is actively exported through the
interaction with a transporter molecule. To investigate if the
migration of U2AF to the cytoplasm depends on RNA transport, HeLa cells
were incubated with the transcription inhibitor actinomycin D for
3 h before fusion to Drosophila SL2 cells. As shown in
Fig. 4, A-C, export of
U2AF65 continues under these conditions. A similar result
was observed when HeLa cells expressing GFP-U2AF35 were
incubated with actinomycin D for 3 h before fusion to mouse 3T3
cells (Fig. 4, D-F). Thus, nuclear export of both U2AF
subunits continues in the absence of RNA synthesis, suggesting that
mRNA traffic to the cytoplasm is dispensable for U2AF shuttling. A direct prediction from this model is that binding of U2AF to RNA is not
required for shuttling. To address this idea, we constructed a GFP
fusion U2AF65 RNA binding mutant (GFP-U2AF65
RRM1mut) (Fig. 5). To obtain a minimal
mutant U2AF65 with compromised RNA binding properties,
amino acid residues 195 (Lys), 197 (Phe), and 199 (Phe) were
substituted by asparagine (195), alanine (197), and alanine (199).
These residues were predicted to form part of the RNP-1 consensus
sequence of U2AF65 RRM1 (10), a prediction then confirmed
by NMR analysis (59). Mutation of these residues in RNP-1 is predicted
to have very detrimental effects in RNA binding by RRM1 (reviewed by
Ref. 60), which in turn makes critical contributions to the overall RNA binding affinity of U2AF65 (10). Consistent with this, Fig.
5G shows that RNA binding was impaired in this mutant when
compared with the wild type protein. Both the wild type construct,
GFP-U2AF65, and the mutant, GFP-U2AF65 RRM1mut,
were analyzed in heterokaryon assays (Fig. 5, A and B, D and E). As depicted in Fig. 5,
D and E, this mutant retains the ability to
shuttle. Incubation of cells at 4 °C confirms that neither
GFP-U2AF65 nor GFP-U2AF65RRM1 chimeras leak
passively to the cytoplasm (Fig. 5, C and F). A
similar result was obtained using HA-tagged U2AF65
RS Domain Requirements for Nucleocytoplasmic Transport of
U2AF--
The finding that the RS domain of shuttling SR splicing
factors is necessary for export of these proteins to the cytoplasm (30)
prompted us to investigate the RS domain requirements for nucleocytoplasmic transport of U2AF. GFP chimeras were constructed using either the RS domain alone (GFP-RS-U2AF65 and
GFP-RS-U2AF35), or deletion mutants spanning the RS domain
of both U2AF subunits (HA-U2AF65
Consistent with the view that U2AF is imported to the nucleus as a
heterodimer requiring only one RS domain, overexpression of either
U2AF65
In conclusion, these data support the view that the presence of an RS
domain in either U2AF subunit is sufficient to target the heterodimeric
complex into the nucleus. Given that the RS domain has been implicated
in both nuclear import and export of SR proteins, it is most likely
that the same model applies to export of U2AF from the nucleus to the cytoplasm.
In this work we show that the U2AF splicing factor shuttles
between the nucleus and the cytoplasm by a carrier-mediated pathway that is independent from mRNA traffic. Several other proteins associated with pre-mRNA processing exhibit shuttling activity, namely hnRNP A1, A2, D, E, I, and K proteins (56, 58, 61) and the SR
proteins ASF/SF2, SRp20, and 9G8 (37). Shuttling of hnRNPs most likely
contributes to export of mRNA to the cytoplasm (58, 62, 63),
whereas SR proteins appear to leave the nucleus as a cargo bound to the
mRNAs (64). In fact, the RS domain is necessary for export of SR
proteins, but unlike the M9 signal of hnRNP A1 (58), it is not capable
of promoting export of an unrelated protein (37). Rather, the RS domain
has to be coupled to a RNA binding domain to give rise to a shuttling
protein (37). Consistent with the view that SR proteins piggyback on
mRNA to exit the nucleus, several members of this family have been
shown to bind to exon sequences (18, 19) and to accompany the mRNA molecules in transit to the nuclear pores (65). The association of SR
proteins with spliced mRNA could therefore serve as a guide for
mature RNAs to cross the nuclear pores (66). Another possibility to be
considered is that SR proteins remain bound to the mRNAs in the
cytoplasm and play a role as regulators of mRNA stability or
translation. Since the transport of SR proteins into the nucleus is
mediated by specific import receptors (35, 36) and regulated by
phosphorylation and transcriptional activity (37, 30), shuttling could
at the same time provide a mechanism to control nuclear availability of
splicing factors in response to external signals. This would allow a
rapid tuning of splicing activity. Consistent with this view,
cytoplasmic accumulation of the shuttling protein hnRNP A1 has been
shown to occur in response to cellular stress and to correlate with
changes in alternative splicing (40).
