* Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208; and Institute of Molecular Medicine, John
Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site
selection in a concentration-dependent manner. They
have a modular structure that consists of one or two
RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have
analyzed the role of the individual domains of these
closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In
contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that
the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although
this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of
the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two
SR proteins that have a single RRM (SC35 and SRp20),
the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2
of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5 splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.
NUMEROUS protein and ribonucleoprotein components are required to catalyze pre-mRNA splicing, which occurs within a macromolecular complex, the spliceosome. The major components of the
spliceosome, which assembles on individual pre-mRNAs,
are the small nuclear ribonucleoprotein particles (snRNPs)1
U1, U2, and U4/6·U5; the polypeptides that associate with
hnRNA to form hnRNP particles (hnRNP proteins); and a
large set of non-snRNP protein splicing factors, which includes the SR family of proteins (for reviews see Dreyfuss
et al., 1993 The SR proteins are essential splicing factors and also
regulate alternative splicing of many pre-mRNAs by affecting the selection of 5 Splicing factors are distributed nonrandomly within the
nucleus. Immunofluorescence localization studies showed
that snRNP particles and SR proteins are organized in the
interphase nucleus in a characteristic speckled pattern.
Two morphologically distinct nuclear structures have been
identified as constituents of the nuclear speckles by electron microscopy: interchromatin granule clusters and perichromatin fibrils (for review see Spector, 1993 The nuclear organization of splicing factors is dynamic,
as shown by the fact that inhibition of transcriptional and/
or splicing activity causes reorganization of the nuclear
speckles (Spector et al., 1983 Several protein kinases capable of phosphorylating SR
proteins on serine residues in vitro have been described:
SRPK1 (Gui et al., 1994a Previous studies showed that the RS domains of two
Drosophila splicing regulators, suppressor-of-white-apricot (SWAP) and Transformer (Tra), direct these splicing
factors to the nuclear speckles (Li and Bingham, 1991 Epitope-tagged Expression Plasmids
Oligonucleotide primers were purchased from GIBCO BRL (Gaithersburg, MD). PCR conditions using Vent polymerase (New England Biolabs, Beverly, MA) were as previously described (Krainer et al., 1991 For the nucleoplasmin fusions, the vector pCGT7-NPc was constructed
by amplifying a Xenopus nucleoplasmin cDNA (Dingwall et al., 1987 Cell Culture and Transfections
HeLa cells were grown in DME supplemented with 10% FCS and transfected with 1 µg of plasmid DNA per 60-mm dish of 60-75% confluent cells, in the presence of 20 µg lipofectin (GIBCO BRL) (Cáceres et al.,
1994 Indirect Immunofluorescence
Cells were fixed for immunofluorescence assays 24 h after transfection.
Cells were washed with PBS and incubated with 3% paraformaldehyde, 0.3% Triton (in PBS) for 5 min at room temperature, followed by incubation with 3% paraformaldehyde for 30 min. The fixed cells were incubated
for 1 h at room temperature with 1:500 anti-T7 monoclonal antibody
(Novagen, Inc.). The cells were washed three times with PBS and incubated for 1 h at room temperature with 1:500 fluorescein-conjugated goat
anti-mouse IgG (Cappel Laboratories, Malvern, PA). Double immunofluorescence labeling was performed by incubation for 1 h at room temperature with 1:500 anti-T7 monoclonal antibody (Novagen, Inc.) and 1:
2,000 human anti-Sm serum. After washing, the cells were incubated with
1:50 Texas red-conjugated antimouse IgG and 1:50 fluorescein-conjugated antihuman IgG (Cappel Laboratories). Samples were observed on a
microscope (model Axiovert 405M; Carl Zeiss, Inc., Thornwood, NY),
and images were acquired with a cooled CCD camera (model NU200;
Photometrics, Inc., Woburn, MA) using Oncor Image software (Gaithersburg, MD). For confocal microscopy, a confocal laser scanning microscope (model LSM410; Carl Zeiss, Inc.) was used. For localization of the
endogenous SF2/ASF protein, a monoclonal antibody against SF2/ASF
was used as culture supernatant at a 1:5 dilution.
Quantitation of Fluorescence Images
Images were acquired under identical conditions, ensuring that the maximal signal was not saturating, and were subjected to contrast stretching,
excluding the top and bottom 1% of pixels. Acquisition of images and
measurements were performed using Oncor Image software 2.0.04. To
measure the intensity of speckles, a modified version of a point-hit
method was used (Weibel et al., 1969 N represents the number of pairs of test points used for one sample. Typically, four to six pairs of test points were chosen arbitrarily on a test line,
and a minimum of three test lines were used for every cell nucleus examined. For each sample, at least four nuclei were examined. All results represent values obtained from two separate experiments, and the results are
average values ± standard deviation from the pooled data.
In Vivo Analysis of Alternative Splicing
Transfections of HeLa cells and purification of total RNA were as previously described (Cáceres et al., 1994 Role of the Structural Domains of SR Proteins in
Cellular Distribution and Subnuclear Localization
First, we analyzed the localization of the endogenous SF2/
ASF protein using an anti-SF2/ASF monoclonal antibody
(Krainer, A.R., unpublished). This antibody does not cross-
react with other SR proteins and detects all phosphorylation variants of SF2/ASF; its detailed characterization will
be published elsewhere. We observed a typical nuclear
speckled pattern and also a diffuse nuclear signal, but no
nucleolar staining (Fig. 1). This immunofluorescence pattern resembles that of endogenous U2B
To determine the role of individual domains of SF2/ASF
and other SR proteins in nuclear and subnuclear localization, we transiently overexpressed in HeLa cells several
epitope-tagged SR protein cDNAs encoding either the
wild-type proteins or several mutant derivatives and determined the cellular distribution and subnuclear localization
of the tagged proteins by indirect immunofluorescence microscopy. We verified the expression of all the transfected cDNAs by Western blot analysis of whole-cell lysates. All
constructs encoded proteins with a bacteriophage T7 gene
10 (T7) epitope tag at their NH2 termini (see Materials
and Methods), allowing detection of the exogenous proteins with a monoclonal antibody that recognizes this
epitope. All expressed proteins accumulated to similar levels in transfected HeLa cells and appeared to be full
length; in most cases, doublets were detected, which may represent different states of phosphorylation (data not
shown). In the case of SF2/ASF wild-type and mutant proteins, the use of an anti-SF2/ASF monoclonal antibody allowed us to compare the levels of exogenous and endogenous SF2/ASF in the cell population. After normalizing
for transfection efficiency, we estimate that the transiently
expressed proteins accumulate in the transfected cells at
5-10-fold higher levels than endogenous SF2/ASF, which is in good agreement with our previous results (Cáceres et
al., 1994 Indirect immunofluorescence using the T7 tag antibody
showed that 24 h after transfection, the transiently expressed wild-type SF2/ASF protein localized exclusively in
the nucleus, with a typical speckled pattern (Fig. 2 a). 48 h
after transfection, SF2/ASF overexpression resulted in the
formation of nuclear aggregates: the speckles were fewer
in number and appeared enlarged (data not shown), in
agreement with previous reports (Hedley et al., 1995
The localization of several transiently expressed SF2/
ASF domain-deletion mutants was then compared to that
of the wild-type protein (SF2-WT). The structures of the
mutant proteins, designated The transiently expressed SF2-WT and SF2-
These experiments demonstrate that all three structural
domains of SF2/ASF are required for exclusive nuclear localization since deletion of the RS domain, or of either
RRM, resulted in proteins with both nuclear and cytoplasmic distribution. However, there appears to be functional
redundancy in the speckle localization signals within SF2/
ASF. At least two domains are necessary for SF2/ASF localization to nuclear speckles: either two copies of the
RRM or one of the two RRMs together with the RS domain.
