From the Division of Nephrology and Clinical
Immunology, University Hospital of Aachen, 52057 Aachen, Germany,
the ¶ Institute of Pathology, Charite, Humboldt University, 10099 Berlin, Germany, and the
Institute for Transplant Diagnostic and
Cell Therapy, Heinrich-Heine University,
40225 Düsseldorf, Germany
Received for publication, December 9, 2002, and in revised form, February 13, 2003
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ABSTRACT |
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The multifunctional DNA- and RNA-associated Y-box
protein 1 (YB-1) specifically binds to splicing recognition motifs and
regulates alternative splice site selection. Here, we identify the
arginine/serine-rich SRp30c protein as an interacting protein of YB-1
by performing a two-hybrid screen against a human mesangial cell
cDNA library. Co-immunoprecipitation studies confirm a direct
interaction of tagged proteins YB-1 and SRp30c in the absence of RNA
via two independent protein domains of YB-1. A high affinity
interaction is conferred through the N-terminal region. We show
that the subcellular YB-1 localization is dependent on the cellular
SRp30c content. In proliferating cells, YB-1 localizes to the
cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus.
After overexpression of YB-1 and FLAG-SRp30c, both proteins are
co-localized in the nucleus, and this requires the N-terminal region of
YB-1. Heat shock treatment of cells, a condition under which SRp30c
accumulates in stress-induced Sam68 nuclear bodies, abrogates the
co-localization and YB-1 shuttles back to the cytoplasm. Finally, the
functional relevance of the YB-1/SRp30c interaction for in
vivo splicing is demonstrated in the E1A minigene model system.
Here, changes in splice site selection are detected, that is,
overexpression of YB-1 is accompanied by preferential 5' splicing site
selection and formation of the 12 S isoform.
The Y-box protein YB-1 is a member of the cold shock protein
family, which exhibits pleiotropic functions. YB-1 specifically binds
to a sequence motif termed Y-box. This motif is characterized by the
presence of a core ATTGG sequence, which represents the inverted
CCAAT-box. YB-1 controls the transcription of numerous genes that among
others include MHC class II antigen, MDR1,
MMP-2, and COL1A1 (1-4). DNA binding specificity
is mediated through the evolutionarily conserved cold shock
domain in conjunction with the adjacent C-terminal protein
residues (5, 6). Interactions of YB-1 with numerous cellular and viral
transcription factors including JC virus antigen (7), AP-2 (8),
Pur In addition to their role in regulating gene transcription, cold shock
proteins exhibit a wide spectrum of activities by virtue of
sequence-specific and -nonspecific RNA binding. YB-1 has been identified as the major component of messenger ribonucleoprotein particles (mRNPs) in mammalian cells, which constitute templates for
the translational machinery (13-15). At higher concentrations Y-box
proteins Xenopus FRGY2 and human YB-1 act as repressors of
translation in a process called mRNA masking (13, 16-18), whereas
at lower YB-1 concentrations mRNA translation is activated (19). In
this regard, specific binding of YB-1 to the 5'-cap structure may be of
importance as mRNA decapping and degradation is inhibited after
binding (20). Sequence-specific mRNA binding by YB-1 occurs through
the evolutionarily conserved cold shock domain, which contains the
RNA-binding motifs RNP1 and RNP2 (21, 22). Upon binding, YB-1
regulates mRNA half-lives, e.g. of the interleukin-2 mRNA during T-cell activation (23) and
granulocyte-macrophage colony-stimulating factor mRNA
in activated eosinophils (24).
Recently, a novel role of YB-1 in splicing has been proposed as YB-1
binds to the A/C-rich exon enhancer element in the CD44 gene
and thereby directs alternative splicing (25, 26). This A/C-rich exon
enhancer element is not directly bound by serine-arginine-rich (SR)1 proteins, whereas the
CD44 alternative splicing was affected through prebinding of
YB-1 to this element (26). Yet, the mechanism by which YB-1 activates
exon recognition in the CD44 gene is unknown. In addition it
has been shown that YB-1 directly interacts with the translocation
liposarcoma protein, which serves as an adapter molecule connecting
gene transcription and RNA splicing and thereby regulates the
adenovirus E1A pre-mRNA splicing (25). In the present study a
direct in vivo interaction of YB-1 with SRp30c is
demonstrated, which has fundamental effects on the subcellular YB-1
localization and is of functional relevance in the alternative splicing process.