In contrast with SR proteins, both U2AF subunits have been shown to
bind only to intronic sequences of pre-mRNA during the early steps
of spliceosome assembly (10-13) and to be later replaced by the U5
snRNP (67). Thus, it is unlikely that U2AF exits the nucleus as a
passive cargo bound to mature mRNA. In agreement with this view,
our data indicate that transport of U2AF to the cytoplasm does not
require binding to mRNA and continues in the absence of mRNA traffic.
What function could U2AF shuttling perform? Very recent data indicate
that U2AF35 interacts with the mRNA export factor TAP
and associates with spliced messenger RNP complexes in the nucleus,
suggesting that the factor participates in coupling of splicing with
export of mRNA to the
cytoplasm.2 Another
possibility is that U2AF plays an as yet undetermined role in the
cytoplasm. Recent observations suggest that the supply of U2AF is
regulated in the cell (68, 69). In this context, shuttling could also
represent a rapid means of controlling the level of U2AF available in
the nucleus for spliceosome assembly. A mechanism of U2AF regulation by
retention has been recently proposed by MacMorris et al.
(70). The Caenorhabditis elegans U2AF65
homologue gene gives rise to two alternatively spliced mRNAs, one
encoding the U2AF protein and another containing an exon with premature
stop codons. This alternative mRNA is retained in the nucleus and
contains 10 repeats of the 3' splice site consensus sequence. MacMorris
et al. (70) propose that U2AF is sequestered by these
repeats and consequently becomes unavailable for splicing.
A unique feature of the splicing factor U2AF is that it is
composed of two subunits that bind tightly to each other and form a
stable complex (9). Copurification of both subunits suggests that at
least 90% of the protein present in HeLa nuclear extracts is in a
heterodimeric complex (14). The interaction between the two subunits
has also been shown to be essential in vivo (71). The data
presented here indicate that deletions of the RS domain of either U2AF
subunit do not alter the distribution and shuttling capacity of the
protein complex. Similar results are obtained after deletion of any of
the other functional domains of U2AF65, suggesting that
both nuclear import and export of this protein complex are mediated by
redundant signals. Furthermore, when the two subunits cannot interact,
each RS domain becomes important for nuclear import. Based on these
results we propose that U2AF enters and leaves the nucleus as a
heterodimer, with the RS domain of each subunit serving as a redundant
nuclear import and export signal. In good agreement with this view,
deletion of the RS domain in either U2AF subunit is viable in
vivo, whereas deletion of the RS region in both subunits
simultaneously is lethal (72).