Next, we analyzed the effect of deleting the RS domain
from another SR protein that has two RRMs (SRp40), and
from two SR proteins that have a single RRM (SC35 and
SRp20). To this end, these epitope-tagged proteins (either
wild-type proteins or mutant proteins lacking the RS domain) were transiently expressed in HeLa cells, and their
cellular localization was analyzed by indirect immunofluorescence. Wild-type SRp40 localized exclusively to the nucleus and showed a typical speckled pattern (Fig. 4 a). Deletion of its RS domain resulted in localization both in the
nucleus (excluding the nucleoli) and in the cytoplasm;
however, nuclear speckles were clearly detected (Fig. 4 b),
as was the case with SF2/ASF lacking its RS domain. Both
SC35 and SRp20 wild-type proteins also localized exclusively to the nucleus and displayed a typical nuclear speckled pattern (Fig. 4, c and e, respectively). In contrast, SC35
The finding that the RS domain of SF2/ASF is not required for speckle localization was unexpected since previous work showed that the RS domains of Drosophila Tra
and SWAP (which have no RRMs) are necessary and sufficient for targeting to speckles (Li and Bingham, 1991 Although the RS domain of SF2/ASF is not required to
target SF2/ASF to the speckles, we tested whether it can
target a different protein to this subnuclear domain. We
assayed the subnuclear localization of a chimeric protein,
A1-RS, which consists of both RRMs from hnRNP A1 and
the RS domain from SF2/ASF replacing the natural COOH-terminal glycine-rich domain (G-domain) of hnRNP
A1 (Mayeda et al., 1994
Next, we compared the nuclear localization signals as
well as the subnuclear targeting properties of both types of
RS domain, i.e., those present in SR proteins with either
one or two copies of the RRM. To this end, we constructed chimeric proteins in which the RS domain of either SF2/ASF or SRp20 was fused to a protein reporter,
the nucleoplasmin core domain. This protein fragment by
itself displays a cytoplasmic distribution (Fig. 6 B, a) since it lacks the nuclear localization signal present in wild-type nucleoplasmin (Dingwall et al., 1987 Role of SF2/ASF Domains in Alternative Splicing
Regulation In Vivo
In addition to their function as general splicing factors, SR
proteins regulate alternative splicing in a concentration-dependent manner (Ge and Manley, 1990
Both constructs with a single RRM and the RS domain
(RRM1/RS and RRM2/RS) were also active in regulating
alternative splicing of a Taken together, the cotransfection results suggest that
RRM2 of SF2/ASF plays a critical role in the specificity of
alternative splicing with certain substrates, such as the
E1A pre-mRNA. Thus, all the functional SR protein constructs that include RRM2 (SF2/ASF WT,
We have studied the role of individual domains of SR proteins in cellular distribution and subnuclear localization, as
well as in alternative splicing activity in vivo. Unexpected
differences were uncovered in the localization pathways
for different SR proteins, despite the close conservation of
structure and biochemical properties among members of
this protein family. SR proteins with a single RRM require
an RS domain for proper localization in the nuclear speckles, whereas SR proteins with two RRMs do not; in the latter, weak speckle targeting signals are present in each of
the three constituent domains and function additively. The
fact that single-domain deletion variants of SF2/ASF localized properly made it possible to determine the role of
each domain in alternative splicing in vivo. Each of the
three domains could be deleted individually without abrogating the ability of SF2/ASF to modulate alternative splice site selection. Remarkably, however, deletion of
RRM2 imparted a distinct activity on the protein, such
that it promoted the selection of a different alternative 5 Nuclear Localization of SR Proteins
The SR protein SF2/ASF has a modular structure, consisting of two RRMs and a COOH-terminal RS domain. Here
we showed that each of these three domains contributes
additively to the nuclear localization of the protein since
deletion of individual domains results in proteins that, unlike the wild-type protein, no longer have an exclusively
nuclear distribution (Fig. 9). In particular, the RS domain
of SF2/ASF contributes to its nuclear localization since deletion of this domain causes nuclear and cytoplasmic distribution of the resulting mutant protein. In agreement
with this finding, when the RS domain of SF2/ASF was
fused to the NPc protein reporter (which on its own localizes in the cytoplasm), the fusion protein (NPc-RSSF2) localized in the nucleus (Table I). Thus, the SF2/ASF RS domain is a nuclear targeting signal.
Table I.
Summary of Localization Of Chimeric Proteins
; Fu, 1995
; Krämer, 1996
; Manley and Tacke,
1996
).
splice sites (Ge and Manley,
1990
; Krainer et al., 1990a
,b; Zahler et al., 1993
; for review
see Horowitz and Krainer, 1994
). Their activity in alternative splicing is antagonized by members of the hnRNP A/
B family of proteins (Mayeda and Krainer, 1992
; Mayeda
et al., 1994
). Thus, the counteracting activities of these two
families of antagonistic factors can regulate alternative splicing, both in vitro and in vivo (Mayeda and Krainer,
1992
; Cáceres et al., 1994
; Yang et al., 1994
). SR proteins
are characterized by the presence of one or two copies of
the RNA-recognition motif (RRM) and a COOH-terminal
domain rich in arginine and serine residues (RS domain)
(Zahler et al., 1992
; for review see Birney et al., 1993
).