Yeast Two-hybrid Screen--
A cDNA library derived from a
human mesangial cell line (27) was subcloned into vector MAB86
(Invitrogen). YB-1 cDNA was PCR-amplified using
5'-CATGCCATGGCAATGAGCAGCGAGGCCGAGACCC-3' and 5'-GCACTAGTTCAGCCTCGGGAGCGGGAATTCTC-3' as primers and inserted into restriction sites NcoI and SpeI of pDBLeu as
bait. In-frame cloning and sequences were verified by full-length
sequencing using the ABI PRISM sequencing reaction (Applied
Biosystems). A yeast two-hybrid screen (ProquestTM,
Invitrogen) (28, 29) was performed with a total of 2 × 106 transformants being plated on 15-cm plates of
Leu Cell Culture and Transient Transfections--
HeLa and HEK-293T
cells were cultured in Dulbecco's modified Eagle medium (Invitrogen)
supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at
37 °C and 5% CO2. For heat-shock experiments, cells
were incubated for 1 h at 42 °C in complete medium and allowed
to recover for 1 h at 37 °C before analysis by
immunofluorescence. Cells were transfected with purified plasmid DNA by
the calcium phosphate precipitation method as described previously
(30).
Expression plasmids for YB-1 (pSG5-YB-1) and SRp30c have been described
previously (1, 31). The plasmids encoding for GFP-YB-1 fusion proteins
were obtained from H. D. Royer and K. Jurchat (Max-Delbrück
Center, Berlin, Germany). In these plasmids full-length YB-1 cDNA
was cloned into the AflII and HindIII restriction sites of vector pcDNA6/V5-His (Invitrogen) that was modified with a
GFP sequence inserted at the protein C terminus. All deletion constructs of YB-1 were generated by PCR amplification of the respective cDNA sequences and subcloning into vector
pcDNA6/V5-His. Sequences were verified by automatic sequencing
reactions (ABI PRISM sequencing reaction, Applied Biosystems).
Immunoprecipitation--
For immunoprecipitation 2 × 106 cells were transfected with a total of 20 µg of
purified plasmid DNA as indicated. 48-h posttransfection cells were
harvested by trypsin digestion and centrifuged, and the cell pellet was
lysed in 1 ml RIPA buffer (50 mM Tris-HCl (pH 7.4), 1%
Nonidet P-40, 0.25% sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3V04, 1 mM NaF) for 15 min at 4 °C. Cell lysates were precleared
for 30 min at 4 °C with 40 µl of pansorbin (Calbiochem, Darmstadt,
Germany) and 4 µl of nonspecific mouse IgG1 (Dako). After
centrifugation at 14,000 rpm for 10 min at 4 °C 200 µl of supernatant was incubated with the indicated antibody (monoclonal anti-GFP, Clontech, Palo Alto, CA; monoclonal M2
anti-FLAG, Sigma) at 4 °C overnight. In separate experiments RNase A
(100 µg/ml, Roche Applied Science) was added to the precleared
lysates for 12 h at 4 °C. To ensure complete degradation of
mRNA by RNase A treatment aliquots of cell lysates were processed
using the RNeasy kit (Qiagen, Hilden, Germany) and subjected to
oligo(dT)-primed reverse transcription by means of superscript II
(Invitrogen). The housekeeping gene GAPDH was amplified by
PCR using the following primer pair: 5'-TTCCATGGCACCGTCAAGGC-3'
and 5'-TCAGGTCCACCACTGACACG-3', yielding a 570-bp amplification
product. To the precleared lysates 20 µl of pansorbin was added and
incubated for 1 h at 4 °C. Pansorbin-bound antibodies and
proteins were pelleted by centrifugation at 14,000 rpm and washed
extensively with PBS on ice followed by two final washing steps using
RIPA buffer. Pellets were resuspended in 50 µl of reducing sample
buffer, boiled at 95 °C for 5 min, and pelleted, and the supernatant
was subjected to SDS-PAGE. Proteins were transferred to nitrocellulose
and detected by suitable primary antibodies anti-GFP (1:5000) or M2
anti-FLAG (1:500) and secondary peroxidase-linked anti-mouse antibody
(Amersham Biosciences) using ECL (Amersham Biosciences) as chemiluminescent.
Immunofluorescence Microscopy--
HeLa cells were grown on
coverslips in six-well plates (105 cells/well). At 60%
confluence cells were transfected with a total of 2.5 µg of plasmid
DNA/well as indicated in the figure legends by means of calcium
phosphate precipitates and cultured for 48 h. After washing with
PBS, cells were fixed with 4% paraformaldehyde in PBS for 30 min,
washed in PBS-CM and incubated with 50 mM ammonium-sulfate in PBS-CM for 10 min. Cells were subsequently permeabilized in buffer A
(0.1% Triton X-100 in PBS-CM) and blocked with 0.2% bovine albumin in
buffer A. M2 anti-FLAG antibody (1:300 in buffer A) was added for 60 min at room temperature in a humidified chamber. After three washes
with buffer A secondary TRITC-conjugated anti-mouse antibody (Dinova,
Hamburg, Germany) was added for 60 min at room temperature (1:600).
