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INTRODUCTION |
Cancer results from the accumulation of multiple genetic
mutations, eventually leading to deregulated growth and/or
differentiation. Chromosomal anomalies such as translocations are a
common mechanism to introduce genetic changes. Many human sarcomas are
characterized by specific chromosomal translocations, which result in
the creation of novel fusion genes; these new genes are thought to be
critical for tumorigenesis. EWS/FLI is a fusion gene created
by the translocation t(11;22)(q24;q12) found in >85% of all Ewing's
sarcomas (1). The resulting fusion protein combines the N terminus of
EWS, an RNA-binding protein of unknown function, with the C terminus of the ETS family transcription factor FLI-1 (2). EWS is fused to related
transcription factors of the ETS family in the remaining cases of
Ewing's sarcoma (3-6). This consistent combination suggests that both
the retained region of EWS, which is capable of transcriptional activation, and the ETS DNA-binding domain of the fusion protein contribute critical functions in the genesis of these tumors. TLS and TAFII-68,
EWS-related genes, are also translocated and fused to
transcription factor gene partners in human sarcomas (7-12).
The transcriptional activity and cellular transformation functions of
EWS/FLI are not completely concordant. Although most of the
transformation potential appears to reside in the N-terminal 82 amino
acids of EWS, the entire EWS domain is required to provide full
transformation efficiency. Interestingly, the region of EWS that
contributes most to transactivation by EWS/FLI does not coincide with
the domain that confers the most efficient transforming activity (13).
In addition, an introduced mutation in EWS/FLI that ablates ETSspecific DNA binding only partly diminishes transformation (14), suggesting an ETS transcription-independent aspect of transformation.
Transcription and post-transcriptional processing are closely coupled
processes in vivo (15), and it is likely that EWS and TLS
participate in both transcription and pre-mRNA splicing. TLS has
the capacity to alter 5'-splice site selection of the adenoviral
E1A gene in certain cell types (16, 17). Both EWS and TLS
interact with the serine/arginine-rich splicing proteins TASR-1 and
TASR-2 (16, 18) and can be copurified with other components of the
splicing machinery such as
hnRNPA11 and hnRNPC1/C2 (19).
EWS interacts with the essential splicing factors SF1 (20) and U1C (21)
as well as with CUG-BP, a protein that is involved with the
alternative splicing of the APP gene and the human cardiac
troponin T gene. The binding of CUG-BP to cardiac troponin T
pre-mRNA is thought to disrupt splicing and to contribute to the
pathogenesis of myotonic dystrophy (22). Interestingly, EWS and TLS
both copurify with the splicing factor PSF (22), which is
commonly involved in chromosomal translocations found in human
papillary renal cell carcinomas (23).
Interaction with the splicing factor U1C represses EWS/FLI-mediated
transactivation (21). The ability of a splicing factor to influence the
transcriptional activity of EWS/FLI suggested that, conversely, EWS/FLI
may play a direct role in splicing or have the ability to alter the
composition of functional splicing complexes. To address this question,
we used an in vivo splicing assay based on the alternative
splicing of the adenoviral E1A gene. This assay is a well
established method to show the ability of proteins such as hnRNPA1 and
SF2 to influence splice site selection. The relative concentrations of
these two proteins to one another determine which splice site in the
E1A gene is favored. High concentrations of hnRNPA1 relative
to SF2 lead to use of distal 5'-sites, whereas higher concentrations of
SF2 favor proximal 5'-splice sites (24). Utilization of different
5'-splice sites leads to the subsequent formation of different amounts
of alternatively spliced isoforms of E1A. We used this assay to show
that although EWS and EWS/FLI have no direct effect on the splicing of
E1A when expressed alone, the tumor-associated EWS/FLI, but
not wild-type EWS, can oppose the change in splicing pattern induced by
expression of hnRNPA1.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The epitope-tagged constructs pCFLAG-EWS and
pCFLAG-EWS/FLI were generated as previously described (21).