These proteins are thus very closely related in their domain structure, primary sequence, and functional properties. Individual SR proteins interact with pre-mRNA during the early stages of spliceosome assembly (Krainer et
al., 1990b
; Fu, 1993
; Staknis and Reed, 1994
; Zahler and
Roth, 1995
) and stimulate binding of U1 snRNP particles
to the 5
splice site (Eperon et al., 1993
; Kohtz et al., 1994
)
and binding of U2AF to the 3
splice site (Zuo and Maniatis, 1996
).
). The
speckled pattern comprises 20-50 regions that are highly
concentrated in splicing factors (Spector, 1990
; Huang and
Spector, 1992
). Immunofluorescence staining and immunoelectron microscopy studies showed that the splicing
factor SC35, a member of the SR family of proteins, colocalizes with snRNPs within the nuclear speckle domains
(Fu and Maniatis, 1990
; Spector et al., 1991
). In some cell
types, snRNPs can be additionally found in another discrete nuclear structure, the coiled body (for review see Lamond and Carmo-Fonseca, 1993
).
; O'Keefe et al., 1994
). The
most intense speckled regions are adjacent to, but not coincident with, transcriptionally active sites, as defined by
fluorescent labeling of nascent RNAs (Wansink et al.,
1993
) and by in situ hybridization (Huang and Spector,
1991
; Xing et al., 1993
, 1995
). Likewise, the interchromatin granule clusters are not sites of tritiated uridine incorporation (for review see Fakan and Puvion, 1980
). The fact that
splicing does not appear to take place at the majority of
sites where splicing factors are most concentrated (Mattaj,
1994
; Zhang et al., 1994
; Huang and Spector, 1996
) led to
the suggestion that splicing factors shuttle between interchromatin granule clusters (sites of storage and/or reassembly) and perichromatin fibrils (sites of active transcription and splicing) (Jiménez-García and Spector, 1993
). Many introns, though not all, are spliced as nascent transcripts in vivo (Beyer and Osheim, 1988
; Bauren and
Wieslander, 1994
; Kiseleva et al., 1994
). An intimate connection between transcription and splicing is further suggested by the observation that a subpopulation of hyperphosphorylated RNA polymerase II localizes to the nuclear speckle domains (Bregman et al., 1995
; Mortillaro et al.,
1996
). The hyperphosphorylated COOH-terminal domain
of the polymerase large subunit appears to mediate interactions with components related to splicing (Mortillaro et
al., 1996
; Yuryev et al., 1996
; Du and Warren, 1997
; McCracken et al., 1997
).
,b), Clk/Sty (Colwill et al., 1996
),
a lamin-B receptor-associated kinase (Nikolakaki et al.,
1996
), and unexpectedly, DNA topoisomerase I (Rossi et
al., 1996
). In addition, it has been shown that addition of
SRPK1 to permeabilized cells (Gui et al., 1994a
) or overexpression of Clk/Sty (Colwill et al., 1996
) results in a diffuse distribution of splicing factors, probably because of
hyperphosphorylation of their RS domains. It has been
proposed that the level of phosphorylation may control
the subnuclear distribution of SR proteins in interphase cells and the reorganization of the speckle domains during
mitosis (Colwill et al., 1996
; Gui et al., 1994a
; Misteli and
Spector, 1996
).
;
Hedley et al., 1995
). We sought to investigate the role of
the structural domains of SR proteins in cellular and subnuclear localization and to study the requirement for individual domains of these proteins for modulation of alternative splicing in vivo. To this end, we transiently overexpressed
in HeLa cells several epitope-tagged SR protein cDNAs
encoding either the wild-type proteins or several mutant
derivatives thereof. We then determined the cellular distribution of the tagged proteins, as well as their activity in
regulating alternative splicing of transcripts expressed from
cotransfected reporter genes. We found that SR proteins
that have either one or two RRMs differ in their requirements for the RS domain as a nuclear speckle targeting signal. We also demonstrate that the presence of RRM2
can affect the alternative splicing specificity of SF2/ASF in
vivo, suggesting that the modular structure of SR proteins
is important for regulated splicing.
Materials and Methods
).
The epitope-tagged, full-length SF2/ASF expression plasmid was constructed by amplifying an SF2/ASF cDNA (Krainer et al., 1991
) with specific primers and subcloning of the resulting PCR product as an XbaI-BamHI fragment into the pCGTHCFFLT7 expression vector (Wilson et
al., 1995
). The resulting vector, pCGT7-SF2, like the previously described
pCG-SF2 vector, is under the control of the cytomegalovirus enhancer/
promoter (Tanaka and Herr, 1990
; Cáceres et al., 1994
) but also includes
an NH2-terminal epitope tag, MASMTGGQQMG. This epitope tag corresponds to the first 11 residues of the bacteriophage T7 gene 10 capsid
protein and is recognized by the T7 tag monoclonal antibody (Novagen,
Inc., Madison, WI). The SF2/ASF mutants and domain deletions were
previously described (Cáceres and Krainer, 1993
) and were subcloned
into the same epitope-tagged expression vector as wild-type SF2/ASF. For
the SRp40, SC35, SRp20, and hnRNP A1 constructs, PCR products were
amplified with specific primers and subcloned into the same expression
vector. In the case of the SRp20 constructs, because of the presence of an
internal XbaI site in the SRp20 cDNA, the amplified fragments were designed with SpeI and BamHI sites and were subcloned into the XbaI-BamHI sites of pCGT7-SF2. In the case of the SRp40 constructs, because
of the presence of an internal BamHI site in the SRp40 cDNA, the amplified fragments were designed with XbaI and BclI sites and were subcloned
into the XbaI-BamHI sites of pCGT7-SF2. The SRp40-
RS protein comprises residues 2-183; SC35-
RS comprises residues 2-94; and SRp20-
RS comprises residues 2-85. Construction of the A1-RS protein was previously described (Mayeda et al., 1994
); this chimeric protein comprises
amino acids 1-185 from hnRNP A1 and 195-248 from SF2/ASF.