After extensive washes with buffer A and a final wash with PBS-CM
coverslips were mounted with immumount (Shannon). Confocal laser
scanning microscopy (Zeiss LSM 510 Meta, Zeiss, Germany) was performed
at 488 nm for GFP fluorescence (detected at 500 nm < In Vivo Splicing Analysis--
15 µg of pMTE1A plasmid DNA
containing the E1A minigene was transfected in HeLa cells (2 × 106 cells) combined with expression plasmids pSG5-YB-1,
pCR-FLAG-30c or control plasmids. Care was taken that the total DNA
amount was equalized to 50 µg in each reaction by inclusion of the
respective amounts of control plasmid. Cells were harvested 48 h
posttransfection and mRNA was extracted using the RNeasy
minikitTM (Qiagen). cDNA synthesis was performed using
oligo(dT) primers (Roche Applied Science) and Superscript II RNase
H Identification of Splicing Factor SRp30c as Partnering Protein of
YB-1--
To identify YB-1-interacting proteins a yeast two-hybrid
screen was set up with full-length YB-1 as bait and a cDNA library generated from a human mesangial cell line (27) as prey. Of 2 × 106 colonies screened 86 fulfilled all five selection
criteria (phenotypes: His+, 3ATR,
As these findings indicate interaction of introduced tagged
proteins FLAG-SRp30c/GFP-YB-1 additional experiments were performed to
also confirm an interaction of endogenous YB-1 with FLAG-SRp30c. For
this purpose HEK-293T cells were transiently transfected with either
FLAG or FLAG-SRp30c expression plasmids, and co-immunoprecipitation studies were performed by means of a polyclonal anti-YB-1 antibody (32). As can be seen in Fig. 1D FLAG-SRp30c was
co-immunoprecipitated with endogenous YB-1 protein (lane
3).
Mapping of YB-1 Protein Domains Involved in SRp30c-Protein
Interaction--
To identify YB-1 protein domains that confer the
interaction with SRp30c a panel of GFP-YB-1 deletion constructs
(depicted in Fig. 2B) were
co-expressed with full-length SRp30c protein in HEK-293T cells, and
co-immunoprecipitations were performed using anti-FLAG antibody. As
shown in Fig. 2A, the N-terminal protein domains of YB-1
containing the evolutionarily conserved cold shock domain (GFP-YB-1
YB-1 and SRp30c Co-localize in Mammalian Cells--
Next we set
out to determine the subcellular localization of YB-1 and SRp30c. For
SRp30c a nuclear localization has previously been described (33),
whereas YB-1 has been reported to localize both to the nucleus and the
cytoplasm, depending on the cell origin and transformation (2, 6, 34).
Transfections of HeLa cells were performed with expression plasmids for
GFP-YB-1, FLAG-SRp30c, and tag control plasmids. In HeLa cells
expressing GFP-YB-1 alone, fluorescence was predominantly localized in
the cytoplasm (Fig. 3a,
panel B), whereas GFP was detected diffusely in all cellular compartments (Fig. 3a, panel A). In contrast,
FLAG-SRp30c protein was localized in a speckled pattern in the nucleus
(Fig. 3a, panel C). A dramatic change of the
subcellular GFP-YB-1 localization was detected with combined expression
of both proteins, GFP-YB-1 and FLAG-SRp30c. Under these conditions
GFP-YB-1 co-localized with SRp30c (Fig. 3a, panel
D), while the subcellular distribution of GFP was unchanged after
co-transfection with FLAG-SRp30c (Fig. 3a, panel
C). The nuclear localization of both proteins in most cells was
not in a speckled pattern. These findings indicate that FLAG-SRp30c
confers shuttling of YB-1 to the nuclear compartment. Transient
transfections of HeLa cells were repeated with expression plasmids for
FLAG or FLAG-SRp30c, and the subcellular distribution of endogenous
YB-1 was assessed by immunohistochemistry using a specific anti-YB-1
antibody (32). These experiments were designed to exclude any
contribution of the GFP tag to the nuclear shuttling of GFP-YB-1
protein. Here, a similar shift of endogenous YB-1 from the cytoplasm to
the nuclear compartment was apparent with introduced FLAG-SRp30c but
not FLAG alone (Fig. 3b).