FLI-1 coding sequence was amplified by PCR from HL-60
cDNA and cloned in-frame into pCFLAG at the EcoRI site.
hnRNPA1 coding sequence was amplified by PCR from pGA1 (a
gift from Dr. Adrian Krainer) (25) and cloned in-frame into pCFLAG at
the EcoRI site. EWS/FLI point mutation constructs
were made by PCR-based strategy (14), and the del54 mutant was
generated as described previously (26). These inserts were cloned into
pCFLAG at the EcoRI site. Amino acid numbers for point
mutations refer to the FLI-1 sequence. The absence of PCR
errors in all plasmids was verified by automated sequence analysis.
The clone containing the E1A minigene, pBSV-E1ATFN, was a generous gift from Dr. James Manley and has been previously described (27).
In Vivo Splicing Assays--
293T cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. 3 × 106 cells were seeded on 100-mm plates and
transfected 16-18 h later. Transfections of only one plasmid contained
12 µg of DNA (pCFLAG vector, pCFLAG-EWS, pCFLAG-EWS/FLI,
pCFLAG-FLI-1, or pCFLAG-hnRNPA1) mixed with 32 µl of FuGene (Roche
Molecular Biochemicals) added to cells according to the manufacturer's
recommendations. Transfections of pCFLAG-hnRNPA1 in combination with
another plasmid contained 12 µg of pCFLAG-hnRNPA1 and 6 µg of the
additional plasmid mixed with 55 µl of FuGene. Cells were harvested
48 h after transfection. Cells were washed with phosphate-buffered
saline, trypsinized with 2 ml of trypsin, and resuspended in 8 ml of
Dulbecco's modified Eagle's medium. Following trypsinization, 90% of
the cells were washed with phosphate-buffered saline and resuspended in
1 ml of Trizol (Life Technologies, Inc.), and RNA was isolated
according to the manufacturer's recommendations. The remaining 10% of
the cells were washed with phosphate-buffered saline and lysed in 100 µl of radioimmune precipitation assay buffer (150 mM
NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0), and protease inhibitor tablets (Roche
Molecular Biochemicals) used according to the manufacturer's
recommendations). Following a brief sonication, lysates were used for
Western blotting. RT-PCR detection of E1A isoforms was performed as
previously described using [
-32P]ATP-labeled
oligonucleotides E1A569 and E1A1315 (28). Detection was carried out by
autoradiography, and isoforms were quantitated with ImageQuant on a
PhosphorImager. The average results of three independent experiments,
with two different PCR cycle numbers for each, are shown.
A673 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. 9 × 105
cells were seeded on 100-mm plates and transfected 16-18 h later. Transfections contained 1.6 µg of pBSV-E1ATFN and 16.4 µg of pCFLAG empty vector, pCFLAG-EWS, pCFLAG-EWS/FLI, or pCFLAG-hnRNPA1 mixed with
72 µl of FuGene according to the manufacturer's recommendations. Cells were harvested, and subsequent experiments were performed as
described above.
Consistent results were obtained for all PCRs in this assay when
repeated with differing numbers of PCR cycles and varying amounts of
input cDNA template to ensure that all results were within the
quantitative range of the assay.
Western Blotting--
Cell extracts were quantitated for total
protein (Bio-Rad protein assay) according to the manufacturer's
recommendations, and 50 µg of protein was resolved by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose. Epitope-tagged proteins were detected with anti-FLAG M2
monoclonal antibody (Babco) at a concentration of 10 µg/ml.
Proteins were visualized with a peroxidase-conjugated anti-mouse
antibody at a dilution of 1:5000, followed by enhanced chemiluminescence (Amersham Pharmacia Biotech) and autoradiography.
