From the Max-Planck Institute of Neurobiology, Am Klopferspitz 18a,
D-82152 Martinsried, the Forschungszentrum Karlsruhe,
Institut für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany,
and the § Eunice Kennedy Shriver Center,
Waltham, Massachusetts 02254
Received for publication, July 31, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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We identified the rat Sam68-like mammalian
protein (rSLM-2), a member of the STAR (signal transduction and
activation of RNA) protein family as a novel splicing regulatory
protein. Using the yeast two-hybrid system, coimmunoprecipitations, and
pull-down assays, we demonstrate that rSLM-2 interacts with various
proteins involved in the regulation of alternative splicing, among them the serine/arginine-rich protein SRp30c, the splicing-associated factor
YT521-B and the scaffold attachment factor B. rSLM-2 can influence the
splicing pattern of the CD44v5, human transformer-2 Prior to export to the cytosol, pre-mRNA generated from most
eukaryotic genes undergoes maturation processes such as splicing, in
which intronic sequences are removed and exonic sequences are rejoined,
as well as polyadenylation and 5'-end capping. There is increasing
evidence that transcription, pre-mRNA processing, and RNA transport
are coupled in a highly coordinated manner (1-3). Recent results
indicate a direct interaction among RNA polymerase II, transcription,
capping, splicing, and polyadenylation factors (3-6). These complexes
are possibly attached to chromatin, for example by the scaffold
attachment factor B (SAF-B1)
(7). This supports the model of a large RNA processing unit (8, 9)
termed RNA factory (3). Pre-mRNA splicing is characterized by a
high fidelity and can be modulated in a cell type- or
development-specific way to use exons alternatively. Although the exact
mechanisms governing splice site selection are still not fully
understood, recent results indicate that loosely defined signals on the
pre-mRNA known as splicing enhancers/silencers, play a crucial role
in splice site selection (10-12). An important class of proteins that recognize splicing enhancers/silencers is the serine/arginine-rich (SR)
and SR-related protein family that is involved in both constitutive and
alternative splicing (13, 14). In addition, it has also been shown that
an increasing number of heterogenous nuclear ribonucleoproteins (hnRNPs) are involved in the regulation of alternative splicing. For
example, splicing regulation of the neuron-specific exon N1 or the
src pre-mRNA is under the control of the hnRNPs
hnRNP I (polypyrimidine tract binding protein), hnRNP F, and
hnRNP H (15, 16).
SR proteins and hnRNPs can change alternative splicing patterns in a
concentration-dependent manner both in vivo and
in vitro (for review, see Refs. 13 and 14). Because the
relative expression levels of SR proteins and hnRNPs show
tissue-dependent variations (17-19), one hypothesis is
that tissue-specific splicing is the result of concentration
differences of ubiquitously expressed proteins that regulate the usage
of a given splice site.
Another way of regulating alternative splicing decisions could be the
presence of factors specific for a tissue type or developmental stage.
A well known example is expression of the sex-specific SR-related
protein transformer (20). In addition, tissue-specific proteins
such as ELAV (embryonic lethal abnormal vision) (21) and NOVA
(22) were described, and we previously found tissue-specific variants
of the splice factor tra2- The nuclear RNA-binding protein Sam68 was first described as a target
of the tyrosine kinase Src during mitosis (27, 28). Sam68 is a member
of the STAR (signal transduction and activation of RNA) protein family
(29), also called GSG protein family (GRP33, Sam68, GLD-1) (30,
31), whose members share a slightly extended hnRNP K homology RNA
binding domain, called maxi-KH domain. Because Sam68 binds to various
proteins involved in signal transduction such as phospholipase
C Here, we demonstrate for the first time the involvement of the
Sam68-like mammalian protein rSLM-2 in the regulation of alternative splicing. This protein interacts with splicing regulatory proteins in vivo and in vitro and influences the splice
site selection of three different minigenes in a
concentration-dependent manner. Using a CD44 minigene, we
show that the rSLM-2-dependent inclusion of exon v5 depends
on a purine-rich sequence to which rSLM-2 binds in
vitro.
Two-hybrid Screening and Cloning--
A yeast two-hybrid screen
and interaction experiments were performed as described (26, 42). Using
rSAF-B as a bait in pGBT9, 200,000 colonies of a rat brain embryonic
day E16 library (Stratagene, pAdGal4-cDNA as prey) were screened.
The DNA of six lacZ-positive clones able to grow on selective
medium containing 10 mM 3-aminotriazole was sequenced as
described (26). An amino-terminal FLAG tag was introduced to the
full-length form or rSLM-2 by polymerase chain reaction followed by
subcloning into pcDNA3.1.
Antiserum Production and Purification--
Peptides specific for
rSLM-2 VVTGKSTLRTRGVTCG and PRARGVPPTGYRPCG were coupled to keyhole
limpet hemocyanin and used to immunize rabbits. After 121 days, serum
was purified by affinity chromatography, employing a mixture of
recombinant GST-rSLM-2 and the two peptides following the manufacturers
instructions (Pierce). Dilution for Western blot was 1:1,000 and for
immunohistochemistry 1:100. Preabsorption of 10 µl of anti rSLM-2
antibody at a concentration of 0.07 mg/ml with 80 µg rSLM-2 peptides
for 30 min at room temperature abolished the specific signal.