In summary, the work presented here reveals a novel property of the
general splicing factor U2AF, i.e. shuttling between the nucleus and the cytoplasm. The RS domain present in either of the two
U2AF subunits is involved in nucleo-cytoplasmic transport and
subnuclear targeting, similarly to what was previously established for
SR proteins. Possibly, higher eukaryotes have evolved a common pathway
to control the availability of RS-containing splicing factors in the
nucleus. Future studies are needed to determine how shuttling of these
factors may contribute to regulate splicing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-150 construct, containing U2AF65
amino acids 151 to 475, was obtained by polymerase chain reaction
amplification of pBluescript-
globin-U2AF65 (10) with the
following primers: primer 1, 5'-CC GGT ACC GAC ACC ATG TAC GAC TAC GAC
GTC CCT GAT TAT GCA CGC CTC TAC GTG GGC-3'; primer 2, 5'-GG ACG CGT CTA
CCA GAA GTC CCG GCG GTG-3' (Life Technologies, Inc.). Primer 1 contains
a KpnI restriction site and encodes an HA tag in-frame with
the U2AF65 sequence from nucleotide 504, corresponding to
the beginning of the RNA binding domain. Primer 2 contains an
MluI restriction site and a Stop codon. After treatment to
obtain phosphorylated blunt ends, the amplification product was
digested with KpnI and cloned in the pCMV5 expression vector
(47) digested with KpnI and SmaI. The ligated
product was amplified and sequenced.
globin-U2AF65 with NotI and
EcoRI and cloning in pEGFP-C2 digested with BglII and EcoRI. NotI and BglII ends were
filled in to generate blunt ends. GFP-RS-U2AF65, containing
U2AF65 amino acids 1 to 55, was obtained by digestion of
GFP-U2AF65 with SmaI and religation. The
U2AF65 RRM1 mutant was obtained by polymerase chain
reaction-based site-directed mutagenesis of pGEX-2T
GST-U2AF65
1-94 (42) as described (48). Mutations
introduced at the RNP-1 octamer sequence of the first RRM consisted of
substitution of lysine 195 by asparagine, phenylalanine 197 by alanine,
and phenylalanine 199 by alanine and were obtained by introducing the
following nucleotide changes: wild type sequence AAG AAT TTT GCC TTT
TTG GAG TTC; RRM1 mutant AAT AAT GCT GCC GCT TTG GAG TTC. GFP-U2AF65 RRM1mut was obtained by digesting both
GFP-U2AF65 and the pGEX-2T GST-U2AF65
1-94
RRM1mut with SgrAI (amino acid 136) and EcoRI and replacing the C-terminal region of the wild type GFP construct from amino acid
136 with the corresponding region of the mutant.
RS,
containing U2AF35 amino acids 1 to 197, was obtained by
digesting GFP-U2AF35 with SmaI and religation.
This mutation has been previously characterized (49).
GFP-RS-U2AF35, containing U2AF35 amino acids
185-238, was obtained by digesting pGEX-3X U2AF35 with
EaeI and EcoRI and cloning the isolated fragment
into pEGFP-C1 digested with HindIII and EcoRI.
The EaeI and HindIII ends were blunt-ended to
allow ligation. GFP-NLS (44) and HA-tagged U2AF65,
U2AF65
RS, U2AF65
RNP2,3, and
U2AF65
84-150 cDNAs (43) have been previously described.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The RS domain of U2AF is a nuclear
localization signal. HeLa cells were either immuno-labeled with
monoclonal antibody MC3 directed against human U2AF65
(A) or transfected with GFP-U2AF65
(B) and GFP-U2AF35 (C). Note that
both U2AF subunits are exclusively detected in the nucleus, excluding
the nucleoli; the proteins are diffuse in the nucleoplasm with
additional concentration in speckles (arrowheads).
Expression of GFP alone results in diffuse staining throughout the
nucleus and the cytoplasm (D) and does not alter the
accumulation of splicing factors in nuclear speckles, as shown by the
anti-SC35 monoclonal antibody (G). Chimeric proteins
resulting from fusion of GFP to the RS domain of either
U2AF65 or U2AF35 are exclusively detected in
the nucleus (E and F). Labeling with anti-SC35
monoclonal antibody shows that these proteins accumulate in nuclear
speckles (H and I, arrowheads). In
contrast with full-length proteins, these polypeptides are localized in
the nucleolus (E and F, arrows).
Bar, 10 µm. J, immunoblotting of transfected
HeLa cell extracts with an anti-GFP antibody shows that a single band
is observed for each GFP construct. Expected molecular masses are GFP
~27 kDa, GFP-U2AF35 ~62 kDa,
GFP-U2AF35 RS ~57 kDa, GFP-RS35 ~33 kDa,
GFP-U2AF65 and GFP-U2AF65 RRM1 mut ~92 kDa,
GFP-RS65 ~33 kDa. wt, wild type.
