[CANCER RESEARCH 64, 8901– 8905, December 15, 2004]
Enhancer-Dependent Splicing of FGFR1 ␣-Exon Is Repressed by RNA
Interference-Mediated Down-Regulation of SRp55
Wei Jin and Gilbert J. Cote
Department of Endocrine Neoplasia and Hormonal Disorders, Unit 435, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
ABSTRACT
The FGFR1 gene transcript is alternatively processed to produce functionally different receptor forms. Previously, we identified a 69-nucleotide
exonic splicing enhancer (ESE) required for ␣-exon inclusion in JEG3
cells. In the present study, we found that this sequence is composed of
three independent elements, two smaller ESE sequences flanking an exonic splicing silencer sequence. Ultraviolet cross-linking and immunoprecipitation identified ESE-specific binding of the splicing regulator SRp55.
A RNA interference-mediated decrease in SRp55 confirmed the significance of this interaction. There was a 6- to 14-fold decrease in exon
inclusion on ablation of SRp55. In SNB19 glioblastoma cells, which normally skip this exon, SRp55 was also demonstrated to play a role in exon
inclusion after the removal of intronic splicing silencer sequences. These
observations indicate that SRp55 plays a major role in maintaining normal FGFR1 ␣-exon inclusion, which is subject to dominant intronic
splicing silencer-mediated and exonic splicing silencer-mediated inhibition
in SNB19 cells.
inclusion or exclusion pathway is the specific target of glial cell
transformation is unclear. Transformation is associated with increased
expression of the splicing repressor polypyrimidine tract-binding protein, but overexpression of this protein in cells that normally include
the exon is unable to induce a complete change in splicing (8, 9). In
the present study, we performed a closer examination of the ESE
sequence required for FGFR1␣ production to ascertain whether exon
inclusion is mediated by SR proteins and whether this process is
targeted during glial cell transformation.
MATERIALS AND METHODS
Plasmid Constructs. The plasmid constructs pFGFR17, ⌬ESE (pFGFR47), ␮AVWT-WT (␣-69), and ⌬ISS (pFGFR104) have been described
previously (6, 7). The ␮AVWT series of deletion plasmids was created via
direct insertion of annealed oligonucleotides or polymerase chain reaction
(PCR)-amplified fragments as described previously (6, 10). Mutations were
introduced through site-specific oligonucleotide-mediated mutagenesis (7).
INTRODUCTION
The construct used to generate the wild-type RNA sequence for ultraviolet
(UV) cross-linking was generated via insertion of a PCR fragment generated
With the complete sequencing of the human genome, it is now clear by primers FP103 and FP104 into pGEMTeasy (Promega, Madison, WI)
that alternative processing of RNA transcripts plays a key role in the followed by a BstXI/BglII deletion to remove extraneous multilinker sequence.
creation of genetic diversity. Accompanying this is a newfound ap- The short hairpin RNA expression plasmids RNA interference (RNAi)-1 and
preciation for the potential role that aberrant RNA processing plays in RNAi-2 were generated according to the methodology described by Brumthe development or progression of human diseases (1). Alternative melkamp et al. (11). A parental expression plasmid, pcDNA3.1/H1A, was
RNA processing of FGFR1 gives rise to two receptor forms that created by replacing the cytomegalovirus promoter in pcDNA3.1 (Invitrogen,
contain either three (FGFR1␣) or two (FGFR1␤) immunoglobulin- Carlsbad, CA) with the human H1A promoter. This was accomplished by first
like extracellular domains. FGFR1␣ has been reported to have lower removing the cytomegalovirus promoter (BglII-NheI deletion) and then insertligand affinity, use a different signal transduction pathway, and have ing a PCR-generated fragment containing the H1A promoter (product of
super-1 and super-2) between the EcoRI and HindIII sites. To generate
altered nuclear localization when compared with FGFR1␤ (reviewed
SRp55-specific short hairpin RNAi (shRNAi) plasmids, two sets of 64-nuclein ref. 2). FGFR1␣ is expressed in normal human pancreatic ductal otide–long oligonucleotides (RNAi-1s, RNAi-1as, RNAi-2s, and RNAi-2as)
epithelium and brain; however, the malignant transformation of these containing BglII and HindIII overhangs were synthesized, purified by PAGE,
tissues results in aberrant production of FGFR1␤ (3, 4).
annealed, and directly inserted into the pcDNA3.1/H1A vector. All of the
The complete mechanism by which alternative RNA processing of plasmids were sequenced to confirm their identities. Specific details on the
FGFR1 is dysregulated during glial cell transformation remains to be oligonucleotides used to create deletion and mutation plasmids are available on
elucidated. Alternative RNA splicing decisions frequently require a request.