)
with specific primers and cloning the resulting PCR product into the XbaI
and BamHI sites of the pCGTHCFFLT7 expression vector. This procedure
results in the insertion of amino acids 2-149 of nucleoplasmin followed by
an XbaI site and stop codon, COOH-terminal to the T7 epitope. This nucleoplasmin fragment is also known as the nucleoplasmin core domain,
NPc. PCR fragments comprising residues 198-248 from the RS domain of
SF2/ASF or 88-164 from the RS domain of SRp20 were subcloned downstream of NPc to generate NPc-RSSF2 and NPc-RSSRp20, respectively.
).
). Random test lines were drawn
over the cell nucleus, several pairs of random test points on the test line
were chosen arbitrarily, and the absolute intensity value of the test points
was measured (X1, X2). A value S for the intensity of speckles was determined as the ratio between the sum of differences and the average intensity value of all points on the test line (Xav):
). Briefly, 1 µg of expression plasmid
was cotransfected into HeLa cells with 6 µg of the adenovirus E1A reporter plasmid pMTE1A (Zerler et al., 1986
) in the presence of 20 µg of
Lipofectin (GIBCO BRL). The cells were grown to 60-75% confluence in
60-mm dishes and harvested 24 or 48 h after transfection, and 200 ng of total RNA was analyzed by reverse transcriptase PCR (RT-PCR), as described (Screaton et al., 1995
). Briefly, first-strand oligo(dT)-primed cDNA synthesized with superscript II (GIBCO BRL) from 200 ng of total
RNA was amplified with Taq DNA polymerase (Perkin-Elmer Corp.,
Norwalk, CT) for 25 cycles, using 5
-end-labeled forward primer. Amplified products separated by urea-PAGE were detected by autoradiography
and quantitated by PhosphorImage analysis (Fujix, BAS2000; Fuji Photo
Film Co., Tokyo, Japan). The PCR primers were previously described
(Cáceres et al., 1994
).
Results
and Sm snRNP
proteins (for review see Spector, 1993
). No staining of
coiled bodies was observed, in agreement with previous
data on SC35 localization (Spector et al., 1991
).
Fig. 1.
Localization of endogenous SF2/ASF. HeLa cells were
fixed, and the cellular localization of SF2/ASF was determined by
indirect immunofluorescence with an anti-SF2/ASF monoclonal
antibody (Krainer, A.R., unpublished). The protein localizes in a
speckled nuclear pattern and is also diffusely distributed throughout the nucleoplasm. (Top) DIC image; (bottom) fluorescence
image of the same field. Bar, 10 µm.
[View Larger Version of this Image (111K GIF file)]
; and data not shown).
; Romac and Keene, 1995
). The remaining localization data were obtained at the shorter time point, when the nuclear
staining pattern of the transiently expressed wild-type protein coincides with that of the endogenous protein in untreated cells. This concordance suggests that transient expression is a valid approach to study the localization
signals of SR proteins. Although the transfected proteins
are expressed at higher levels, we note that the natural abundance of individual SR proteins fluctuates considerably in different cell types and that modulation of the levels of these proteins appears to be the key to their role in
alternative splicing (Ge and Manley, 1990
; Krainer et al.,
1990a
; Zahler et al., 1993
; Cáceres et al., 1994
).
Fig. 2.
Role of SF2/ASF
structural domains in cellular
localization and distribution
in the nuclear speckles. The
structure of the SF2/ASF domain-deletion mutants was
previously described (Cáceres and Krainer, 1993). (a)
SF2-WT, (b) SF2-
RS, (c)
RRM1/RS, (d) RRM2/RS,
(e) RRM1, (f) RRM2. HeLa
cells were transfected with plasmids encoding the respective T7-tagged proteins
and fixed 24 h after transfection. The localization of the
expressed proteins was determined by indirect immunofluorescence with anti-T7
monoclonal antibody and
FITC-conjugated secondary
antibody. Bar, 5 µm.
[View Larger Version of this Image (123K GIF file)]
RS, RRM1/RS, RRM2/RS,
RRM1, and RRM2, have been previously described (Cáceres and Krainer, 1993
). (RRM2 has sometimes been
referred to as pseudo-RRM,
RRM, or RRM-homology,
because of its lack of canonical RNP-1 and RNP-2 submotifs; see Birney et al., 1993
.) In contrast to wild-type SF2/
ASF, the
RS mutant protein localized mostly in the nucleus (excluding the nucleoli) but also in the cytoplasm;
unexpectedly, nuclear speckles were still clearly detectable (Fig. 2 b). This observation demonstrates that the RS domain of SF2/ASF, although required for exclusive nuclear
localization, is not essential for subnuclear localization to
the speckles. The RRM1/RS and RRM2/RS mutant proteins displayed a similar cellular distribution to that observed with the
RS mutant protein, and they also localized to speckles (Fig. 2, c and d). In contrast, when
individual domains were expressed (RRM1 or RRM2),
the mutant proteins localized throughout the cell, and no
nuclear speckles were detected (Fig. 2, e and f). Expression of just the epitope-tagged RS domain from SF2/ASF
was very inefficient and could not be detected by immunofluorescence (data not shown).
RS proteins colocalized with snRNP particles in the endogenous
speckles, as shown by confocal laser scanning microscopy
using the anti-T7 tag mouse monoclonal antibody (red)
(Fig. 3, a and d) and a human autoimmune serum specific
for the Sm core proteins of snRNP particles (green) (Fig.
3, b and e). Colocalization of SF2/ASF proteins and snRNPs results in a yellow color (Fig. 3, c and f). We conclude that transiently expressed SF2-WT and a fraction of
SF2-
RS proteins localize properly to nuclear speckles
containing endogenous snRNPs.
Fig. 3.
Colocalization of transiently expressed SF2/ASF, with or without its RS domain, with endogenous snRNPs. HeLa cells were
transiently transfected with plasmids expressing T7-tagged, wild-type SF2/ASF (SF2-WT; top row) or a derivative lacking the RS domain (SF2-RS; bottom row). The cells were fixed 24 h after transfection and analyzed by double-label immunofluorescence using laser
scanning confocal microscopy. SF2/ASF or SF2-
RS were detected using an anti-T7 mouse monoclonal antibody followed by Texas
red-conjugated secondary antibody (a and d). Endogenous snRNPs were detected in the same cell using an anti-Sm human serum followed by FITC-conjugated secondary antibody (b and e). Both SF2-WT and SF2-
RS colocalized with endogenous snRNPs in nuclear
speckles, as shown by the yellow color when the two signals were superimposed (c and f). Bar, 5 µm.