To exclude a cell-specific effect of this protein shuttling analogous
experiments were performed with HEK-293T cells yielding similar results
(data not shown).
Since the N-terminal YB-1 protein domains were mapped as high affinity
interacting domains with FLAG-SRp30c, the corresponding GFP-YB-1
deletion construct, GFP-YB-1 Reversal of SRp30c-dependent Nuclear YB-1 Shuttling by
Heat Shock--
As the nuclear co-localization of YB-1 and SRp30c is
dependent on the cellular SRp30c content, we subsequently addressed the question whether a shift of nuclear SRp30c into stress-induced Sam68 nuclear bodies (33) by heat shock treatment affects the subcellular YB-1 localization. As can be seen in Fig.
5, the localization of SRp30c (Fig. 5,
panel A) and GFP-YB-1 (Fig. 5, panel B) remained unchanged after 1 h heat shock treatment at 42 °C when both
proteins were overexpressed separately. Remarkably, the nuclear
co-localization was no longer present with FLAG-SRp30c and GFP-YB-1
overexpressed in combination when heat shock treatment was performed.
Under these conditions GFP-YB-1 was detected in the cytoplasm (Fig. 5,
panel C), whereas FLAG-SRp30c remained in the nuclear
compartment.
YB-1 Affects Alternative Splicing of SRp30c--
Next we tested
for the functional relevance of the YB-1/SRp30c interaction in the
alternative splicing process. The adenovirus E1A pre-mRNA minigene
was chosen as a model system in which
concentration-dependent changes of the splicing
pattern by SR proteins has been described (35, 36). In vivo
splicing was monitored by reverse transcription PCR analysis using the
pMTE1A-sense and pMTE1A-antisense primers with mRNA collected from
HeLa cells that were transiently transfected with the E1A-minigene and
a combination of SRp30c expression vector and/or increasing amounts of
YB-1 expression vector. Care was taken to ensure that equal DNA amounts
were introduced in all transfections. The E1A minigene contains three
different 5' splicing sites resulting in three major isoforms 13 S, 12 S, and 9 S (Fig. 6A). As can
be seen in Fig. 6B, co-transfection of increasing amounts of
YB-1 resulted in the preferential formation of the 12 S isoform,
whereas the 9 S isoform decreased in a
concentration-dependent manner (quantification depicted in
Fig. 6C).
In contrast to this finding overexpression of SRp30c alone lead to the
preponderance of 13 S transcripts with concomitant decreased appearance
of the 9 S isoform (Fig. 6B, lane 5), as has been
described previously (33, 36). When both expression plasmids were
introduced in combination, that is SRp30c at a fixed and YB-1 at
increasing concentrations, the relative intensity of the 12 S isoform
was increased, whereas 13 S transcripts were slightly decreased at
lower YB-1 concentrations. At the same time isoform 9 S was decreased
(Fig. 5D). Taken together these changes in splicing indicate
YB-1-dependent selection of splice site selection that is a
shift from the 13 S toward the more distal 12 S splicing site. Thus,
YB-1 influences alternative splice site selection of the adenovirus E1A
pre-mRNA in vivo and interferes with the SRp30c-dependent splicing pattern in a
concentration-dependent manner.
Here we report the identification of SRp30c as an interacting
protein of YB-1 by a two-hybrid screen against a human mesangial cell cDNA library. The identified SRp30c clones encoded for
full-length protein. Co-immunoprecipitation studies with tagged
endogenous proteins expressed in HEK-293T and HeLa cells confirm this
protein-protein interaction. Although both proteins, SRp30c and YB-1,
have been shown to specifically bind to mRNA sequences (26, 37),
binding properties of SRp30c and YB-1 to each other were independent of the presence of mRNA, as shown by RNase A digestion. Interestingly, two independent protein domains of YB-1 are involved in SRp30c partnering, a high affinity binding region in the N terminus and a low
affinity binding domain in the C terminus. Similar findings have been
reported for YB-1 partnering with heterogeneous nuclear ribonucleoprotein K (hnRNP K) (38).
SRp30c is a member of the SR protein family, that has multiple
functions in constitutive and alternative splicing processes. It is
expressed in most tissues (36) and exhibits structural similarities
with the more intensely studied alternative splicing factor ASF/SF2.
SRp30c may directly contact RNA via two independent RNA recognition
motifs that are linked by a glycine-hinge. This protein family has been
named SR-proteins due to arginine/serine dipeptides within the C terminus.