Immunoprecipitation and Western Blot--
These were performed
as described (26). The following antibodies were used: anti-rSLM-2
(1:1,000); anti-SAF-B (1:1,000); anti-p62/SAM68 C20 (Santa Cruz
Biotechnology, 1:2,000); anti-htra2- In Vitro Protein Interaction Assay--
The cDNA of
potential interactors of rSLM-2 was cloned into pCR3.1 (Invitrogen) and
used for an in vitro reticulocyte lysate transcription/translation (TNT, coupled reticulocyte lysate system, Promega) to obtain the corresponding 35S-labeled proteins.
For the binding experiments, 2 µl of the reactions were incubated
with 1 µg of GST or GST-rSLM-2 coupled to glutathione-Sepharose 4B
(Amersham Pharmacia Biotech) in the presence of 200 µl of HNTG buffer
and 0.1% Triton X-100 (43) for 2 h at 4 °C. Washing and detection were as described (43).
RNA Gel Shift Assay--
RNA gel shift assays were performed as
described (24). End labeling of the RNA oligonucleotides was performed
with T4 polynucleotide kinase (New England Biolabs) and
[
r-rich, GAGGAGGAAAGAGGAGAGAGAAAGGAGGAA; y-rich,
CUCCUCCUUUCUCCUCUCUCUUUCCUCCUU;
v5, AGUAUCAGGAUGAAGAGGAGACCCCACAUGCUACAAGCACAA; v5ls9, AGUAUCAGGcgacgcgucgGACCCCACAUGCUACAAGCACAA.
In Vivo Splicing Assays--
These assays were
essentially performed as described (59) employing the CD44v5 (44), the
htra2-
For the CD44v5 and clathrin light chain B minigenes, polymerase chain
reaction conditions were as described (24, 44). For the htra2-
For the tau minigenes, polymerase chain reaction was carried out
for 22 cycles with 94 °C for 1 min, annealing at 65 °C for 1 min,
and extension at 72 °C for 1 min. The resulting splicing pattern was
quantified using the Herolab EASY system.
Molecular Cloning and Sequence of rSLM-2--
SAF-B was initially
cloned because of its strong interaction with scaffold/matrix
attachment regions (47). Recently, we described the interaction of
SAF-B with RNA polymerase II as well as with splicing factors (7). In
addition, SAF-B changed the adenovirus E1A alternative splicing pattern
in a concentration-dependent manner (7).
To find new proteins involved in the regulation of alternative
pre-mRNA splicing, we used the yeast two-hybrid system to screen an
embryonic (E16) rat brain library with rSAF-B (7) as an interacting
partner. From about 200,000 colonies screened, six lacZ-positive
clones were able to grow in the presence of 5 mM 3-aminotriazole. Two clones contained an open reading frame bearing 67% homology to human Sam68 (27). The predicted protein shares the
typical domain structure with members of the STAR family (29) and shows
96% sequence identity to the recently identified mouse proteins
etoile/Sam68-like mammalian protein SLM-2 (39-41). It was
therefore named rSLM-2. The protein has a maxi-KH homology RNA binding
motif (shadowed box, Fig. 1).
Similar to Sam68, rSLM-2 has a central region containing multiple
arginine/glycine (RG) dipeptides, followed by a stretch of six prolines
which matches the class I consensus SH3 domain binding site (48). The
carboxyl-terminal part of rSLM-2 is rich in tyrosine residues and
represents a potential SH2 domain binding site (48, 49).
Identification of Novel Interactors of rSLM-2 Using the Yeast
Two-hybrid Screen--
To obtain information on the function of
rSLM-2, we performed yeast two-hybrid screens with rSLM-2 as an
interacting partner in an embryonic rat brain library. It has been
shown previously that SLM-2 interacts with the highly homologous
protein Sam68 (39), with itself as well as with hnRNP G family members
(40). Our two-hybrid results with rSLM-2 confirmed these interactions because we isolated the rat homologs of Sam68 and hnRNP G. In addition,
among the lacZ-positive yeast colonies that were able to grow on
selective medium containing 5 mM 3-aminotriazol, we identified four novel interactors of rSLM-2: rSAF-B, which has initially been used to clone rSLM-2; the novel splicing-associated protein YT521-B (26, 50); hnRNP L (51); and the SR-protein SRp30c (52)
(Fig. 2A). In addition, we
tested its interaction with another KH domain containing RNA-binding
protein, SF1 (53). Although Sam68 was shown to bind to other KH
domain-containing proteins such as Bicaudal C or Grp33 (54), rSLM-2 did
not interact with the splicing factor SF1, indicating the specificity
of the observed interactions.