Real-time PCR--
RT-PCR detection of total cellular
E1A message was performed on a Model 7700 sequence
detector machine (Applied Biosystems) using the TaqMan Gold RT-PCR kit
(Applied Biosystems) according to the manufacturer's
recommendations. Briefly, 10 µl of cDNA synthesized from 500 ng
of total RNA (isolated as described above) was mixed with 1× TaqMan
Universal PCR Master Mix (Applied Biosystems), 5 µM probe
(6FAM-ACCGAAGAAATGGCCGCCAG-TAMRA, Applied Biosystems), 300 nM final concentration E1A forward primer
(ATTATCTGCCACGGAGGTGTT), and 300 nM final concentration E1A
reverse primer (TCGATCAGCTGGTCCAAAAG) for a final reaction volume of 50 µl. Samples had dual annealing cycles at 55 and 60 °C for 1 min each, consecutively. Samples underwent 40 rounds of
thermocycling. Samples included COS cDNA and mock cDNA
synthesized without reverse transcriptase as negative controls;
untransfected, A1+vector, A1+EWS, A1+EWS/FLI, A1+FLI-1, A1+EF
del54, A1+EF W321R, and A1+EF I347E as experimental 293T cDNAs; and pBSV-E1ATFN plasmid as a positive control. The probe and
primers were contained within the common 5'-exon that is included in
all spliced isoforms. TaqMan PCR was repeated on duplicate samples
using glyceraldehyde-3-phosphate dehydrogenase primers and probe
(Applied Biosystems) according to the manufacturer's recommendations.
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RESULTS |
EWS and EWS/FLI Do Not Directly Affect 5'-Splice Site Selection of
E1A--
We performed in vivo splicing assays to see if EWS
or EWS/FLI could affect 5'-splice site selection of the adenoviral
E1A gene. Alternative splicing of E1A
pre-mRNA generated three major isoforms (13 S, 12 S, and 9 S) and
two minor isoforms (11 S and 10 S) (Fig.
1) (25). Proteins such as hnRNPA1, SF2,
and TLS have the ability to generate these isoforms in differing
amounts by altering 5'-splice site selection (16, 17, 24). In this established assay, the proteins of interest are coexpressed with an
E1A minigene. Alternatively spliced isoforms of E1A are
detected by RT-PCR using the primers shown in Fig. 1. 293T cells are
human embryonic kidney cells immortalized with the adenoviral
E1A gene (29) and therefore contain endogenous
E1A. To analyze alternative splicing of E1A in 293T cells,
we amplified E1A mRNA by RT-PCR (Fig.
2A) and quantitated the
relative proportions of the three major isoforms (Fig. 2B).
The endogenous representation of the three major E1A isoforms in these
cells comprised 51% of the 13 S isoform, followed by the 12 S isoform
at 41% and then the 9 S isoform at only 5% (Fig. 2, A,
lane 1; and B). Introduced expression of EWS,
EWS/FLI, or FLI-1 did not significantly affect the alternative splicing of E1A (Fig. 2, A, lanes 2 and
3; and B) (data not shown). In contrast,
overexpression of hnRNPA1 caused increased usage of the distal
5'-splice site, resulting in greatly increased levels of the 9 S
isoform and decreased levels of the relative proportion of the 13 S
isoform, consistent with previous reports (Fig. 2, A,
lane 4; and B). Therefore, we concluded that EWS,
EWS/FLI, and FLI-1 did not directly affect E1A splice site selection in these cells, despite expression at levels comparable to hnRNPA1, as
shown by Western blotting (Fig. 2C) (data not shown). All
PCRs were repeated at differing cycle numbers and with varying amounts of input cDNA to verify that results were reproducible and
quantitative.

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Fig. 1.
E1A isoforms generated by in vivo
splicing. Shown is a diagrammatic representation of the
alternatively spliced isoforms of E1A pre-mRNA. The
major isoforms (9 S, 12 S, and 13 S) were generated by alternative
selection of the 5'-splice site. The minor isoforms (10 S and 11 S)
involved usage of an additional internal 3'-splice acceptor site.
Dashed lines represent sequences removed by splicing. Sizes,
in base pairs (bp), of the corresponding RT-PCR products
obtained using the E1A569 and E1A1315 primers (represented by
arrows) are shown.
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Fig. 2.