Previously, interaction of SRp30c with nuclear proteins involved in
nuclear architecture maintenance has been reported, that is, nuclear
and nucleolar protein (Nop30) (39), heterogeneous nuclear
ribonucleoprotein A1-interacting protein HAP (33, 40), and
src activated during mitosis (Sam68) like mammalian
protein (SLM2) (31). For HAP and SLM2 a function in the organization of
the nuclear architecture and RNA-trafficking has been described, and
these are localized in Sam68 nuclear bodies (41). By changing local
concentrations of SRp30c or by recruiting SRp30c to other nuclear
compartments, e.g. stress-induced bodies (33), splicing patterns are regulated, as has been determined with the E1A minigene (33).
Changes in local concentrations of splicing factors are a
common regulatory mechanism. From this standpoint it is an important observation that YB-1 shifts from its cytoplasmic localization to the
nucleus when the cellular SRp30c content is increased by protein
overexpression. It cannot be ruled out that this effect is mediated by
another protein or an alternatively expressed isoform(s) due to
elevated SRp30c concentrations. However, some data support the notion
that a direct YB-1/SRp30c interaction is responsible for this shift.
First, the same protein domains of YB-1 that interact with SRp30c are
required for the nuclear YB-1 localization. Secondly, heat shock
treatment, a condition under which SRp30c is recruited to
stress-induced Sam68 nuclear bodies (33), leads to a reversal of
the nuclear YB-1 shift within 1 h.
Stein et al. (34) recently reported on a nuclear shift of
YB-1 with heat stress treatment, whereas in our experiments a mainly
cytoplasmatic localization of YB-1 was observed. These differences may
be explained by a cell-specific regulation of YB-1 or by
differences in the experimental setup. Stein et al. (34)
have used two human colon carcinoma cell lines that possess different
sensitivities to multiple drug resistance-related drugs. By
means of immunofluorescence microscopy, YB-1 was detectable in the
cytoplasm of most cells under stress-free conditions; however, it was
also observed in the nuclei of untreated cells exhibiting higher
intrinsic drug resistance, which corresponds to higher intrinsic
expression of MDR1 and MRP1. It was concluded that hyperthermia-induced effects are cell type-specific and dependent on the intrinsic resistance status (34). Given our observations of a nuclear YB-1 shift
that depends on the cellular SRp30c content it may be of interest to
determine the SRp30c content in human carcinoma cell lines to correlate
this with multiple drug resistance.
To further determine the functional meaning of the YB-1/SRp30c
interaction an in vivo splicing assay using the adenoviral E1A minigene was performed. Both YB-1 and SRp30c are reported to affect
the splicing pattern of the E1A minigene (25, 33, 36). For YB-1 an
increase of the major 13 S isoform was found by RNase protection assay
in NIH/3T3 cells but not in Ewing sarcoma cells (25). In our hands a
similar increase of the 13 S and even more the 12 S isoform could be
detected by in vivo splicing assay. According to results by
Screaton et al. (36) SRp30c overexpression also enhances the
13 S isoform. A similar change of the splicing pattern was observed
with overexpression of FLAG-SRp30c, suggesting that the FLAG tag does
not affect the splicing properties of SRp30c (Fig. 6, B and
D). With combined overexpression of FLAG-SRp30c and
increasing amounts of YB-1 a concentration-dependent
increase of the 12 S isoform was observed. This result reflects a
change in splice-site selection from the most proximal 5' splice site toward a more distal splice site and may hint at a role for YB-1 as an
adapter protein linking RNA binding and splicing.
Further evidence for YB-1 acting as an adapter protein that recruits
splicing factors is provided by Stickeler et al. (26) who
described YB-1 binding to exonic splicing enhancers in the human
CD44 gene and observed exon inclusion. The function of YB-1 may thus resemble the one of a slightly different member of the SR
protein family, Tra 2, found in Drosophila melanogaster (26) or human Tra2 In summary our findings reveal a direct interaction of the
multifunctional protein YB-1 with splicing factor SRp30c. This interaction may have a direct influence on the subcellular YB-1 localization. A nuclear co-localization is found in
SRp30c-overexpressing cells with subsequent changes in the splicing
pattern. However, the splicing pattern was also changed in cells
overexpressing only YB-1, most likely due to minute amounts of protein
being shuttled to the nucleus. These results suggest a model in which YB-1 acts as an adapter molecule between RNA splicing recognition motifs and splicing factor(s). Furthermore, given the other known functional properties of YB-1, which include gene regulation and mRNA masking in the cytoplasm, a link between transcription and mRNA-processing events may be provided. YB-1 as well as SRp30c are
strongly regulated by cellular stress events (33, 42-44). It will be
of great interest to unravel the function of this interaction in
cellular stress responses as this interaction may contribute to
oncogenetic and metastatic processes. Future experiments will also
address the potential role of YB-1 in mRNA export due to its
presence in the cytoplasmic and nuclear compartment as well as to its
regulating functions in transcription, splicing, and translation. A
common feature of proteins involved in the formation of
ribonucleoprotein complexes shuttling between the nucleus and cytoplasm
are alternating basic and acidic protein residues. Such alternating
residues are present in Y-box proteins (22), although little is known
about the mechanisms underlying the YB-1 shuttling observed under
certain conditions, e.g. after exposure to UV irradiation or
anticancer agent exposure (44).