Cell Culture, Transfection, and Reverse Transcription-Polymerase
balance of positive and negative selection, with the serine/arginine
(SR) family of proteins having clearly demonstrated roles as media- Chain Reaction. The human glioblastoma cell line SNB19 and human chotors of exon inclusion and members of the heterogeneous nuclear riocarcinoma cell line JEG3 were maintained as described previously (6).
Transfection experiments were performed in 6-well plates using 2 ␮g of
ribonucleoprotein (hnRNP) family inhibiting exon inclusion (5).
plasmid DNA (1 ␮g of each plasmid for cotransfections) per well with
Whereas these protein families are ubiquitously expressed, altered GenePORTER 2 transfection reagent (Gene Therapy Systems, San Diego,
expression of specific proteins has been associated with aberrant CA). Forty-eight hours after transfection, total RNA was isolated using the
splicing (reviewed in ref. 1). Production of FGFR1␣ occurs via mRNA capture kit (Roche Molecular Biochemicals, Indianapolis, IN), and
inclusion of one additional exon. The normal inclusion of this exon reverse transcription-PCRs (RT-PCRs) were performed as described previrequires an exonic splicing enhancer (ESE) sequence (6). Studies ously (6). The ␣-exon inclusion was quantified using a Bio-Rad molecular
using glioblastoma cells have identified two intronic splicing silencer imager (Model GS-363; Bio-Rad, Hercules, CA) by measuring the incorpora32
(ISS) sequences that flank this exon and appear to play a critical role tion of P– end-labeled primer. For detection of endogenous SRp55 and
32
in excluding the exon to produce FGFR1␤ (7). Whether the exon glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, P– endlabeled reverse primers (SRp55-R and GAPDH-2) were used to perform either
18 (SRp55) or 12 (GAPDH) cycles of PCR.
Received 2/26/04; revised 8/12/04; accepted 10/3/04.
Ultraviolet Cross-Linking and Immunoprecipitation. UV cross-linking
Grant support: National Institutes of Health grant CA67946 (to G. Cote).
experiments used JEG3 cell nuclear extracts with incubations performed under
The costs of publication of this article were defrayed in part by the payment of page
in vitro splicing conditions as described previously (8). [32P]UTP-labeled RNA
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
transcripts were generated through SP6-mediated in vitro transcription of
Requests for reprints: Gilbert J. Cote, Department of Endocrinology, Unit 435, The
BamHI-digested plasmid. Immunoprecipitation of the RNA/protein complexes
University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,
was performed after UV cross-linking and RNase digestion using the 1H4
TX 77030. Phone: 713-792-2840; Fax: 713-794-4065; E-mail: [email protected].
antibody (Covance, Berkeley, CA) or ␣SC35 antibody (BD Biosciences, San
©2004 American Association for Cancer Research.
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ALTERNATIVE SPLICING OF THE FGFR1 IS REGULATED BY SRP55
Diego, CA) and GammaBind G Sepharose (Amersham Pharmacia Biotech,
Piscataway, NJ).