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Fig. 2.
U2AF65 and U2AF35
shuttle between nucleus and cytoplasm. HeLa cells were fused with
Drosophila SL2 cells to form heterokaryons. HeLa cells were
treated with emetine for 3 h before fusion. After fusion the cells
were kept in culture for 7 h in the presence of emetine.
Heterokaryons were fixed and immuno-labeled with either monoclonal MC3
directed against human U2AF65 (B) or monoclonal
4F4 directed against the nonshuttling hnRNP C protein (C).
The MC3 antibody does not react with SL2 nuclei (A).
Alternatively, HeLa cells expressing GFP-U2AF35 were fused
to murine 3T3 cells in the presence of emetine (D). At
4 h after fusion the heterokaryons were immuno-labeled for hnRNP C
protein (E). Panel F shows the same cells labeled
with the DNA stain DAPI; note that mouse cells are easily distinguished
by the presence of numerous heterochromatin clumps in the nucleoplasm
(arrows). Bar, 10 µm.
View larger version (60K):
[in a new window]
Fig. 3.
Temperature-sensitive nuclear export of
U2AF. HeLa cells were either transfected with GFP-NLS
(A and B), immuno-labeled with
anti-U2AF65 antibody (C and D), or
transfected with GFP-U2AF35 (E and
F). The cells were treated with a protein synthesis
inhibitor (20 µg/ml emetine) for 3 h and then incubated in
medium containing emetine for another 3 h at 37 or 4 °C, as
indicated. At 37 °C all proteins are exclusively detected in the
nucleus due to active import. At 4 °C, GFP-NLS was detected in the
cytoplasm because it passively diffused out of the nucleus while active
nuclear import was blocked; in contrast, U2AF65 and GFP-
U2AF35 remained restricted to the nucleus, indicating that
these proteins do not leak to the cytoplasm by passive diffusion.
Bar, 10 µm.
RNP2,3, a deletion mutant that covers the RNP consensus 2 and 3 of
U2AF, showing impaired RNA binding activity (data not shown). Taken
together, these results strongly suggest that U2AF shuttling is
independent from RNA binding.
View larger version (93K):
[in a new window]
Fig. 4.
U2AF shuttling is independent from ongoing
transcription. HeLa cells were co-cultured with
Drosophila SL2 cells and treated with inhibitors of protein
synthesis and transcription (20 µg/ml emetine and 5 µg/ml
actinomycin D) for 3 h (A-C). After polyethylene
glycol-induced fusion, the cells were incubated in medium containing
both inhibitors for another 7 h. The resulting heterokaryons were
double-labeled with anti-U2AF65 (A) and hnRNP C
antibodies (B). Panel C shows the corresponding
phase contrast image. HeLa cells expressing GFP-U2AF35 were
similarly treated with emetine and actinomycin D, fused to mouse 3T3
cells, and observed 4 h after fusion (D-F). As a
control, heterokaryons were immunolabeled with anti-hnRNP C antibodies
(E) and stained with DAPI (F). DAPI binds to DNA
and facilitates the identification of human and murine cells due to
different heterochromatin patterns. Bar, 10 µm.
View larger version (82K):
[in a new window]
Fig. 5.
RNA binding is dispensable for U2AF
shuttling. HeLa cells were transfected with either
GFP-U2AF65 (A-C) or GFP-U2AF65
RRM1mut (D-F). Heterokaryons were formed with murine 3T3
cells and incubated for 4 h in the presence of 20 µg/ml emetine
(A and D). As a control, the cells were
immunostained with anti-hnRNP C antibodies (B and
E). To exclude that nuclear export of U2AF65 is
due to diffusion, cells expressing these chimeras were treated with
emetine for 3 h and then incubated at 4 °C for another 4 h
(C and F). Bar, 10 µm. RNA binding
properties of U2AF65 wild type (WT) and mutant
RRM1mut proteins were analyzed in a gel retardation assay
(G). The apparent dissociation constant
(Kd) was estimated from the protein concentration
that results in retardation of 50% of the RNA. The results show that
although the wild type protein has an apparent Kd of
5 × 10 9, almost no binding was detected
at a 20-fold higher concentration for the RRM1mut protein. Titration
over a wider range of concentrations indicated that the difference in
apparent affinity between the two proteins was at least 100-fold.