[View Larger Version of this Image (44K GIF file)]
RS and SRp20
RS localized throughout the cytoplasm and nucleoplasm, and no nuclear speckles were detected (Fig. 4, d and f); this cellular distribution is very similar
to that observed with the RRM1 and RRM2 single-domain
mutants of SF2/ASF (Fig. 2). These observations indicate
a significant difference in the localization signals present
in two SR proteins that have two copies of the RRM (SF2/
ASF and SRp40), and two SR proteins that have only one RRM (SC35 and SRp20), since only in the latter case is the
RS domain absolutely required for localization to the speckle
domains. Quantitation of the fluorescence signals confirmed these qualitative observations. The relative intensity of nuclear speckles was reduced by 30% in constructs
lacking one of the three domains and further reduced by
80% in those constructs lacking two domains. Loss of one
domain from two-domain SR proteins had the same effect
as loss of two domains from a three-domain SR protein.
The relative intensity values were similar for wild-type
SRp40, SC35, and SRp20 as compared to SF2/ASF, and
were similarly decreased for SC35-
RS and for SRp20-
RS, as compared to SF2/ASF RRM1 or RRM2 mutants (Fig. 5).
Fig. 4.
Cellular localization of other transiently expressed SR proteins with or
without their RS domains.
HeLa cells were transfected with plasmids encoding the
following epitope-tagged
proteins: (a) wild-type SRp40,
(b) SRp40-RS, (c) wild-type SC35, (d) SC35-
RS, (e) wild-type SRp20, and (f)
SRp20-
RS. The cells were
fixed 24 h after transfection
and the localization of the expressed proteins was determined as in Fig. 1. Bar, 5 µm.
[View Larger Version of this Image (103K GIF file)]
Fig. 5.
Quantitative analysis of localization of transfected proteins in nuclear speckles. The relative fluorescence intensity was calculated as described in Materials and Methods. Note that the relative intensity of speckles decreased by ~30% for SF2/ASF
lacking one of its three domains, and by ~80% when two of the
three domains were missing. Results represent average values ± standard deviation from the pooled data of at least two experiments.
[View Larger Version of this Image (51K GIF file)]
;
Hedley et al., 1995
).
). This domain of hnRNP A1 contains a short signal for bidirectional transport between the
nucleus and the cytoplasm (Michael et al., 1995
; Siomi and
Dreyfuss, 1995
; Weighardt et al., 1995
). The hnRNP A1
wild-type protein gave a diffuse nucleoplasmic staining
rather than a speckled pattern (Fig. 6 A, a), in agreement with published data (for review see Piñol-Roma and Dreyfuss, 1993
). Replacing the G-domain of hnRNP A1 by the
RS domain of SF2/ASF did not target the chimeric protein
to the nuclear speckles (Fig. 6 A, b), demonstrating that, at
least in this context, the RS domain of SF2/ASF is not sufficient to direct a heterologous protein to the nuclear
speckles. On the other hand, the A1-RS protein localized
exclusively in the nucleus, whereas hnRNP A1 lacking a
COOH-terminal domain is known to localize throughout the cell (Siomi and Dreyfuss, 1995
). Therefore, the RS domain of SF2/ASF is a nuclear localization signal, but not a
subnuclear speckle localization signal.
Fig. 6.
Cellular localization of transiently expressed
chimeric proteins. HeLa cells
were transfected with plasmids encoding the following epitope-tagged proteins: (A)
(a) hnRNP A1; (b) A1-RS, a
chimeric protein with both
RRMs from hnRNP A1
fused to the COOH-terminal RS domain of SF2/ASF.
(B) (a) NPc, the nucleoplasmin core domain; (b) NPc-RSSF2, a chimera consisting
of NPc fused to the RS domain from SF2/ASF; (c)
NPc-RSSRp20, NPc fused to
the RS domain of SRp20. The cells were fixed 24 h after transfection and the localization of the expressed proteins was determined as in
Fig. 1. (C) Colocalization of
transiently expressed NPc-RSSRp20 with endogenous snRNPs. HeLa cells were transiently transfected with plasmid expressing T7-tagged NPc-RSSRp20. The cells were
fixed 24 h after transfection
and analyzed by double-label
immunofluorescence using
confocal laser scanning microscopy. NPc-RSSRp20 was
detected using an anti-T7 mouse monoclonal antibody followed by Texas red-conjugated secondary antibody (a). Endogenous snRNPs were detected in the same
cell using an anti-Sm human serum followed by FITC-conjugated secondary antibody (b). NPc-RSSRp20 colocalized with endogenous snRNPs in nuclear speckles, as shown by the yellow color when the two signals were superimposed (c). Bar, 5 µm.
[View Larger Versions of these Images (28 + 34 + 53K GIF file)]
). Both types of RS
domain act as nuclear localization signals, as demonstrated
by the fact that both NPc-RSSF2 and NPc-RSSRp20 localized
to the nucleus (Fig. 6 B, b and c). However, a clear difference was evident in the ability of these RS domains to target a protein reporter to the nuclear speckles. Whereas a
diffuse nuclear staining was observed with the SF2/ASF
RS domain (Fig. 6 B, b), the RS domain of SRp20 was sufficient to direct the NPc reporter to the nuclear speckles (Fig. 6 B, c). The nuclear speckles observed with the NPc
reporter fused to the RS domain of SRp20 colocalized
with endogenous human Sm (Fig. 6 C), as shown by confocal laser microscopy using the mouse anti-T7 tag monoclonal antibody (red) (Fig. 6 C, a) and human autoimmune
serum specific for the Sm core proteins of snRNP particles
(green) (Fig. 6 C, b). Colocalization of NPc-RSSRp20 protein and snRNPs results in a yellow color (Fig. 6 C, c). We conclude that the transiently expressed NPc-RSSRp20 protein localizes properly in nuclear speckles containing endogenous snRNPs, demonstrating that this RS domain is
sufficient to determine subnuclear localization in the
speckle domain. These experiments demonstrate the existence of two distinct pathways for localization of different
SR proteins to the nuclear speckles and also show the
unique properties of individual RS domains.