Primers. The DNA primers used were as follows: FP103, 5⬘-GAAGTGAGATCTTCCTGGTC-3⬘; FP104, 5⬘-CCCCGTCCCGGATCCAGTTGATG3⬘; super-1, 5⬘-CCATGGAATTCGAACGCTGACGTC-3⬘; super-2, 5⬘-GCAAGCTTAGATCTGTGGTCTCATACAGAACTTATAAGATTCCC-3⬘; RNAi-1s, 5⬘-GATCCCCATGGGTACGGCTTCGTGGATTCAAGAGATCCACGAAGCCGTACCCATTTTTTGGAAA-3⬘; RNAi-1as, 5⬘-AGCTTTTCCAAAAAATGGGTACGCCTTCGTGGATCTCTTGAATCCACGAAGCCGTACCCATGGG-3⬘; RNAi-2s, 5⬘-GATCCCCGCAGATCCAGGTCTCGATCTTCAAGAGAGATCGAGAC CTGGATCTGCTTTTTGGAAA-3⬘; RNAi-2as,
5⬘-AGCTTTTCCAAAAAGCAGATCCAGGTCTCGATCTCTCTTGAAGATCGAGACCTGGATCTGCGGG-3⬘; SRp55-F, 5⬘-AAGATAAGCCACGCACAAGC-3⬘; SRp55-R, 5⬘-TAGATTTCCTGCCTTTTGAT-3⬘; GAPDH-1, 5⬘ACTTTGGTATCGTGGAAGGA-3⬘; and GAPDH-2, 5⬘-CTCAGTGTAGCCCAGGATGC-3⬘.
RESULTS AND DISCUSSION
In previous studies, we established that transient transfection of the
reporter plasmid pFGFR17 can mimic endogenous FGFR1 ␣-exon
splicing (6). Expression of the pFGFR17 minigene in the SNB19
glioblastoma cell line results predominantly in skipping of the ␣-exon,
whereas the exon is predominantly included (80 –90%) in the human
choriocarcinoma-derived cell line JEG3 (Fig. 1). Deletion of a 69-bp
fragment within the ␣-exon coincides with a dramatic decrease in
exon inclusion in JEG3 cells, identifying the presence of an ESE.
Given that many ESE sequences are typically short and found in
multiple copies within some exons, we sought to better characterize
the functional properties of the 69-nucleotide region.
To exclude the potential peripheral effects of other regulatory
sequences, we used the heterologous splicing reporter ␮AVWT,
which was originally used to characterize purine-rich (GAR) exonic
enhancers (11). The ␣-exon enhancer sequence was able to activate
␮AVWT RNA splicing in transfected JEG3 cells to a level comparable with that of the control plasmid, which included four purine-rich
ESE elements (ref. 6; data not shown). Deletion analysis suggested the
presence of multiple regulatory elements (Fig. 2). Removal of the 3⬘
sequence demonstrated biphasic enhancement of splicing. Whereas
construct D1, which removes 9 nucleotides, retained enhancer activity, removal of the additional sequence (construct D2) greatly reduced
splicing activity. This observation suggests that the 17-bp sequence
contains crucial sequences required for ESE function, which is sup-
Fig. 1. Cell-specific recognition of the ␣-exon requires an exon sequence. Schematic
of the FGFR1 splicing reporter pFGFR17. The gray-shaded region indicates the general
location of the ESE sequence, and the arrows indicate the positions of the RT-PCR
primers. SNB19 and JEG3 cells were transfected with pFGFR17 splicing reporter plasmid
or a construct lacking the ESE (⌬ESE). The graph shows the average percentage of
RT-PCR products containing the ␣-exon sequence in three independent transfections
(⫾SD).
ported by deletion construct D6. Surprisingly, further removal of
sequence (construct D3) restores enhancer activity, which is ultimately lost on additional deletion (construct D4). Removal of 16
nucleotides from the 5⬘ end (construct D5) did not affect splicing and
defines a 54-nucleotide sequence that maintains maximal enhancer
activity. Combined, these findings suggest the presence of two ESE
sequences, which we have termed ESE region 1 (ESE1) and ESE
region 2 (ESE2), flanking an exonic splicing silencer (ESS) sequence.
Results of additional mutation analysis support this assignment (Fig.
2). Mutation of the first 5 nucleotides of the D5 construct (construct
M2) clearly reduced splicing. Targeted mutation of two TGC sequences within ESE2 (construct M9) also was able to reduce splicing.
Finally, two different 6-nucleotide mutations targeting the ESS region
(constructs M7 and M8) enhanced overall splicing.
To confirm a regulatory role for the elements defined using the
␮AVWT construct, mutations scanning the same regions were introduced into pFGFR17. Construct M1, which introduced mutations
immediately upstream of ESE1, had no effect on ␣-exon inclusion and
therefore defines the 5⬘ boundary of ESE1 (Fig. 3A). Five constructs
(M2–M6) contain mutations targeting the ESE1 region. Construct M2
contains mutations identical to those introduced into ␮AVWT construct D5 and shows an analogous reduction in splicing (Fig. 3A).