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(9), CTCF (10), and p53 (11, 12) have been demonstrated. These
interactions may in part explain cell-specific gene regulation, that
is, stimulation and repression of transcription, even of the same gene
(3). In addition, it has been proposed that YB-1 plays a role as an architectural protein by its propensity to sequence specifically unwind DNA duplexes and stabilize single-stranded templates,
thereby altering sequence recognition motifs (1, 4, 8).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, Trp
, and His
synthetic
complete medium containing 40 mM 3-amino-1,2,4-triazole (Sigma). 86 His+ clones were isolated and tested for
fulfillment of all five selection criteria, including growth in media
containing 60 mM 3-amino-1,2,4-triazole (3-AT, Sigma),
ura+ 5-fluoro-orotic-acid (Invitrogen) metabolism and
-galactosidase expression.
FITC < 540 nm) and 543 nm for excitation of
TRITC-conjugated antibody (detected at 550 nm <
TRITC < 600 nm).
reverse transcriptase (Invitrogen). For PCR the
E1A-specific primers pMTE1A-sense 5'-ATTATCTGCCACGGAGGTGT-3' and
pMTE1A-antisense 5'-GGATAGCAGGCGCCATTTTA-3' were used with 25 cycles of
amplification (90 s at 94 °C, 120 s at 50 °C, 120 s at
72 °C). Amplification products were separated on 3% agarose gels
containing ethidium bromide with band intensities being quantified
by OptiQuantTM software. Band intensities of all splicing
isoforms combined were set as 100%, and relative band intensities
were determined.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase, Ura+, and
5-fluoro-orotic-acid+), and of these two colonies encoded
for full-length splicing factor SRp30c. To confirm the result of the
two-hybrid screen co-immunoprecipitation studies were performed
with both proteins co-expressed as tagged fusion proteins in HEK-293T
cells. GFP was fused to the C terminus of YB-1 and FLAG was fused to
the N terminus of SRp30c (Fig.
1A, GFP: <1 in lane
1; GFP-YB-1: <2 in lane 2, FLAG-SRp30c: <3 in
lane 3). Cell lysates were subsequently used for
co-immunoprecipitation studies with monoclonal antibodies directed
against the respective tags. As can be seen in Fig. 1A FLAG-tagged SRp30c protein was co-immunoprecipitated with anti-GFP antibody when cells were co-transfected with GFP-YB-1 (lane
5, indicated by <4); however, not in cells expressing GFP protein alone (lane 4). Conversely, GFP-YB-1 was detected with
immunoprecipitated FLAG-SRp30c (lane 8, indicated with <5);
however, not in control reactions with expressed FLAG protein (Fig.
1A, lane 7). The co-immunoprecipitations were
nearly quantitative and suggested a direct association of YB-1 and
SRp30c. To exclude the possibility that the contact of both proteins is
mediated by indirect association via mRNA binding, co-immunoprecipitation studies were repeated following RNase A treatment. RNase A treatment did not change the amount of
immunoprecipitated protein, indicating direct YB-1/SRp30c partnering
(Fig. 1B, compare lanes 6 and 12). In
the latter experiment control reactions were set up to test for
consistent and complete degradation of messenger RNA by reverse
transcription and amplification of the housekeeping gene GAPDH. As can
be seen in Fig. 1C untreated cell extracts contained
abundant amounts of GAPDH mRNA (lanes 2-4), whereas no
GAPDH mRNA was present in RNase A-treated extracts (lanes
5-7).
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Fig. 1.
A, interaction of GFP-YB-1 with
FLAG-SRp30c in vivo. FLAG-tagged SRp30c plasmid was
co-introduced with GFP protein or with GFP-tagged YB-1 into HEK-293T
cells. Cell lysates were separated by SDS-PAGE, and expressed proteins
were visualized with anti-GFP (GFP and GFP-YB-1 bands indicated by <1
and <2 in lanes 1 and 2) or M2 anti-FLAG
antibody (FLAG-SRp30c band indicated by <3 in lane 3).