Similar differences were observed with different uridine-rich RNA
substrates both in the presence and in the absence of the RS domain
(data not shown). Molar concentrations of protein in the different
lanes: lane 1, no protein; lanes 2 and
6, 10
7; lanes 3 and
7, 3 × 10
8; lanes
4 and 8, 10
9; lanes
5 and 9, 3 × 10
9.
RS and
GFP-U2AF35
RS). HeLa cells expressing
GFP-RS-U2AF65 or GFP-RS-U2AF35 were fused to
murine 3T3 cells in the presence of the protein synthesis inhibitor
emetine. At ~4 h after fusion, both chimeras were readily detected in
the mouse nuclei, confirming their ability to be exported from the HeLa
nuclei (Fig. 6, A and
B, D and E). However, when these cells
are treated with emetine and incubated at 4 °C, the two GFP fusion
proteins accumulate in the cytoplasm (Fig. 6, C and
F). Thus, the U2AF RS domains fail to retain the GFP
chimeras in the nucleus, and as a consequence of their small size, both
proteins diffuse passively through the pores to the cytoplasm. In
contrast, after deletion of the RS domain in either U2AF65
or U2AF35, the proteins remain in the nucleus at 4 °C
but are exported at 37 °C (Fig. 6, G-L). This implies
that, unlike the situation observed in SR proteins, in U2AF, the RS
domain is dispensable for nuclear export. However, because U2AF is a
heterodimer and the region of interaction between U2AF65
and U2AF35 does not overlap with the RS domain, there is
the possibility that the presence of this domain in only one of the two
subunits is sufficient to direct the nucleocytoplasmic shuttling of the complex. To address this idea, two additional deletion mutants of
U2AF65 were analyzed (Fig.
7). HA-U2AF65
84-150 has a
deletion of amino acids 84-150, spanning the region of interaction
with U2AF35 (43), and HA-U2AF65
1-150 has a
deletion of amino acids 1-150, spanning both the region of interaction
with U2AF35 and the RS domain. The
HA-U2AF65
84-150 protein is exclusively localized in the
nucleus and retains shuttling ability (Fig. 7A and data not
shown), whereas HA-U2AF65
1-150 shows significant
accumulation in the cytoplasm (Fig. 7B). Thus, when
U2AF65 cannot interact with U2AF35, its RS
domain becomes important for nuclear import.
View larger version (78K):
[in a new window]
Fig. 6.
The RS domain in each U2AF subunit is
sufficient for shuttling of the heterodimeric complex. HeLa cells
were transfected with GFP-RS-U2AF65 (A-C),
GFP-RS-U2AF35 (D-F), HA-U2AF65 RS
(G-I), and GFP-U2AF35
RS (J-L).
Heterokaryons were formed with murine 3T3 cells and incubated for
4 h in the presence of emetine. Heterokaryons were either stained
with anti-hnRNP C protein antibody (B, E, and
K) or DAPI (H). To determine whether the
expressed proteins could passively diffuse to the cytoplasm, cells were
treated with emetine for 3 h and then incubated at 4 °C for
another 4 h (C, F, I, and
L). Bar, 10 µm.
View larger version (46K):
[in a new window]
Fig. 7.
The RS domain is necessary for nuclear import
of U2AF65 in the absence of binding to
U2AF35. HeLa cells were transfected with
either HA-U2AF65 84-150 (A) or
HA-U2AF65
1-150 (B) and immunostained with
an anti-HA antibody. In contrast with HA-U2AF65
RS (Fig.