These novel two-hybrid interactions were tested subsequently using
coimmunoprecipitations (Fig. 2B). We generated a specific antibody against rSLM-2 which did not cross-react with the related proteins Sam68 or rSLM-1.2 To
exclude nucleic acid-mediated interactions, benzonase was present in
all experiments. In addition, the RNA-binding protein SF1 (53) was used
as a negative control (Fig. 2B, bottom row). We
expressed EGFP-tagged proteins in HEK293 cells and precipitated them
using a monoclonal anti-GFP antibody. Overexpressed EGFP was used as a
negative control. The efficiency of the immunoprecipitation was
analyzed in Western blots with a polyclonal anti-GFP antibody (Fig.
2B, left column). The coimmunoprecipitating
proteins and cell lysates were analyzed by Western blot (Fig.
2B, middle column) using the appropriate antibody
(right column). Using this method, we found endogenous
hSAF-B to bind to rSLM-2 (Fig. 2B, top row). In
agreement with previous data (39, 40) and our data obtained in yeast,
cotransfected hnRNP G and endogenous Sam68 interacted with rSLM-2 (data
not shown). Finally, we confirmed the interaction of rSLM-2 with hnRNP
L (second row), with the splicing associated protein YT521-B
(third row), and the interaction of rSLM-2 with the splicing
factor SRp30c (fourth row).
In summary, EGFP-rSLM-2 was able to homomultimerize and to bind to a
subset of splicing and splicing-associated factors in the yeast
two-hybrid system as well as in coimmunoprecipitations.
rSLM-2 Interacts with hnRNPs and SR Proteins in Vitro--
To
confirm further the observed interactions of rSLM-2, we performed
pull-down experiments using in vitro translated rSAF-B, hnRNP L, YT521-B, SRp30c, and tra2- rSLM-2 Forms a Complex with SR Proteins in Vivo--
It has been
shown previously that the rSLM-2 interactor SAF-B binds to htra2- rSLM-2 Can Change Alternative Splicing Patterns--
The
association of rSLM-2 with splicing factors as well with the
splicing-associated protein YT521-B and hnRNPs points to a role in
pre-mRNA processing. Therefore, we asked whether rSLM-2 could
modulate splice site selection in different cell lines in a
concentration-dependent manner, analogous to several
proteins involved in splicing, such as SR proteins (14, 58), hnRNPs (58), SAF-B (7), and YT521-B (26). To investigate this possibility, we
employed a CD44 reporter gene, which contains the alternative exon v5
(44). This minigene was transfected with increasing amounts of
EGFP-rSLM-2 in HEK293 cells. Vector DNA (pEGFP) was added to ensure
that comparable amounts of DNA were transfected in each experiment
(59). Increasing the amount of transfected pEGFP-rSLM-2 resulted in an
increased incorporation of exon v5, as shown in Fig.
5A, suggesting a rSLM-2
concentration-dependent modulation of splice site
selection. Western blots with lysates from the transfections confirmed
an increase in EGFP-rSLM-2 expression (data not shown).
Next, we determined the role of various rSLM-2 domains and tested
deletion variants for their influence on splice site selection. As
shown in Fig. 5B, deletion of either the RG-rich region and the potential SH2 and SH3 binding domains (rSLM-2
We therefore wondered whether proteins binding to rSLM-2 can influence
the regulation of alternative splicing of the CD44 minigene and
analyzed the interactors SRp30c, hnRNP G, and SAF-B. First, we tested
them separately with the CD44 minigene and found that SRp30c slightly
repressed exon v5, whereas hnRNP G and SAF-B had no effect on exon v5
usage (Fig. 5D). Then, we analyzed these interactors in the
presence of rSLM-2 and found that all of them led to a significant
decrease of the exon v5 stimulation by rSLM-2 (Fig. 5E).
These data provide evidence that the rSLM-2-dependent inclusion of exon v5 can be antagonized by SRp30c, hnRNP G, and SAF-B.
The rSLM-2-dependent Exon Inclusion Depends on a
Purine-rich Exonic Splicing Enhancer--
Pull-down assays on
immobilized RNA with HEK293 cell lysates containing overexpressed
rSLM-2 showed that rSLM-2 selectively binds to purine-rich RNA
(39).3 Previously, a
purine-rich splice enhancer has been characterized in exon v5 of the
CD44 gene using linker-scan mutations (44). Because the analysis of
rSLM-2 deletion variants demonstrated the necessity of its RNA binding
domain in splice site regulation (Fig. 5B), we were
interested in determining the target sequence of the CD44 pre-mRNA.
We compared the influence of rSLM-2 on three different CD44 minigene
mutants ls8-ls10 (Fig. 6A).
In the ls9 variant, the purine-rich enhancer sequence is replaced.
Similar to the wild type minigene, EGFP-rSLM-2 was able to induce exon v5 inclusion with the ls8 and ls10 minigene constructs in HEK293 cells
(Fig. 6B). However, when 10 purine-rich nucleotides have been replaced (ls9), we detected a drastic decrease in the
rSLM-2-dependent exon v5 inclusion rate (Fig.
6A). Instead of 80% (± 4.8) exon v5 inclusion, we only
observed 42% (± 5.1%) exon inclusion. With EGFP alone, the default
splicing pattern was exon v5 skipping in each case.