Constructs M4 and M6 define the 3⬘ boundary of ESE1. The M4
construct dramatically reduced ␣-exon inclusion, whereas only a
partial reduction in ␣-exon inclusion from 90% to 52% was seen for
construct M6. To control for the possibility that mutations created
ESS elements rather than destroying ESE elements, two additional
constructs, M3 and M5, were tested. Both demonstrated parallel
reductions in ␣-exon inclusion (Fig. 3A). The ESS1 and ESE2 regions
were targeted with mutations identical to those introduced into
␮AVWT construct D5. These mutations had an analogous effect in
pFGFR17, with constructs M7 and M8 showing enhanced inclusion in
transfected JEG3 cells, and construct M9 showing reduced ␣-exon
inclusion in transfected JEG3 cells (Fig. 3A). Furthermore, mutations
targeting the ESS1 region were also found to enhance ␣-exon inclusion in SNB19 cells, highlighting the potential importance of this
sequence (data not shown). For these regions, however, we cannot
rule out effects resulting from creation of regulatory sequence. Overall, these mutation results confirm the assignment of the ESE1, ESS1,
and ESE2 regions identified using ␮AVWT and strongly support a
requirement for the presence of both ESE elements to maintain
␣-exon inclusion.
Members of the SR family of RNA-binding proteins have a demonstrated role in ESE-mediated splicing (5). To help identify the
specific SR proteins that may interact with the ␣-exon sequence to
enhance splicing, we used ESEfinder (12) to scan for known SR
protein-binding motifs. The 45-nucleotide sequence defined by the
ESE1, ESS1, and ESS2 regions was found to contain four SRp55,
three SF2/ASF, and two SC35 binding sites with scores above the
default thresholds (data not shown). Unfortunately, additional analysis
failed to clearly associate specific mutation-mediated changes in
␣-exon inclusion with loss of predicted SR protein-binding sites.
Therefore, UV cross-linking experiments using JEG3 nuclear extract
were performed to determine whether specific interactions indeed
occurred. Cross-linking to wild-type sequence identified a Mr 50,000
band, consistent with SRp55 interaction (Fig. 3B). Introduction of
either the M4 or M5 mutation, predicted by ESEfinder to impact
SRp55 binding, specifically reduced binding of the Mr 50,000 band.
These mutations were also associated with an increased binding of
three smaller proteins (approximate molecular weights of 29,000,
32,000, and 38,000). It is unclear whether these proteins represent
nonspecific recruitment of splicing inhibitory proteins (e.g., hnRNPs)
or specific binding to the mutated sequence. It is interesting to note
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ALTERNATIVE SPLICING OF THE FGFR1 IS REGULATED BY SRP55
Fig. 2. Fine mapping of the ESE sequence. The heterologous splicing enhancer reporter plasmid ␮AVWT is
shown schematically, with the relative position of the
RT-PCR primers indicated by the arrows. The wild-type
plasmid (WT) contains the ESE sequence identified in
Fig. 1 inserted between the BglII and SpeI sites. The
inserted sequence of the plasmids containing deletions
(D1–D6) or mutations (M2–M9) is shown below the
wild-type sequence, with the mutated nucleotides underlined. The relative splicing level compared with that of
the wild-type plasmid is indicated to the right of each
insert sequence. A representative autoradiograph depicting the splicing for all of the plasmids is shown in the
bottom panel. The mapped boundaries of the ESE and
ESS sequences are indicated.
that whereas the mutations are predicted to create SRp40- and SC35binding sites, they are clearly not associated with enhanced ␣-exon
inclusion. A similar reduction in the Mr 50,000 band was obtained
with the M9 mutation, which targets two SRp55-binding consensus
sites within the ESE2 region (data not shown). Because of the lack of
SRp55-specific antibody and the potential for nonspecific protein
binding to obscure important differences in binding profiles, we
immunoprecipitated cross-linked complexes using the anti-SR monoclonal antibody 1H4. Although Western blot analysis of JEG nuclear
extract confirmed the presence of all of the major SR proteins, the Mr
50,000 band was the only detectable protein that remained bound to
wild-type sequence after immunoprecipitation (Fig. 3C; data not
shown). Similar experiments performed using anti-SC35 monoclonal
antibody failed to demonstrate binding (data not shown). Based on the
size of the Mr 50,000 band and the observation that binding was
disrupted by introduction of the M4 mutation, we concluded this
protein to be SRp55, a well-characterized enhancer of exon inclusion.