6G) and HA-U2AF65
84-150 (Fig.
7A), HA-U2AF65
1-150 accumulates in the
cytoplasm even at low expression levels. The structural organization of
U2AF65 is schematically illustrated at the bottom of the
figure. The protein contains an N-terminal RS-like domain (amino acids
23-65) followed by a region of interaction with U2AF35
(amino acids 84-150, hatched area) and three tandem
RNA-recognition motifs (RRM1, RRM2, RRM3, dark boxes).
Bar, 10 µm.
RS or U2AF35
RS results in
significant accumulation in the cytoplasm (Fig. 8, A and E). As
expected, these two deletion mutants become exclusively localized in
the nucleus of cells that simultaneously overexpress the wild type
forms of U2AF35 or U2AF65, respectively (Fig.
8, B and F). In contrast, overexpressed
U2AF65
RS remains in the cytoplasm of cells that
simultaneously overexpress either wild type U2AF65 (Fig.
8C) or the splicing factor SC35, which contains an RS domain but does not interact with U2AF65 (Fig. 8D).
Furthermore, overexpressed U2AF35
RS remains in the
cytoplasm of cells that simultaneously overexpress the deletion mutant
HA-U2AF65
84-150, which lacks the region of interaction
with U2AF35 (Fig. 8G) or U2AF65
RS
(Fig. 8H).
View larger version (96K):
[in a new window]
Fig. 8.
The RS domain in either U2AF subunit is
sufficient to target the heterodimeric complex to the
nucleus. A-D, HeLa cells were either
single-transfected with HA-U2AF65 RS (A,
red staining) or double-transfected with
HA-U2AF65
RS (B-D, red staining)
plus GFP-U2AF35 (B, green staining),
GFP-U2AF65 (C, green staining), or
GFP-SC35 (D, green staining). E-H,
cells were either single-transfected with GFP-U2AF35
RS
(E, green staining) or double-transfected with
GFP-U2AF35
RS (F-H, green
staining) plus HA-U2AF65 (F, red
staining), HA-U2AF65
84-150 (G,
red staining), or HA-U2AF65
RS (H,
red staining). Overlapping of red and green
staining produces a yellowish color. Note that all
cells depicted are overexpressing the cDNAs used for transfection
at levels that do not produce aberrant protein distribution patterns.
Bar, 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Thomas Blumenthal (University of Colorado) and Dr. Elisa Izaurralde (EMBL, Heidelberg, Germany) for stimulating discussions. We are grateful to Dr. Vladimir Benes and Wilhelm Ansorge (EMBL, Heidelberg, Germany) for help with sequencing. We also wish to acknowledge J. David-Ferreira (University of Lisbon, Portugal) for encouragement and Inês Condado (University of Lisbon, Portugal) for technical support. We are also grateful to Prof. M. R. Green (University of Massachusetts, Worcester, MA) for pGEX-3X U2AF35, Prof. G. Dreyfuss (University of Pennsylvania, Philadelphia, PA) for monoclonal antibody 4F4 (anti-hnRNP C), Prof. T. Maniatis (Harvard University, Massachusetts) for monoclonal antibody anti-SC35, and Prof. G. Akusjärvi (Uppsala University, Sweden) for GFP-SC35.
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FOOTNOTES |
---|
* This study was supported by a grant from Fundação para a Ciência e Tecnologia (Portugal) and European Union Grant BMH4-98-3147.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: Institute of Histology and Embryology, Faculty of Medicine, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal. Tel.: 351 21 7934340; Fax: 351 21 7951780; E-mail: m.gamacarvalho@fm.ul.pt.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M008759200
2 A. Zolotukhin and B. Felber, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: snRNP, small nuclear ribonucleoprotein; hnRNP, heterogeneous nuclear RNP; U2AF, U2 snRNP auxiliary factor; RRM, RNA recognition motif; NLS, nuclear localization signal; PBS, phosphate-buffered saline; HA, hemagglutinin; GFP, green fluorescent protein; DAPI, 4,6-diamidino-2-phenylindole; RS, arginine-serine.
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