EWS and EWS/FLI do not directly alter E1A
splice site selection. A, shown is the in
vivo alternative splicing of endogenous E1A
pre-mRNA in 293T cells. 293T cells were transiently transfected
with 12 µg of pCFLAG (lane 1), pCFLAG-EWS
(lane 2), pCFLAG-EWS/FLI (lane 3), or
pCFLAG-hnRNPA1 (lane 4) expression plasmid. Radiolabeled PCR
fragments corresponding to E1A isoforms were resolved on polyacrylamide
gel and detected by autoradiography. B, the E1A iso- forms were quantified with ImageQuant on a PhosphorImager. The
relative percentage of each major isoform is shown: 13 S
(gray bars), 12 S (white bars), and 9 S (black bars). Percentages of 13 S, 12 S, and 9 S isoforms
shown are the averages of three representative independent experiments
with two different PCR cycle numbers for each. Error bars
represent S.D. values. C, 50 µg of total protein isolated
from 293T cell lysates used in A was resolved by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose. Immunoblotting was performed with the anti-FLAG M2
monoclonal antibody, and proteins were visualized by enhanced
chemiluminescence and autoradiography. D, ImageQuant
PhosphorImager quantification was carried out in vivo in
A673 cells. A673 cells were transiently cotransfected with 1.6 µg of
pBSV-E1ATFN and 16.4 µg of pCFLAG vector, pCFLAG-EWS, pCFLAG-EWS/FLI,
or pCFLAG-hnRNPA1. The in vivo splicing assay was performed,
quantitated, and presented as described for B.
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One potential limitation of this assay was that the low levels of the 9 S isoform in 293T cells could preclude detection of an increase in
proximal splice site selection and an associated decrease in the 9 S
isoform. Therefore, we repeated the assay in the Ewing's sarcoma cell
line A673. These cells were cotransfected with the E1A
minigene and empty vector or EWS, EWS/FLI, or
hnRNPA1 expression plasmid, and subsequent results were
compared with those seen in 293T cells. In vivo splicing of
E1A in this cell background gave increased levels of the 9 S isoform
relative to 293T cells. However, overexpression of EWS or EWS/FLI still
did not affect splice site selection. As before, expression of hnRNPA1 increased distal splice site selection, resulting in increased levels
of the 9 S isoform (Fig. 2D). Thus, EWS and EWS/FLI do not
directly affect alternative splicing of E1A in either cell background. The E1A in vivo splicing assay was used first to
demonstrate that the relative concentrations of hnRNPA1 and SF2 to one
another determined splice site selection. These relative concentrations were manipulated by experimental overexpression (24). To detect an
effect on splice site selection, the introduced proteins must be
expressed at levels higher than the endogenous proteins to increase the
relative concentration within the cell. Under our experimental
conditions, EWS/FLI was expressed at ~6-fold higher levels than
endogenous EWS/FLI protein in a Ewing's sarcoma cell line, and hnRNPA1
was expressed at 2-3-fold higher levels than endogenous protein in
293T or Ewing's sarcoma cells (data not shown).
EWS/FLI, but Not EWS, Alters hnRNPA1-dependent Splice
Site Selection--
Because EWS has been shown to co-immunoprecipitate
with hnRNPA1 (19), we considered the possibility that EWS or EWS/FLI may influence hnRNPA1-dependent splice site selection by
changing the composition of splicing complexes. As expected,
cotransfection of hnRNPA1 with empty vector gave a distinct
increase in the 9 S isoform of E1A as compared with levels in
untransfected 293T cells (Fig. 3,
A, lanes 1 and 2; and B).
Cotransfection of EWS with hnRNPA1 did not change
the pattern of isoforms seen with hnRNPA1 and empty vector
(Fig. 3, A, lanes 2 and 3; and
B). However, the addition of EWS/FLI to the
transfection with hnRNPA1 significantly decreased the
relative amount of 9 S isoform generated by 25%, with a concomitant
increase in the 12 S and 13 S isoforms (Fig. 3, A,
lanes 2 and 4; and B). The
interference with hnRNPA1-mediated splice site selection was likely to
come from the FLI-1 portion of the fusion protein since wild-type EWS
did not demonstrate this activity. To confirm this, a plasmid
containing the full coding sequence of FLI-1 was
cotransfected with hnRNPA1, and the resulting alternative
splicing pattern was analyzed. FLI-1 expression at levels similar to
EWS/FLI did not affect the E1A splicing pattern (Fig. 3, A,
lanes 2 and 5; and B). However,
elevated FLI-1 expression opposed the hnRNPA1-dependent
effect on E1A differential splicing in the same manner as EWS/FLI, with
a dramatic decrease in the 9 S isoform and a compensatory increase in
both the 12 S and 13 S isoforms (Fig. 3, A, lanes
2, 4, and 6; and B). The effects seen with EWS/FLI and FLI-1 were not a result of less hnRNPA1 expression in these transfected cells, as Western blot analysis showed
that hnRNPA1 levels were consistent in all samples (Fig. 3C).