Lysed proteins were immunoprecipitated with either anti-GFP
(lanes 4 and 5) or anti-FLAG (lanes
6-8) antibody and immunoblotted with anti-FLAG (lanes
4 and 5) or anti-GFP (lanes 6-8) antibody.
Co-immunoprecipitated FLAG-SRp30c is indicated by <4 in lane
5 and GFP-YB-1 by <5 in lane 8, whereas control
reactions were negative (lanes 4, 6, and
7). B, the YB-1/SRp30c protein interaction is not
mediated by RNA. GFP or GFP-YB-1 were co-expressed with FLAG or
FLAG-SRp30c in HEK-293T cells. RNase A treatment of cell lysate was
performed as indicated (lanes 7-12) followed by
immunoprecipitation with the indicated antibodies and immunoblotting
with anti-GFP antibody. Co-immunoprecipitated GFP-YB-1 is indicated by
<1 and <2. C, determination of RNA-degradation by RNase A
treatment. To assure complete degradation of mRNA by RNase A
treatment extracts were subjected to reverse transcription and
amplification of GAPDH cDNA by PCR. Consistent degradation of RNA
was apparent (lanes 5-7) compared with untreated extract
(lanes 2-4). In lane 1 the DNA ladder is given.
D, endogenous YB-1 interacts with FLAG-SRp30c.
Co-immunoprecipitation studies were repeated with lysates from cells
that express FLAG or FLAG-SRp30c protein. Following immunoprecipitation
of endogenous YB-1 with specific anti-YB-1 antibody (lane 3)
FLAG-SRp30c could be co-immunoprecipitated (size indicated by
<1).
1, aa 21-147) are sufficient for association with FLAG-tagged
SRp30c (Fig. 2A, lanes 1 and 2,
indicated by <1). In comparison to full-length GFP-YB-1 wild type a
similarly high percentage of expressed GFP-YB-1
1 (Fig.
2A, lane 3) was co-immunoprecipitated by
anti-FLAG antibody (Fig. 2A, lane 4, indicated by
<2). After removal of the YB-1 N-terminal protein domains aa 1-147
(GFP-YB-1
3) only a weak interaction with SRp30c could be detected
(Fig. 2A, lanes 7 and 8, indicated by
<4). This low-level binding affinity was mapped to aa 260-317 of the
C terminus (GFP-YB-1
4), whereas GFP-YB-1
2 comprised of
aa 147-225 was not co-immunoprecipitated. Taken together, these
results demonstrate that YB-1 strongly interacts with SRp30c via aa
21-147, whereas a low affinity binding domain resides in the C
terminus (aa 260-317).
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Fig. 2.
Mapping of the YB-1 domain required for
SRp30c interaction. A, full-length (GFP-YB-1 wild type)
and truncated GFP-tagged YB-1 proteins (GFP-YB-1 1-4) were analyzed
for protein interaction with FLAG-tagged SRp30c by
co-immunoprecipitation of overexpressing HEK-293T cell lysates.
Expressed proteins before (L) and after immunoprecipitation
(I.P.) with anti-FLAG antibody were separated by SDS-PAGE
and immunoblotted with anti-GFP antibody. B, schematic
illustration of GFP-YB-1 constructs with amino acid residues given on
top of the bars. Arrow head numbers indicate
protein bands as indicated in A.
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Fig. 3.
a, GFP-YB-1 and SRp30c
co-localize in the nucleus of HeLa cells. HeLa cells are co-transfected
with either GFP or GFP-YB-1 expression plasmid and either FLAG or
FLAG-SRp30c vector and stained with monoclonal M2 anti-FLAG antibody.
The YB-1 distribution was visualized by its GFP tag fluorescence,
whereas FLAG-SRp30c was detected with a rhodamine-conjugated goat
anti-mouse antibody by confocal laser scanning microscopy.
Co-localization of the two antigens is revealed by yellow in
the merged images. Panel A, GFP and FLAG-antigen is
diffusely detected throughout the cells. Panel B, GFP-YB-1
predominantly localizes in the cytoplasm, whereas FLAG-SRp30c is
detected in the nucleus in a speckular pattern (panel C).
With co-expression of both proteins, GFP-YB-1 and SRp30c, a nuclear
co-localization is detected (panel D). b, nuclear
shuttling of endogenous YB-1 with overexpressed FLAG-SRp30c. Upon
transiently overexpressing FLAG or FLAG-SRp30c protein in HEK-293T
cells immunohistochemistry was performed using a specific anti-YB-1
antibody. Here, a nuclear shuttling of endogenous YB-1 protein was only
present with overexpressed FLAG-SRp30c protein (B) but not
the FLAG alone (A).