; Krainer et al.,
1990a
; Zahler et al., 1993
). We sought to investigate the
role of individual domains of SF2/ASF in the regulation of
alternative splicing in vivo. To this end, we overexpressed
several of the constructs described above and assayed
changes in the patterns of alternative splicing of an adenovirus E1A splicing reporter. It was previously shown that
SF2/ASF strongly activates the use of the proximal 13S
E1A 5
splice site in vivo (Cáceres et al., 1994
; Wang and
Manley, 1995
) and that overexpression of hnRNP A1 results in activation of the distal 9S 5
splice site (Cáceres et
al., 1994
; Yang et al., 1994
). Compared to wild-type SF2/
ASF, which gave the expected activation of the 13S 5
splice site (Fig. 7 B, lane 2), the
RS protein led to only
slightly lower relative levels of 13S mRNA (Fig. 7 B, lane
3), demonstrating that the RS domain of SF2/ASF is not
required for alternative splicing modulation in vivo. This result is consistent with previous findings (Wang and Manley, 1995
), except that in the earlier study the 9S mRNA
levels remained constant for both wild-type and
RS proteins, whereas in our hands they decreased, as expected
from in vitro studies. The SF2/ASF mutants with a single
RRM and the RS domain had strikingly different functional properties: both proteins influenced alternative
splicing, but whereas RRM2/RS strongly activated the 13S
5
splice site (Fig. 7 B, lane 5), RRM1/RS strongly and reproducibly stimulated the 12S 5
splice site (Fig. 7 B, lane
4). The unexpected altered specificity observed upon deletion of RRM2 demonstrates that the nature of the RRM
can influence the selection of a particular 5
splice site. Activation of the E1A 12S 5
splice site by the RRM1/RS mutant is similar to the effect of SRp20, which naturally lacks
RRM2 (Fig. 7 B, lane 6; Screaton et al., 1995
). Quantitation of the relative use of the E1A 5
splice sites upon overexpression of the different proteins is shown in Fig. 7 C.
Fig. 7.
Role of SF2/ASF
structural domains in regulating alternative splicing of
adenovirus E1A pre-mRNA.
(A) Diagram of the E1A reporter gene. The alternative
5 splice sites and splicing
events that generate 13S,
12S, and 9S mRNAs are
shown schematically. The location of the exon primers
used for RT-PCR analysis is
shown. (B) Alternative splicing activity of the SF2/ASF
domain-deletion mutants.
Each of the indicated SF2/
ASF mutant and control proteins was overexpressed from
plasmids cotransfected with
the E1A reporter gene. RNA
was harvested 24 h after
transfection and analyzed by
RT-PCR with a labeled forward primer, denaturing
PAGE, and autoradiography, as described in Materials and Methods. The positions of the unspliced pre-mRNA, and of 13S, 12S, and
9S spliced mRNAs are indicated on the right. The background products obtained from mock-transfected cells are shown in lane 7. End-labeled DNA size markers are shown in lane M. (C)
Quantitation of E1A mRNA isoforms in transfected cells. The relative amounts of 13S, 12S, and 9S E1A mRNAs were calculated from
the data in B, using a PhosphorImager, and the percentage of each isoform is shown.
[View Larger Version of this Image (37K GIF file)]
-thalassemia splicing reporter,
leading in both cases to activation of the most proximal
cryptic 5
splice site (data not shown); this is a similar pattern as that obtained with wild-type SF2/ASF (Cáceres et
al., 1994
). We and others previously reported that deletion
of RRM1 or RRM2 in SF2/ASF abolished the alternative splicing activity of this protein in vitro (Cáceres and
Krainer, 1993
; Zuo and Manley, 1993
). This apparent discrepancy with our present results may reflect incorrect
folding of the mutant proteins in vitro, but not in vivo,
and/or the different pre-mRNA substrates used in the two
types of assays. Wang and Manley (1995)
reported that
small deletions within RRM2 inactivated the protein in
vivo; since we find that deletion of the entire domain imparts a novel specificity on the protein in a similar assay, it
is likely that the small deletions disrupted the folding of
the RRM, which has a highly conserved tertiary structure
(for review see Birney et al., 1993
).
RS, and
RRM2/RS) favored the 13S 5
splice site, whereas those
that lack a second RRM (RRM1/RS and SRp20) selected
the 12S 5
splice site (Fig. 8). We conclude that there is a
different functional requirement for individual domains of
SR proteins in the regulation of alternative splicing in living cells. Whereas the RS domain is dispensable for this
activity, as was previously demonstrated in vitro (Cáceres
and Krainer, 1993
; Zuo and Manley, 1993
), the presence of
a particular RRM can promote selection of a specific
splice site and also influence substrate specificity.
Fig. 8.
Summary of the role of SR protein domains on E1A alternative splicing specificity in vivo. The domain structures of
SF2/ASF and domain-deletion mutants thereof, and that of
SRp20, are shown schematically. The predominant E1A isoform
generated upon overexpression of each protein is shown on the
right. The presence of RRM2 of SF2/ASF correlates with use of
the proximal 13S 5 splice site, whereas proteins that lack this
atypical RRM cause preferential use of the 12S 5
splice site.
[View Larger Version of this Image (19K GIF file)]
Discussion
splice site in the adenovirus E1A pre-mRNA.
Fig. 9.
Summary of the localization of SR protein variants. The domain structures
of the SF2/ASF wild-type
and mutant proteins are shown schematically above
the line, and those of SRp40,
SC35, and SRp20, with or
without an RS domain, are
shown below the line. The
observed cellular location of
the expressed proteins is indicated as N (nuclear) or C
(cytoplasmic). In all cases,
the expressed proteins were
excluded from nucleoli. Subnuclear accumulation in
speckles is indicated by + or signs, with the number of + signs reflecting the quantitations shown in Fig. 5. Proteins that did not accumulate
in speckles gave a diffuse nuclear pattern.
[View Larger Version of this Image (25K GIF file)]
We do not know at present whether the nuclear and cytoplasmic distribution observed with several mutants represents incomplete nuclear import and/or incomplete retention of these proteins in the nucleus. Two alternative
explanations, which are not mutually exclusive, can be
proposed on the basis of our findings. In the first model,
there are multiple nuclear localization signals distributed throughout SF2/ASF. These signals have additive effects,
such that deletion of either RRM, or of the RS domain, reduces the overall steady-state accumulation in the nucleus.