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Fig. 3.
EWS/FLI and FLI-1 oppose hnRNPA1-mediated
5'-splice site selection. A, shown is the in
vivo alternative splicing of endogenous E1A
pre-mRNA in 293T cells. 293T cells were transiently cotransfected
with 12 µg of pCFLAG-hnRNPA1 and 6 µg of pCFLAG vector (lane
2), pCFLAG-EWS (lane 3), or pCFLAG-EWS/FLI (lane
4); 0.3 µg of pCFLAG-FLI-1 and 5.7 µg of pCFLAG vector
(lane 5); or 6 µg of pCFLAG-FLI-1 (lane 6).
Untransfected cell lysate is represented in lane 1.
Resolution of radiolabeled PCR fragments corresponding to E1A isoforms
was performed on polyacrylamide gel and detected by autoradiography.
B, the E1A isoforms were quantified with ImageQuant on a
PhosphorImager as described for Fig. 2B. C, 25 µg of total protein isolated from 293T cell lysates used in
A was resolved by SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose. Immunoblotting was performed with the
anti-FLAG M2 monoclonal antibody, and proteins were visualized by
enhanced chemiluminescence and autoradiography. All proteins were
FLAG-tagged and resolved on the same gel.
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EWS/FLI Mutants Show Association between Ability to Alter Splice
Site Selection and Transforming Activity--
Several EWS/FLI mutant
proteins were previously analyzed for their ability to bind DNA and to
transform NIH-3T3 cells (14). The activities of the mutants and the
wild-type counterparts are reviewed in Table
I. The del54 mutant contains a gross
deletion of 54 of the 80 amino acids comprising the ETS domain
of EWS/FLI. This deletion ablates DNA-binding activity and should
abolish protein-protein interactions or any other processes
mediated by the ETS domain. Two point mutations that ablate ETS
DNA-binding activity, replacement of tryptophan 321 with arginine
(W321R) and replacement of isoleucine 347 with glutamic acid (I347E), were also tested in transformation. Although the del54 and W321R mutant
proteins lack transforming activity, I347E retains a somewhat reduced
transforming activity. The differential ability of I347E to transform
cells may be related to the structural integrity of the ETS domain.
Tryptophan 321 is absolutely conserved in all ETS family members, and
this substitution may disrupt protein structure. The substitution of
I347E, however, occurs naturally in the ETS protein PU.1. Therefore,
this change can be accommodated within the structural constraints of
the ETS domain. The transforming activity of the I347E mutant
demonstrates that a component of EWS/FLI-mediated transformation is
independent of ETS DNA binding. For this reason, we looked at the
effects of this mutant protein, as well as the del54 and W321R mutant
proteins, on the alternative splicing pattern of E1A mediated by
hnRNPA1. 293T cells were cotransfected with hnRNPA1 and the
various forms of EWS/FLI, and the resulting splicing
patterns were analyzed. As expected, cotransfection of hnRNPA1 and empty vector gave a large increase in the 9 S
isoform, with compensatory decreases in the 13 S and 12 S isoforms
compared with untransfected cells (Fig.
4, A, lanes 1 and
2; and B). This effect was opposed by the
addition of EWS/FLI (Fig. 4, A, lanes 2 and
3; and B). The EWS/FLI del54 mutant protein
failed to affect the splicing pattern induced by hnRNPA1, giving the
same isoform profile as the addition of empty vector (Fig. 4,
A, lanes 2 and 4; and B).