To determine whether recombinant GST-rSLM-2 binds directly to CD44 exon
v5 RNA, we performed gel mobility shift assays with several RNA
oligomers. First, we determined that GST-rSLM-2 binds to the
purine-rich oligonucleotide r-rich. With increasing amounts of rSLM-2,
a super shift is visible which is probably due to the ability of rSLM-2
to multimerize (Fig. 6C). This is consistent with our
two-hybrid and immunoprecipitation data and the observation that
GST-rSLM-2 migrates as a complex of about 450 kDa upon gel filtration
(data not shown). Next, we employed the oligonucleotide v5, which
contains the regulatory sequence of the CD44 exon v5 and observed
binding of rSLM-2 to it (Fig. 6D, lanes 1-3).
Then we used an oligonucleotide (v5ls9) containing the mutation
ls9, which abolished the effect of rSLM-2 on CD44 exon v5 regulation in vivo (Fig. 6B). As shown in Fig.
6D, lanes 4-6, no binding is observed with the
v5Rls9 oligonucleotide. Furthermore, we observed binding to the
purine-rich oligonucleotide r-rich (Fig. 6D, lanes 7-9), but no specific binding to the pyrimidine-rich
oligonucleotide y-rich could be detected (Fig. 6D,
lanes 10-12). No binding of these oligonucleotides was
observed when GST was used (Fig. 6D, lanes
13-16). We conclude that rSLM-2 regulates inclusion of CD44 exon
v5 by binding to a purine-rich exonic enhancer (see Fig. 8).
Effect of rSLM-2 on Other Minigenes--
Next, we tested the
effect of rSLM-2 on the alternative splicing of the htra2-
Next, we analyzed the effect of rSLM-2 expression on a tau
minigene in COS cells. The tau minigene contains the
alternatively spliced tau exon 3 (SV
Finally, overexpression of rSLM-2 did not stimulate inclusion of the
pyrimidine-rich neuron-specific exon EN of the clathrin light chain B
transcript (data not shown) and did not repress exon EN usage when this
exon is activated by improving its 5'-splice site (Fig. 7C).
In contrast, htra2- rSLM-2 Interacts with Proteins Involved in Splice Site
Selection--
SAF-B has been shown to associate with the splicing
factors SRp30c, ASF/SF2, and htra2-
To identify novel proteins involved in mRNA processing, we
performed a yeast two-hybrid screen using SAF-B as a bait. This led to
the identification of rSLM-2 as a novel interactor of SAF-B.
rSLM-2 and its close relatives Sam68 and rSLM-1 are members of the STAR
protein family (29), also called GSG protein family (30). These
nuclear proteins combine a slightly modified hnRNP K homology RNA
binding domain, called maxi-KH domain, with binding sites for proteins
containing phosphotyrosine or proline binding domains like SH2, SH3, or
WW domains. To investigate the function of rSLM-2, we searched for
novel interactors using yeast two-hybrid screens and further confirmed
these interactions with coimmunoprecipitations and in vitro
interaction assays using recombinant rSLM-2 protein. Interestingly, all
of the rSLM-2-interacting proteins found within these three independent
systems are implicated in the regulation of alternative splicing. In
addition to SAF-B, which has initially been used to isolate rSLM-2, the
splicing-associated protein YT521-B has been found to interact with
rSLM-2. Using the SRp20 and htra2-
It is notable that rSLM-2, SAF-B, and YT521-B all interact with the
splicing factor SRp30c. The physiological significance of this complex
formation of rSLM-2 has been demonstrated by endogenous coimmunoprecipitation using the pan anti-SR protein antibody mAb104 (Fig. 4). However, in contrast to SAF-B and YT521-B, no direct binding
could be detected between rSLM-2 and the splicing factors htra2-
Finally, a growing number of hnRNPs have been characterized as
molecular players in the regulation of alternative splicing, among them
hnRNP A1 (58), hnRNP F, hnRNP H (15), hnRNP I (polypyrimidine tract
binding) (16), hnRNP L (66), and a testis-specific member of
the hnRNP G family, RNA-binding motif (RBM) (67, 68). Using a
two-hybrid screen with RBM as a bait, Venables et
al. were previously able to isolate the human homologue of
rSLM-2, T-STAR (40). Now we provide evidence that rSLM-2 also interacts
with the ubiquitously expressed hnRNP G and with hnRNP L.
rSLM-2 Regulates Alternative Splice Site Selection--
The
binding properties of rSLM-2 indicate a role in pre-mRNA
processing. We tested this hypothesis by performing transient transfection assays with several reporter minigenes that contained purine-rich sequences in their alternative exons. As a control, a
pyrimidine-rich exon of clathrin light chain B was employed. We found
that rSLM-2 changes the splicing patterns of several alternatively
spliced exons in a concentration-dependent manner. It
induced inclusion of exon v5 of CD44 (44) (Fig. 5A), of exon 3 of neurofilament tau (Fig. 7B) (69), but caused
exon II skipping of the htra2-
Our deletion analysis showed that binding of rSLM-2 to RNA is not
sufficient to influence splice site selection. The deletion mutant
lacking a functional KH domain rSLM-2(
Binding of rSLM-2 to a purine-rich RNA sequence is necessary to
regulate splice site selection because either deleting its RNA binding
domain or changing the purine-rich sequence abolishes an effect on
usage of exon v5 (Figs. 5B and 6B).