Thus, when all three structural domains are present, the
protein has an exclusively nuclear distribution. When only
two of the three domains are present, a fraction of the protein accumulates in the cytoplasm, and when only one domain is present, the proportion of cytoplasmic protein greatly increases. In the second model, partition between
the nucleus and the cytoplasm can be attributed to incomplete nuclear retention of SF2/ASF mutants lacking either
RRM repeat or the RS domain. In support of this model,
we have shown that fusing the RS domain to a cytoplasmic
reporter, NPc, directs the fusion protein exclusively to the
nucleus. NPc is thought to have a propensity to be retained
in the nucleus, once it gets there, because of oligomerization (Michael et al., 1995). In contrast, the SF2/ASF mutant proteins that lack one of the two RRMs (RRM1/RS,
RRM2/RS) display mostly nuclear but also cytoplasmic localization, and they have been shown to have reduced
RNA binding, compared to the wild-type protein. SF2/ASF
RRM domains expressed individually distribute evenly
throughout the whole cell (Fig. 2) and bind RNA very
poorly (Cáceres and Krainer, 1993
; Zuo and Manley, 1993
).
In the case of SR proteins that in their natural form have a single RRM (e.g., SC35 and SRp20), deletion of the RS domain resulted in mutant proteins with nuclear and cytoplasmic distribution (Fig. 9). Therefore, the nuclear localization mechanism appears to be different for the two SR protein subfamilies: in the case of SC35 and SRp20, a single RRM together with the RS domain resulted in exclusive nuclear localization. In contrast, in the case of SF2/ASF, either RRM together with the RS domain resulted in nuclear and cytoplasmic distribution (RRM1/RS and RRM2/RS; Fig. 2), and exclusive nuclear localization was only achieved when both RRMs were present together with the RS domain.
Localization of SR Proteins to Nuclear Speckles
The process of nucleo-cytoplasmic transport has been extensively studied, and several components of this pathway
have been identified (for reviews see Silver, 1991; Dingwall and Laskey, 1991
; Görlich and Mattaj, 1996
). In contrast, the mechanisms involved in localization of proteins
within specific subnuclear regions are poorly understood.
They may involve active transport mediated by subnuclear
targeting signals, or alternatively, passive diffusion and
binding to the nuclear matrix or its components. It has
been proposed that the speckle domains are anchored directly or indirectly to the nuclear matrix (Spector et al.,
1983
). In agreement with this hypothesis, a role for the nuclear matrix in splicing has been postulated (Zeitlin et al.,
1987
), and antibodies raised against components of the nuclear matrix cross-react with SR proteins (Blencowe et al.,
1994
, 1995
).
The RS domain is the most prominent feature shared by
splicing factors that localize in a speckled pattern. As such,
it is the best candidate domain to mediate this subnuclear
localization, as first proposed and tested with Drosophila
proteins by Bingham and coworkers (Li and Bingham,
1991). In the case of human SF2/ASF, we have shown that
the RS domain is neither necessary nor sufficient for localization to the speckles. In marked contrast, deleting the
RS domain of two human SR proteins that have a single
RRM caused the mutant proteins to distribute throughout
the cell, and accumulation in the speckled region was no
longer observed. A similar result was obtained when expressing individual domains of SF2/ASF (RRM1 and RRM2
proteins). In addition, by fusing the RS domain of SRp20
to a cytoplasmic protein reporter, we demonstrated that
this RS domain is necessary and sufficient to target a protein to the nuclear speckle domains (Table I). These results demonstrate the existence of two different mechanisms for localization to the speckle domains, based on the
different behavior of two types of RS domains.
The lack of speckle-targeting signals in the RS domain
of human SF2/ASF was unexpected, in light of previous
findings with the RS domains of the Drosophila SWAP
and Tra splicing regulators, which are necessary and sufficient to target reporter proteins to the speckled region (Li
and Bingham, 1991). A recent study further defined the
specific elements within the Tra RS domain required for
localization in the speckled region (Hedley et al., 1995
). In
addition to a classical bipartite nuclear localization sequence, a novel motif was defined, which is necessary and
sufficient for subnuclear localization. The subnuclear targeting signal of Tra and homologous motifs present in
Drosophila Tra2 and SWAP, and in human U1-70K and
SC35, consist of three or four basic amino acids (generally
arginine and histidine) followed by a run of RS dipeptides
(Hedley et al., 1995
). Interestingly, this motif is present in
human SR proteins that have a single RRM (SC35, SRp20
and 9G8) (Ayane et al., 1991
; Fu and Maniatis, 1992
; Vellard et al., 1992
; Cavaloc et al., 1994
), but it is absent from
some, though not all, of the SR proteins with two RRMs
(SF2/ASF, SRp40 and SRp30c) (Ge et al., 1991
; Krainer et
al., 1991
; Screaton et al., 1995
).
The Drosophila Tra and SWAP alternative splicing factors have RS domains, but they lack RRMs. In contrast,
the modular structure of SR proteins, with a COOH-terminal RS domain of variable length and one or two
RRMs, is likely to allow multiple protein-protein and protein-RNA interactions. The mechanism of nuclear speckle localization may be a complex process, by analogy to the
pathways described for localization in coiled bodies or in
nucleoli (Schmidt-Zachmann and Nigg, 1993; Bohmann et
al., 1995
; Scheer and Weisenberger, 1995), which involve
complex signals rather than simple sequence motifs.
Studies with the Drosophila splicing regulators suggested a mechanism for subnuclear localization based on
protein-protein interactions mediated by the RS domains.
Recent studies pointed to a role for the RS domains of
Drosophila and/or human Tra, Tra2, U2AF35, U1-70K,
and those of several SR proteins in mediating specific protein-protein interactions, which may be modulated by
phosphorylation of many of the serine residues (Wu and
Maniatis, 1993; Amrein et al., 1994
; Kohtz et al., 1994
;
Xiao and Manley, 1997
). In particular, the RS domains of
SC35 and SF2/ASF are thought to mediate specific interactions with the RS domains of both the U1-70K polypeptide and with the 35-kD subunit of the splicing factor
U2AF (Wu and Maniatis, 1993
; Kohtz et al., 1994
). These
interactions are thought to be involved in defining and
bridging the splice sites during spliceosome assembly, but
in principle they could also play a role in specifying subnuclear localization. Although both snRNP polypeptides and
SR proteins colocalize in a speckled distribution, their localization appears to involve different molecular interactions since the speckled distribution of snRNP proteins is
sensitive to RNase A treatment, while the SC35 distribution is not affected (Spector et al., 1991
).