To confirm a role for SRp55 in the regulation of ␣-exon inclusion,
we obtained a SRp55 expression plasmid (13) and created two SRp55
shRNAi expression plasmids. The shRNAi target sites are illustrated
in Fig. 4A. Cotransfection of the SRp55 expression plasmid with
pFGFR17 did not significantly increase the level of ␣-exon inclusion
(86% versus 85% using a vector control; Fig. 4B). This lack of
response may have been due in part to the relative abundance of
SRp55 in JEG3 cells because transfection did not lead to a dramatic
increase in expression. However, shRNAi-mediated reductions in
SRp55 were clearly correlated with a reduction in ␣-exon inclusion. In
JEG3 cells cotransfected with the RNAi-1 plasmid, exon inclusion
was reduced from 85% to 15%; an even greater reduction in exon
inclusion was observed for the RNAi-2 plasmid (Fig. 4B). JEG3 cells
transfected with control shRNAi showed no effect on the level of
SRp55 expression or FGFR1 splicing (data not shown).
The observation that reduced SRp55 expression correlates with a
loss of ␣-exon inclusion strongly supports a role for this protein in
JEG3 cell-mediated splicing. It is unclear, however, if a loss of SRp55
function, either through direct effects on SRp55 level or activity or
mediated through ESS1, plays a role in the exclusion of the ␣-exon
observed in glioblastoma cells. Western blot and RT-PCR analysis
detected no apparent differences in the SRp55 level between JEG3
and SNB19 cells (data not shown). Cotransfection of the pFGFR17
splicing reporter with either RNAi-2 or SRp55 expression plasmid
was also not associated with substantial changes in the level of ␣-exon
inclusion, although the trends were in the appropriate direction (Fig.
4C). Therefore, either the splicing inhibitory pathway is dominant
over the ␣-exon inclusion pathway or SRp55 produced in SNB19 cells
is not able to mediate enhanced splicing. To address the latter possibility, a FGFR1 minigene with both ISS elements deleted (⌬ISS) was
cotransfected into SNB19 cells. We showed previously (7) that deletion of both ISS elements led to a dramatic increase in ␣-exon
inclusion in SNB19 cells (Fig. 4C, compare Lanes 1 and 4). A
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ALTERNATIVE SPLICING OF THE FGFR1 IS REGULATED BY SRP55
Fig. 3. Definition of the FGFR1 ␣-exon ESE region and its binding
proteins. A, schematic of the splicing reporter pFGFR17 identifying the
relative position and sequence of the three regulatory motifs defined in Fig.
2. A series of plasmid constructs (M1–M9) containing the specific mutated
sequence is indicated below the wild-type sequence. A representative autoradiograph examining the alternative splicing in transfected JEG3 cells by
RT-PCR for the plasmids listed is shown, with the percentage of ␣-exon
inclusion indicated below each lane. I, inclusion product; E, exclusion
product. B. In vitro transcribed 70-nucleotide labeled RNAs of either wildtype or the indicated mutant sequences were incubated with JEG3 nuclear
extract under splicing conditions. After UV cross-linking, samples were
digested with RNase, proteins were separated by 10% SDS-PAGE, and
RNA-binding proteins were identified by autoradiography. C. Immunoprecipitation (IP) of UV cross-linked complexes with the monoclonal antibody
1H4 identified a Mr 50,000 protein as the major SR protein associated with
the wild-type (WT) sequence. A lane of total UV cross-linked complexes (T)
is provided for comparison.
reduction of SRp55 mediated through transfection of RNAi-2 caused
a decrease in ␣-exon inclusion from 83% to 46%, whereas cotransfection of ⌬ISS and the SRp55 expression plasmid had no effect on
splicing. RNAi-2 was also able to mediate a similar reduction ␣-exon
inclusion when cotransfected with FGFR1 minigene plasmids containing ESS mutations (data not shown). These results clearly demonstrate that endogenous SRp55 is capable of enhancing ␣-exon
inclusion and therefore is not targeted for inactivation in the SNB19
cell line. Instead, it appears that the ␣-exon inclusion pathway is
primarily suppressed by the presence of splicing inhibitors that interact with the ISS elements and possibly with the ESS1 element.