1, was tested for co-localization with
SRp30c. As can be seen in Fig.
4A (images c and
d) construct GFP-YB-1
1 co-localized with SRp30c in the
nucleus, whereas construct GFP-YB-1
3 lacking aa 1-147 localized in
the cytoplasm when co-expressed with SRp30c (Fig. 4B,
images c and d). These results support the notion
that the N-terminal YB-1 protein domains are responsible for the
interaction with SRp30c.
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Fig. 4.
A and B,
the N-terminal region of YB-1 co-localizes with SRp30c in the
nucleus. GFP-coupled deletion constructs of YB-1 (GFP-YB-1 1 or
GFP-YB-1
3, compare with Fig. 2B) were co-transfected
with FLAG-SRp30c in HeLa cells. SRp30c distribution was detected by
staining with an M2 anti-FLAG antibody and visualized by
rhodamine-conjugated anti-mouse antibody. A, confocal laser
scanning microscopy revealed that GFP-YB-1
1 alone is localized in
the cytoplasm (a), whereas it co-localizes with FLAG-SRp30c
in the nucleus in cells overexpressing both proteins (b,
TRITC; c, FITC; d,
merge). B, with GFP-YB-1
3 alone a similar
cytoplasmic localization is present (a), which did not
change after co-expression of GFP-YB-1
3 and FLAG-SRp30c
(b, TRITC; c, FITC;
d, merge).
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Fig. 5.
Heat shock treatment reverses
co-localization of GFP-YB-1 and SRp30c in HeLa cells. HeLa cells
were co-transfected with either GFP or GFP-YB-1 combined with either
FLAG- or FLAG-SRp30c vector. Cells were heat-stressed for 1 h at
42 °C and allowed to recover for 1 h at 37 °C. By confocal
laser scanning microscopy GFP and GFP-YB-1 were visualized (FITC
filter). At the same time FLAG- and FLAG-SRp30c protein were detected
by TRITC filter. SRp30c reveals a predominant nuclear localization
after heat stress (panel A) with diffuse pattern of GFP
throughout the cells. The cytoplasmatic localization of GFP-YB-1 is not
changed by heat stress (panel B) when co-transfected with an
empty FLAG-vector. Heat shock treatment prevents nuclear
GFP-YB-1/SRp30c co-localization with both proteins overexpressed in
combination (panel C).
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Fig. 6.
A-D, YB-1 affects alternative
splicing of the E1A minigene. An in vivo splicing assay
was performed using the adenovirus E1A minigene model system.
A, schematic presentation of the alternative spliced
isoforms of the E1A pre-mRNA. The major isoforms 13 S, 12 S, and 9 S are generated by three different 5' splice sites. B, the
E1A minigene was introduced into HeLa cells, and splicing was analyzed
by quantitative reverse transcription PCR. cDNA was analyzed by
electrophoresis in 3% agarose ethidium bromide gels and quantified
densitometrically. C and D, results are expressed
as relative intensities of corresponding bands compared with minigene
alone, the intensities of which was set as 1. Given are means of a
representative experiment that was confirmed in seven independent
experiments. B and C, upon overexpression of
increasing YB-1 amounts a concentration-dependent change in
splicing pattern toward the 12 S isoform is detected. At the same time
the 9 S isoform is reduced. B and D,
overexpression of SRp30c alone leads to preferential formation of the
13 S isoform, whereas combined expression of YB-1 at increasing
concentrations and SRp30c at a fixed concentration leads to a minor
reduction of 13 S formation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
that strengthens the binding of SRp30c to AG-rich exonic splicing enhancers within exon 7 of the human survival motoneuron gene to increase exon 7 inclusion (37).
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ACKNOWLEDGEMENTS |
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We are grateful for plasmid donations by S. Stamm, Max-Planck Institute of Neurobiology, Martinsried, Germany (pCR-FLAG-30c) and A. R. Krainer, Cold Spring Harbor Laboratories, New York, NY (E1A minigene pMTE1A). Expert technical assistance was provided by Marina Wolf El-Houari.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft SFB 542, Project C4 (to P. R. M.)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.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed: Medizinische Klinik II, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany. Tel.: 49-241-8089532; Fax: 49-241-8082446; E-mail: pmertens@ukaachen.de.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M212518200
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ABBREVIATIONS |
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The abbreviations used are: SR, serine-arginine-rich; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; CM, conditioned medium; TRITC, tetramethylrhodamine isothiocyanate; aa, amino acids; FITC, fluorescein isothiocyanate.
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