Because rSLM-2 binds directly to purine-rich RNA (Fig. 6, C
and D), it is likely that it regulates splice site selection
through interaction with purine-rich sequences in vivo. The
in vitro binding of rSLM-2 to exon v5 RNA, but not to the
mutant v5ls9 (Fig. 6D) is in complete agreement with the
regulation of CD44 exon v5, but not CD44 exon v5ls9 by rSLM-2 (Fig.
6B). In addition, rSLM-2 cannot regulate the pyrimidine-rich
exon EN of clathrin light chain B (Fig. 7C), even when this
exon is activated by a 5'-splice site improvement. A sequence
comparison between the regulated exons of CD44, tra2-
Our data provide for the first time evidence for a role of a STAR
protein in the regulation of alternative splicing. In addition, there
is increasing evidence that the STAR protein Sam68 is also involved in
alternative splice site selection. First, Sam68 has been found to
cross-link to an intronic regulatory RNA sequence of the tropomyosin
pre-mRNA (71). Second, Bedford et al. recently demonstrated the binding of Sam68 to the spliceosome-associated protein
FBP21 (72). Finally, like rSLM-2, Sam68 interacts with the
testis-specific splicing factor RBM (67). However, a direct effect of Sam68 on alternative splice site selection remains to be shown.
In addition to the role of Sam68 in viral replication (73, 74), two
possible functions of rSLM-2 and Sam68 have been investigated to date.
First, there is evidence that rSLM-2 and Sam68 are important for cell
cycle progression (27, 28, 36, 41), but a direct molecular link is
still missing. We speculate that STAR proteins influence the cell cycle
by regulating mRNAs necessary for its progression.
Furthermore, Sam68 has been proposed to act as an adapter protein
within signal transduction pathways. Upon T cell or insulin receptor
stimulation, Sam68 is tyrosine-phosphorylated (33-35). Phosphorylation
changes its binding affinities to phospholipase C and tau
minigenes in cotransfection experiments. This effect can be reversed by
rSLM-2-interacting proteins. Employing rSLM-2 deletion variants, gel
mobility shift assays, and linker scan mutations of the CD44 minigene,
we show that the rSLM-2-dependent inclusion of exon v5 of
the CD44 pre-mRNA is dependent on a short purine-rich sequence.
Because the related protein of rSLM-2, Sam68, is believed to play a
role as an adapter protein during signal transduction, we postulate
that rSLM-2 is a link between signal transduction pathways and
pre-mRNA processing.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(23) which have a different effect on a
given splice site (24). Finally, several proteins involved in
pre-mRNA splicing, judged to be ubiquitously expressed by Northern
blot analysis, show cell type-specific expression when examined by
histochemical methods. These include tra2-
1 (23, 25), hnRNP L (19),
and YT521-B (26).
or p85 phosphatidylinositide 3-kinase, this protein has
been proposed to play a role as an adapter protein in fibroblast and
lymphocyte signaling (32-35). In addition, there is increasing
evidence that Sam68 is important for cell cycle progression (36-38).
Recently, two nuclear Sam68-like mammalian proteins (SLM-1 and SLM-2,
also called T-STAR/etoile/Salp) have been described (39-41).
However, the exact function of Sam68 and its relatives in the nucleus
still remains elusive.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (1:2,000) (25), PY20
(1:10,000), anti-Src (1:1,000) and anti GFP (Boehringer 1:5,000).
32P]ATP followed by purification using the nucleotide
removal kit (Qiagen). Indicated amounts of recombinant GST-rSLM-2 were
incubated with 30 pmol of the 32P-labeled RNA
oligonucleotides for 15 min at 30 °C. The oligonucleotide sequences
were:
1 (23), tau minigenes containing exon 2 or exon 3 (45),
or clathrin light chain B minigenes (24, 46). Transfection of the
CD44v5 and clathrin light chain B minigenes occurred in HEK293 cells,
whereas the htra2-
1 and tau minigenes were analyzed in HN10
cells and COS cells, respectively.
minigene, 20-s denaturation at 94 °C, 20-s annealing at 65 °C,
and 40-s extension at 72 °C for 33 cycles were used followed by a
final extension at 72 °C for 20 min.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
cDNA and protein sequence of rSLM-2.
The coding cDNA sequences are indicated in uppercase,
untranslated regions in lowercase. Start and stop codons are
shown in bold. The protein sequence is shown underneath the
cDNA sequence. The KH RNA binding domain is shown as a
shadowed box and is flanked by the QUA1 and QUA2 regions
(indicated as open boxes). The arginine and glycine
dipeptides clustered in the central part of the protein are
boxed. The stretch of six prolines and tyrosine residues
located in the carboxyl-terminal part of the protein is indicated in
bold.
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Fig. 2.
Analysis of proteins binding to rSLM-2.
Panel A, yeast two-hybrid interactions of rSLM-2. rSLM-2 was
fused to the Gal4 activation domain. The rSLM-2 interacting proteins
were fused to the Gal4 DNA binding domain (in pGBT9). Plus
signs indicate growth; minus signs indicate no growth
on His plates containing 5 mM
3-aminotriazole. None of the constructs showed self-activation.