Proteins that lack RS domains, such as the splicing factor PSF, are thought to localize to the speckled region by
interacting with snRNPs or with RS domain-containing
proteins (Patton, J., personal communication; Hedley et
al., 1995). Deletion mutants of Tra lacking the RS domain
can still localize in the speckles, provided that they are
able to interact with Tra2 (Hedley et al., 1995
). Thus, two
mechanisms may operate to target splicing components to
the nuclear speckles: a direct one, in which the presence of a targeting signal determines the proper subnuclear localization, and an indirect one, in which proteins lacking a
targeting signal localize to the speckle domains by binding
to splicing components that have a targeting signal. If SF2/
ASF is targeted to the speckles indirectly, by protein-protein or RNA-protein interactions, the interaction regions
must be redundant since we showed that each of the three
constituent domains can be deleted individually without a
complete loss of localization in the speckle domains. Currently, the RS domain of SF2/ASF is the only region of the
protein known to be involved in protein-protein interactions, but this domain is neither necessary nor sufficient
for speckle localization of SF2/ASF. Targeting by interaction with RNA is a distinct possibility; to explain the
present localization data, this model would require that a
single RRM derived from an SR protein be unable to interact stably with RNA, and that either a second RRM or
an RS domain be required to stabilize interactions with
RNA. In vitro RNA-binding experiments showed that
these are indeed the properties of SF2/ASF (Cáceres and
Krainer, 1993
; Zuo and Manley, 1993
; Jamison et al.,
1995
). The direct targeting model is more likely for single-domain SR proteins, such as SC35, whose localization is
RNase resistant (Spector et al., 1991
), and which appear to
have short sequence motifs that are sufficient for correct
subnuclear localization (Hedley et al., 1995
).
Alternative Splicing Activity of SR Proteins
We analyzed the role of the structural domains of SF2/
ASF in regulation of alternative splicing in living cells (Fig.
8). We found that the RS domain of SF2/ASF is not required for regulation of alternative splicing in vivo, since a
mutant protein lacking this domain regulates alternative
splice site selection of an E1A splicing reporter in a manner very similar to the wild type. This finding is consistent
with previous observations made in vitro and in vivo
(Cáceres and Krainer, 1993; Zuo and Manley, 1993
; Wang
and Manley, 1995
). Whereas the in vitro studies also showed that the RS domain of SF2/ASF is required for its
full activity in constitutive splicing, the observation that it
is not required for concentration-dependent effects on alternative splicing may suggest that for this particular function, the lack of the RS domain can be compensated by interactions with other SR proteins. As noted above,
however, the only protein-protein interaction region of
the protein known so far is, in fact, the RS domain (Wu
and Maniatis, 1993
; Amrein et al., 1994
; Kohtz et al.,
1994
).
We showed that the presence of a particular domain,
RRM2 of SF2/ASF, confers selectivity for a specific alternative 5 splice site. Thus, all the constructs we analyzed
that included RRM2 favored the most proximal 5
splice
site in E1A pre-mRNA (13S), whereas natural (SRp20) or
mutant (SF2-RRM1/RS) proteins lacking RRM2 selected
the middle 5
splice site (12S). This remarkable difference
in splice site selection demonstrates that the RRMs of
SF2/ASF function as modules that contribute to specificity in alternative splicing, and that the functions of the two
RRM modules can be separated. Moreover, this effect is
substrate specific since both the mutant SF2/ASF lacking
RRM2 and SRp20 had altered specificity (compared to
the other proteins) with the E1A pre-mRNA but showed
normal specificity with a
-thalassemia pre-mRNA (Screaton et al., 1995
; data not shown). We conclude that the selection of alternative splice sites depends on both the
properties of the pre-mRNA as well as on the presence of
particular domains in the SR proteins. However, not all
SR proteins that possess or lack RRM2 will necessarily
have the same alternative splicing specificities as SF2/ASF
and SRp20, respectively. For example, overexpression of
SC35, which has a single RRM, fails to activate 12S splicing (Wang and Manley, 1995
; data not shown). These substrate differences among SR proteins are consistent with
the notion that they may function as global regulators of
alternative splicing for distinct classes of pre-mRNAs in
vivo (Screaton et al., 1995
).
The mechanistic relation between general or specific
RNA binding by SR proteins and their activity in alternative splicing regulation has not been established. Although
both RRMs in SF2/ASF are required for efficient binding
to RNA (Cáceres and Krainer, 1993; Zuo and Manley,
1993
) and to high affinity sites (Tacke and Manley, 1995
),
our present data show that mutants of SF2/ASF lacking either RRM1 or RRM2 are nevertheless active in alternative splicing in vivo. The fact that alternative splicing activity can be observed in vivo with SF2/ASF mutants lacking
any one of its three constituent domains suggests that this
activity of the protein does not require highly sequence-specific binding to RNA. On the other hand, the different
splice site activation specificity of the SF2/ASF mutants,
depending on which of the two RRMs was deleted, suggests that sequence-specific interactions may play a role in
selecting particular splice sites for activation. The alternative splicing activity of SF2/ASF has been shown to correlate with its ability to promote multiple occupancy of alternative 5
splice sites by U1 snRNP (Eperon et al., 1993
).
Interestingly, SF2/ASF mutants lacking either RRM are
still able to form a ternary complex with U1 snRNP and
pre-mRNA containing a functional 5
splice site (Jamison
et al., 1995
). The activity of similar mutants in alternative
splicing in vivo is consistent with this finding.
In summary, we have shown that the modular structure
of SR proteins has profound consequences for their cellular localization, alternative splicing activity, and also their
specificity in alternative 5 splice site selection.
Received for publication 21 February 1996 and in revised form 16 May 1997.
T. Misteli was supported by the Swiss National Science Foundation and the Human Frontiers Science Program. G.R. Screaton was supported by the Wellcome Trust and the Arthritis and Rheumatism Council. D.L. Spector is supported by a grant from NIH/NIGMS (GM 42694). J.F. Cáceres and A.R. Krainer were supported by grants GM42699 from NIH/ NIGMS and CA13107 from NCI and by the Pew Charitable Trusts.We thank Angus Wilson for the T7-expression vector, C. Dingwall for the nucleoplasmin cDNA, and Mario Gimona and Sui Huang for helpful discussions.
G-domain, glycine-rich domain; RRM, RNA-recognition motif; RS domain, arginine/serine-rich domain; RT-PCR, reverse transcriptase PCR; snRNP, small nuclear ribonucleoprotein particle; SWAP, suppressor-of-white-apricot; Tra, Transformer.
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