The EWS/FLI W321R mutant protein significantly altered this profile
(p = 0.02), decreasing the amount of the 9 S isoform by
only 12%, whereas the I347E mutant protein more dramatically decreased
the 9 S isoform by 25% (p = 0.002) (Fig. 4,
A, compare lanes 2, 5, and
6; and B). The effect of the I347E mutant on
alternative splicing did not differ significantly from that seen with
the wild-type EWS/FLI protein. Therefore, there was a correlation between transforming activity and the ability to influence alternative splicing in the absence of DNA-binding activity (Table I). Western blotting confirmed that hnRNPA1 was made at comparable levels in all
samples (Fig. 4C, lower panel) and that EWS/FLI
mutants were expressed at least as highly as the wild-type EWS/FLI
protein (upper panel). To ensure that changes in splicing
were not a secondary effect from changes in the levels of
E1A transcript, we quantitated the levels of E1A
using TaqMan real-time PCR. The transfected constructs did not affect
levels of E1A transcription (data not shown).
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Table I
Transforming versions of EWS/FLI interfere with hnRNPA1-mediated
5'-splice site selection
"DNA binding" indicates the capacity to bind to the FLI-1-binding
consensus sequence and activate transcription. "Transformation"
refers to the capacity of the protein to induce anchorage-independent
growth of NIH-3T3 colonies in soft agar. "Effect on splice site
selection" indicates the ability to interfere with the distal
5'-splice site selection favored by hnRNPA1. +++, activity of EWS/FLI;
+, diminished activity relative to EWS/FLI; , no activity.
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Fig. 4.
Transforming EWS/FLI mutant protein opposes
hnRNPA1-mediated 5'-splice site selection, whereas non-transforming
mutants do not. A, shown is the in vivo
alternative splicing of endogenous E1A pre-mRNA in 293T
cells. 293T cells were transiently cotransfected with 12 µg of
pCFLAG-hnRNPA1 and 6 µg of pCFLAG vector (lane 2),
pCFLAG-EWS/FLI (lane 3), pCFLAG-EWS/FLI del54 (lane
4), pCFLAG-EWS/FLI W321R (lane 5), or pCFLAG-EWS/FLI
I347E (lane 6). Untransfected cells are represented in
lane 1. Radiolabeled RT-PCR fragments corresponding to E1A
isoforms were resolved on polyacrylamide gel and detected by
autoradiography. B, the E1A isoforms were quantified with
ImageQuant on a PhosphorImager as described for Fig. 2B.
C, 25 µg of total protein isolated from 293T cell lysates
used in A was resolved by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose. Immunoblotting was
performed with the anti-FLAG M2 monoclonal antibody, and proteins were
visualized by enhanced chemiluminescence and autoradiography.
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DISCUSSION |
The recurrent examples of splicing proteins associated with EWS,
EWS/FLI, and the related TLS protein (16, 17, 20-22) suggest that
alterations in pre-mRNA splicing may contribute to EWS/FLI-mediated cellular transformation. We used an in vivo splicing assay
with the E1A gene to test the hypothesis that EWS or EWS/FLI
may have the ability to affect alternative splicing. We showed that
although EWS and EWS/FLI do not directly affect the splicing pattern of E1A, the tumor-associated EWS/FLI opposed alternative 5'-splice site
selection induced by hnRNPA1. Furthermore, analysis of mutant proteins
showed an association between the ability to affect alternative splicing and transforming activity, independent of ETS DNA binding. Thus, the splicing-associated functions of EWS/FLI may contribute to transformation.
Although the contribution of aberrant splicing to tumorigenesis remains
unclear, abnormal splicing profiles have been associated with tumors.
For example, two novel aberrantly spliced transcripts of fibroblast
growth factor receptor 3 were identified in a large percentage of both
primary tumors and colorectal carcinoma cell lines. It is thought that
these may confer a selectable growth advantage to cells contributing to
the progression of colorectal tumors (30). Similarly, a spliced variant
of vascular endothelial growth factor is associated with greater
tumorigenicity and increased angiogenic properties in breast carcinoma
cells compared with other isoforms (31). Alternatively spliced forms of
the key cell cycle regulators cyclin D1 and p53 have also been
identified in human tumors and cell lines (32, 33).