Panel B, coimmunoprecipitations with rSLM-2. EGFP fusion
proteins were overexpressed in HEK293 cells and immunoprecipitated
using a monoclonal anti-GFP antibody. The immunoprecipitated protein is
indicated by a superscript IP. EGFP alone was used as a
negative control in each experiment. The immunoprecipitates were
analyzed with a polyclonal anti-EGFP antibody (left column)
to verify successful immunoprecipitation. The immunoprecipitates were
also analyzed with interactor-specific antibodies (middle
column) that are listed in the right column.
Arrows indicate the protein signals observed. IG,
immunoglobulin signal. Immunoprecipitated rSLM-2 interacts with
endogenous SAF-B (top row). Immunoprecipitated
EGFP-hnRNP L (second row), EGFP-YT521-B (third
row), and EGFP-SRp30c (fourth row) bind to FLAG-rSLM-2.
Precipitated EGFP-SF1 did not interact with FLAG-rSLM-2 (fifth
row, middle column). The ratio of immunoprecipitation
to lysate loaded on the gel was 20:1 in each case.
1 (Fig.
3, lanes 1-5), as well as
recombinant GST-rSLM-2 protein (Fig. 3, lanes 6-10). As shown in lanes 6-9 in Fig. 3, the hnRNPs rSAF-B and hnRNP
L, as well as the splicing-associated protein YT521-B and the SR
protein SRp30c, bound directly to rSLM-2. In contrast, htra2-
1 did
not bind to GST-rSLM-2 (Fig. 3, lane 10), which is in
agreement with our data obtained in yeast and demonstrates the
specificity of the interactions.
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Fig. 3.
In vitro interaction assay using
recombinant GST-rSLM-2 and radiolabeled hnRNPs or SR proteins.
rSAF-B, hnRNP L, YT521-B, SRp30c, and htra2- 1 were translated
in vitro in the presence of [35S]Met using a
coupled transcription/translation reaction in reticulocyte lysates.
These proteins were incubated with GST-rSLM-2 coupled to
glutathione-Sepharose 4B. The input of radiolabeled proteins is shown
in lanes 1-5. Retained proteins were analyzed on a 15%
SDS-PAGE (lanes 6-10). GST alone was used as a negative
control (lanes 11-15).
1,
SRp30c, SF2/ASF, and hnRNP A1 and can change alternative splice sites
in vivo (7, 55). Therefore, we asked whether rSLM-2 is
present in a complex with splicing factors in vivo.
Screening of several cell lines revealed that SLM-2 is expressed in
HN10 cells. These cells are derived from hippocampal neurons (56) that
express SLM-2 (Fig. 4A,
Lysate). We performed an immunoprecipitation in HN10 cell
lysates with the pan SR antibody mAb104 (57) in the presence of
benzonase and found rSLM-2 to be present in this complex (Fig.
4A, IP). One-third of the corresponding
immunoprecipitates were analyzed with the mAb104 antibody (Fig.
4B) to verify the successful immunoprecipitation.
Furthermore, mAb104 did not cross-react with rSLM-2, even when we
overexpressed EGFP-rSLM-2 (Fig. 4C). We conclude that
endogenous rSLM-2 forms an in vivo complex with factors
implicated in pre-mRNA splicing.
View larger version (19K):
[in a new window]
Fig. 4.
Interaction of endogenous rSLM-2 with
splicing factors. Panel A, interaction of
endogenous rSLM-2 with SR proteins. Immunoprecipitation using the pan
SR protein antibody mAb104 (IP) was performed with lysates
from HN10 cells. rSLM-2 was detected with the rSLM-2 antiserum. The
ratio of immunoprecipitate to lysate loaded on the gel was 20:1.
Panel B, verification of the immunoprecipitation analyzed in
panel A. One-third of the mAb104 immunoprecipitate was
analyzed with the mAB104 antibody (IP) and compared with the
HN10 lysate. Panel C, mAb104 does not cross-react with
overexpressed EGFP-rSLM-2. HEK293 cells were transfected with
EGFP-rSLM-2. Lysates were probed with the rSLM-2 antiserum
(left) or mAb104 (right), indicating that mAB104
does not cross-react with rSLM-2.
View larger version (45K):
[in a new window]
Fig. 5.
rSLM-2 influences alternative splicing
patterns in transient transfection assays. The CD44 minigene
containing an alternatively spliced exon was cotransfected with
increasing amounts of rSLM-2 followed by reverse
transcriptase-polymerase chain reaction analysis of the total RNA using
minigene-specific primers flanking the alternative exon. The results of
three independent experiments were quantified, and the percentage of
exon inclusion is shown below each gel. M, 100-base pair
ladder. Panel A, rSLM-2 promotes the inclusion of the CD44
exon v5. The structure of the minigene is shown schematically on the
top. INS2 and INS3 are constitutive exons from the
preproinsulin gene (44). An increasing amount of pEGFP-rSLM-2 was
cotransfected with 2 µg of the CD44v5 minigene. In each titration
point, pEGFP was added to keep the total amount of DNA constant.