These studies reported the identification and detailed mapping of
ESE1, ESE2, and ESS1 elements within the FGFR1 ␣-exon. The
organization of ESE elements in close proximity to or overlapping
Fig. 4. SRp55 mediates ␣-exon inclusion. A, schematic
of the SRp55 protein structure (15). RNA recognition
motif (RRM) domains and their relative ribonucleoprotein
motif (RNPs) are indicated. The primers used for endogenous SRp55 RT-PCR are indicated by the horizontal
arrows, and the positions targeted by RNAi are indicated
by the vertical arrows. B. The top panel shows a representative autoradiograph examining the alternative splicing
in JEG3 cells by RT-PCR after cotransfection with pFGFR17 and either a vector, SRp55 expression vector,
RNAi-1 expression vector, or RNAi-2 expression vector.
The average percentage of ␣-exon inclusion in three independent transfections is indicated below each lane. The
bottom panels show the expression levels of endogenous
SRp55 and GAPDH in the same RNA samples as detected
via low-cycle RT-PCR. C, schematic of the FGFR1 splicing reporter pFGFR17 with the gray-shaded regions indicating the general locations of the ISS sequences. Bottom
panel, a representative autoradiograph examining alternative splicing in SNB19 cells after cotransfection with pFGFR17 or pFGFR17 with the ISS elements deleted (⌬ISS)
and either a vector, RNAi-2 expression vector, or SRp55
expression vector. The average percentage of ␣-exon inclusion in three independent transfections is indicated below each lane. I, inclusion product; E, exclusion product.
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ALTERNATIVE SPLICING OF THE FGFR1 IS REGULATED BY SRP55
ESS elements has been proposed as a generalized mechanism for
regulation of exon recognition. In this model, the binding of negative
regulators to ESS elements suppresses SR protein binding to ESE
elements (5). The observation that ESS mutations enhance SRp55dependent ␣-exon inclusion suggests a similar mechanism may exist
for FGFR1. However, because ␣-exon inclusion was also SRp55
dependent in the ⌬ISS plasmids, we cannot rule out the possibility that
mutations targeting the ESS create ESE sequences. Additionally, we
have clearly identified SRp55 as a key regulator of ESE-mediated
enhancement of splicing. This is the first description of a RNAimediated reduction in mammalian SRp55 leading to an exon-skipping
phenotype. This finding is somewhat unexpected, given that the SR
family of proteins is known to have partial functional redundancy, but
it is not without precedent. Previously, an antisense-mediated reduction of SRp55 was able to increase skipping of CD45 exon 4 in
transfected COS cells (13). Additionally, in Drosophila mutants lacking B52, the homolog of human SRp55, lethality was thought to arise
from aberrant splicing in tissues in which B52 is the predominant SR
protein, such as the brain (14). Given that ␣-exon inclusion occurs
predominantly in the brain, it is easy to speculate that production of
SRp55 or a brain-specific isoform plays a critical role in normal
FGFR1 splicing. In the few glioblastoma cell lines that we have
examined, functional SRp55 continues to be expressed, suggesting
that this protein is not a target of the transformation process. Whether
this is true in glioblastoma tumors remains to be addressed.
ACKNOWLEDGMENTS
We thank R. Lafyatis for generously providing the SRp55 expression
plasmid. We also thank L. J. Sanger, H. Marks, and L. D. Morales for their
excellent technical contribution to this work and I. Bruno, H. Cheung, and D.
Norwood for critical reading.
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Research.
Enhancer-Dependent Splicing of FGFR1 α-Exon Is
Repressed by RNA Interference-Mediated Down-Regulation
of SRp55
Wei Jin and Gilbert J. Cote
Cancer Res 2004;64:8901-8905.
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