There are several examples of splicing factor involvement in
tumor-derived fusion proteins. DEK, which is fused to CAN in a
subset of acute myeloid leukemia, associates with serine/arginine-rich splicing proteins in vitro and in vivo. DEK
remains bound to exon-product RNA after prior removal of an
intron, suggesting a role for DEK in pre-mRNA processing (34). In
papillary renal cell carcinomas, the splicing proteins PSF and
p54NRB/NonO are fused to DNA-binding domains from TFE3
(23). Additionally, EWS is fused to WT-1 in chimeric proteins from
desmoplastic small round cell tumors (10). WT-1 exists as two different
isoforms, one of which binds to DNA at specific promoter sequences,
whereas the other binds more efficiently with splicing factors
(35).
Studies on the PU.1 protein illustrate a precedent in which the ETS
domain regulates both transcription and splicing. Fli-1 and
Pu.1 are the two genes most frequently targeted by viral
integration in Friend virus-induced mouse erythroleukemias (36). PU.1
interacts with TLS and modulates TLS-mediated splice site selection
(17). PU.1 also interacts with the RNA-binding protein
p54NRB and can inhibit splicing of
-globin mRNA. It
is thought that PU.1 may disturb gene regulation at the
post-transcriptional level through sequestration of important
RNA-binding proteins like p54NRB (37). It is important to
note that the ETS domain of PU.1 alone could inhibit splicing (37),
suggesting that this domain is critical for mediating such sequestering interactions.
Our data support a similar role for EWS/FLI in altering 5'-splice site
selection. Interactions between EWS/FLI and hnRNPA1, or other proteins
required for hnRNPA1-mediated splice site selection, may alter the
composition of the active spliceosome. EWS/FLI was more potent than
FLI-1 in its effect on splice selection, perhaps due to the
contribution of EWS interactions with splicing factors (20, 21).
However, the ETS domain was required to influence splicing. Deletion of
the ETS domain ablated the effect, and the point mutation W321R, which
is likely to disrupt the structure of the ETS domain, decreased the
effect substantially. The diminished activity of EWS/FLI W321R in the
splicing assay may reflect a failure to interact with a subset of
proteins involved in splice site selection. The EWS/FLI I347E
substitution is found in wild-type PU.1 and therefore probably does not
greatly alter the structure of the ETS domain. Thus, it is possible
that EWS/FLI I347E maintains critical interactions with RNA-binding
proteins mediated by this domain and therefore retains the full effect
on hnRNPA1-dependent splice site selection.
An alternative possibility is that EWS/FLI may alter the composition of
the spliceosome by influencing the transcription of other splicing
factors. Although it is formally possible that the mutant proteins
I347E and W321R retain the ability to bind a variant DNA sequence, they
lack the expected ETS DNA-binding activity that is well characterized
for proteins containing this modular DNA-binding domain. Therefore, it
seems quite unlikely that the I347E mutant would regulate specific
transcriptional targets in a manner that produces splicing effects
equivalent to those induced by EWS/FLI.
Consistent with our findings, EWS/FLI and TLS/ERG, a fusion protein
found in acute myeloid leukemia, also interfere with the alternative
splicing patterns of E1A mediated by serine/arginine-rich proteins. The
RNA-binding regions of TLS and EWS interact with the
serine/arginine-rich proteins TASR-1 and TASR-2, perhaps recruiting them to sites of transcription. EWS/FLI and TLS/ERG inhibit splicing mediated by TASR proteins, presumably due to loss of the interaction (18, 38). Unlike the effect on hnRNPA1-mediated splicing, wild-type
FLI-1 does not affect splicing by TASR proteins. Our study illustrates
that alterations in splicing can also be mediated by the ETS domain,
adding another level of complexity and specificity to the involvement
of EWS/FLI in splicing.
Altogether, the evidence indicates that EWS/FLI has the ability to
influence the splicing process. The domains of the protein required and
the precise effect depend on the complement of splicing proteins
present and will almost certainly vary with the RNA substrate examined.
E1A is an established experimental splicing substrate, but is not a
physiological substrate for EWS/FLI in Ewing's sarcoma. New
technologies such as intron sequence information system (ISIS), an intron information system used to detect alternative splicing in the
human genome (39), could help to identify such transcripts in the
future. Transcriptional activity contributes substantially to the
transforming activity of EWS/FLI, although it is not absolutely required. Identification of relevant target genes that are
transcriptionally regulated by EWS/FLI may also provide key substrates
to dissect the specific effects of EWS/FLI on splicing.