Panel B, the CD44v5 minigene was cotransfected with 2 µg
of pEGFP, pEGFP-rSLM-2, or its deletion variants (shown on the
left). Panel C, Western blot analysis
demonstrating equal amounts of pEGFP-rSLM-2 and its deletion variants
in cellular lysates of the transfections. 20 µl of the lysates was
analyzed with an anti-GFP antibody. Panel D, the CD44v5
minigene was cotransfected with 2 µg of the rSLM-2 interactors
SRp30c, htra2- 1, and SAF-B to determine their influence on exon v5
regulation. Panel E, the CD44v5 minigene was cotransfected
with 2 µg of rSLM-2 in the presence of 2 µg of its
interactors.
2), the
tyrosine-rich SH2 domain binding site (rSLM-2
3), or the KH domain
(rSLM-2
4) abolished the influence on splice site selection. All
deletion variants were expressed at similar levels in the transfected
cells (Fig. 5C). We conclude that the ability of rSLM-2 to
bind to RNA is necessary for splice site selection. However, in
addition to RNA binding, the potential protein interaction sites are
also necessary for the splice site switch. This suggests that rSLM-2 acts in a complex with other proteins on pre-mRNA splicing.
View larger version (42K):
[in a new window]
Fig. 6.
rSLM-2-mediated inclusion of exon v5 is
dependent on a purine-rich sequence. Panel A,
schematic diagram of CD44 exon v5. The locations of the 10-base pair
linker scan mutations ls8, ls9, and ls10 within exon v5 are indicated
(44). In the mutant ls9 the purine-rich sequence ATGAAGAGGA is replaced
by CGACGCGTCG. Panel B, the effect of EGFP-rSLM-2 on three
different linker scan mutations (ls8-10) in exon v5 of the CD44
minigene has been compared with the effect of EGFP. With the ls8 and
ls10 minigenes, 2 µg of transfected EGFP-rSLM-2 increased exon v5
inclusion similar to the wild type minigene. In contrast, only a minor
effect of EGFP-rSLM-2 was detected with the ls9 construct. HEK293 cells
were used for transient transfections. Panel C, recombinant
rSLM-2 binds to RNA in vitro. An increasing amount of
GST-rSLM-2 was incubated with a 32P-labeled polypurine RNA
oligonucleotide (r-rich) and separated on a nondenaturing gel. The
RNA·rSLM-2 complex is indicated by a closed arrow, a
larger complex observed at higher rSLM-2 concentration with an
open arrow. Panel D, recombinant rSLM-2 binds the
purine-rich exon v5 enhancer in vitro. 30 pmol of GST-rSLM-2
was incubated with the RNA oligonucleotides v5, v5ls9, r-rich, and
y-rich. Each oligonucleotide was competed with the 20- and 40-fold
excess of unlabeled v5 oligonucleotide (lanes 2 and
3; 5 and 6; 8 and
9; 11 and 12). Upon longer exposure,
binding of the v5ls9 oligonucleotide is observed only in the absence of
competitor. The dot indicates an unspecific complex seen
with the y-rich oligonucleotide that is competed by excess of r-rich
oligonucleotide. No shift is observed with GST (lanes
13-16) or without protein (lanes 17-20).
1 gene in
HN10 cells (23). Transfection of pEGFP alone leads to 40% inclusion of
alternative exon 2. Increasing amounts of rSLM-2 caused a
decrease in the
4 isoform and led to an increase in the htra2-
1
isoform (Fig. 7A).
View larger version (41K):
[in a new window]
Fig. 7.
The influence of rSLM-2 on the alternative
splicing of the htra2- , tau,
and clathrin light chain B pre-mRNA. The structure of
the minigenes is shown on the top of each panel.
A statistical evaluation of three independent experiments is shown
below representative ethidium bromide gels. Error bars
indicate the S.D. Panel A, an increasing ratio of
pEGFP-rSLM-2 to pEGFP was cotransfected with 2 µg of the htra2-
minigene in HEK293 cells. This increase leads to exon II skipping.
Panel B, the influence of rSLM-2 concentration on
tau minigenes was tested in COS7 cells. 4 µg of the
tau minigenes was cotransfected with indicated amounts (µg)
of pEGFP-rSLM-2. rSLM-2 leads to an increase in exon 3 inclusion.
X, phiX HaeIII digest; M, 100-bp
ladder. Panel C, up to 4 µg of pEGFP-rSLM-2 did not
promote exon skipping of clathrin light chain B exon EN in the
construct pJS85. In pJS85, exon EN is activated in fibroblasts by an
optimized 5'-splice site (24). In contrast, this exon is regulated by
htra2-
1 (right).
2/3) inserted in an
insulin expression vector (45). Exons 2 and 3 exhibit the rare
incremental combinatorial splicing pattern because exon 3 never appears
independently of exon 2. Exons 2 and 3 are adult-specific in the
central nervous system (60) but seem to be constitutive in the
peripheral nervous system (61, 62). Upon cotransfection, expression of
rSLM-2 stimulates exon 3 usage, whereas EGFP alone has no effect (Fig. 7B). Interestingly, a sequence stretch in exon 2 of
htra2-
and a sequence stretch in exon 3 of the tau transcript
show a significant homology to the purine-rich sequence of exon v5 of
the CD44 minigene, which is necessary for the
rSLM-2-dependent exon inclusion.
1 can regulate splice site usage of this
construct (Fig. 7C), which emphasizes the specificity of
rSLM-2-mediated splice site selection. Taken together, this indicates
that rSLM-2 can change the alternative splicing pattern of specific
substrates in a concentration-dependent manner by binding to
purine-rich sequences.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 as well as with the
carboxyl-terminal domain of the largest subunit of RNA polymerase II,
which has also been shown to stimulate pre-mRNA splicing (7, 63).
In addition, SAF-B binds to matrix attachment regions and stimulates the generation of the 10S E1A transcript in a
concentration-dependent manner (7).
minigenes, we recently
demonstrated the influence of YT521-B on alternative splicing (26).
Like rSLM-2, YT521-B interacts with Sam68 and is
tyrosine-phosphorylated upon overexpression of the tyosine kinases Src
and Fyn (26). It remains to be determined whether rSLM-2 is
tyrosine-phosphorylated by nuclear tyrosine kinases, such as SIK/BRK,
which was shown to phosphorylate Sam68 (64, 65).
1
(Figs. 2A and 3) or SF1 (Fig. 2). This demonstrates the specificity of the analyzed interactions, which have been carried out
in the presence of benzonase to avoid RNA-dependent protein interactions.
exon II (23) (Fig. 7A).
The opposing effect of rSLM-2 on different minigenes is reminiscent of
the effects of splicing factors on natural minigenes in
vivo. For example, SF2/ASF promotes exon EN inclusion when tested
with the clathrin light chain B minigene (58) but causes exon 4 skipping with the SRp20 minigene (70).
4) is exclusively nuclear
(data not shown). In contrast, mutants lacking parts of the carboxyl
terminus (rSLM-2
2 and
3) are also present in the cytosol (data
not shown). This indicates that potential protein binding sites such as
the tyrosine-rich carboxyl terminus or the proline-rich region are also
necessary for activity and proper localization of rSLM-2.
Interestingly, the rSLM-2-interacting proteins SAF-B, hnRNP G, and
SRp30c inhibited exon v5 inclusion mediated by rSLM-2 (Fig.
5D), although these three interactors alone exerted only a
minor or no effect on the CD44 minigene. Because SAF-B, hnRNP G, and
SRp30c are ubiquitously expressed but rSLM-2 is predominantly expressed
in muscle, brain, and testis (40 and data not shown), this suggests
that cell type-specific combinations of rSLM-2 and some of its
interactors may contribute to different splicing patterns in different
cell types. This supports the model that different concentrations of
antagonizing factors govern splice site selection.
1 and
tau (Fig. 8) also suggests a
purine-rich motif as the likely site of action.
View larger version (11K):
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Fig. 8.
Comparison of purine-rich sequences present
in the CD44 exon v5 (44), htra2- exon2 (23),
and tau exon 2 (45). Identical nucleotides are
indicated in bold.
1, to the
regulatory p85 subunit of phosphatidylinositide 3-kinase (32), to
itself, and to RNA (54, 75). The p85 phosphatidylinositide 3-kinase
also binds to the human homolog of rSLM-2 (41). There are numerous
examples for the regulation of alternative splicing by extracellular
stimuli (for review, see Ref. 76). Because this list of stimuli which
includes insulin (77), nerve growth factor (78), cytokines
(79), or neuronal activity (25) is rapidly growing, we are now
investigating whether rSLM-2 and similar adapter proteins are part of a
signal transduction cascade from receptors toward the spliceosome.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Claudia Cap for sequencing and James Chalcroft for artwork.
![]() |
FOOTNOTES |
---|
* This work was supported by the Max Planck Society, the Human Frontier Science Program, Deutsche Forschungsgemeinschaft Grants RG562/96 and Sta399/3-1 (to S. S.), and National Institutes of Health Grant R01 NS38051 (to A. A.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF152547.
¶ To whom correspondence should be addressed: University of Erlangen, Institute of Biochemistry, Fahrstraße 17, 91054 Erlangen, Germany. Tel.: 49-9131-85-24622; Fax: 49-9131-85-22485; E-mail: stefan@stamms-lab.net.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M006851200
2 O. Stoss, M. Olbrich, A. M. Hartmann, and S. Stamm, manuscript in preparation.
3 O. Stoss, M. Olbrich, A. M. Hartmann, H. König, J. Memmott, A. Andreadis, and S. Stamm, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
, SAF-B, scaffold
attachment factor B;
EGFP, enhanced green fluorescent protein;
GST, gluthatione S-transferase;
HEK, human embryonic kidney;
htra2-, human transformer-2-beta;
hnRNP, heterogenous nuclear
ribonucleoprotein;
KH domain, hnRNP K homology domain;
mAb, monoclonal
antibody;
Sam68, Src-associated in mitosis;
SLM, Sam68-like mammalian
protein;
SR protein, serine/arginine-rich protein;
STAR, signal
transduction and activation of RNA.
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