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[Cancer Research 64, 8901-8905, December 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Enhancer-Dependent Splicing of FGFR1 {alpha}-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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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 {alpha}-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 {alpha}-exon inclusion, which is subject to dominant intronic splicing silencer-mediated and exonic splicing silencer-mediated inhibition in SNB19 cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
With the complete sequencing of the human genome, it is now clear that alternative processing of RNA transcripts plays a key role in the creation of genetic diversity. Accompanying this is a newfound appreciation for the potential role that aberrant RNA processing plays in the development or progression of human diseases (1) . Alternative RNA processing of FGFR1 gives rise to two receptor forms that contain either three (FGFR1{alpha}) or two (FGFR1ß) immunoglobulin-like extracellular domains. FGFR1{alpha} has been reported to have lower ligand affinity, use a different signal transduction pathway, and have altered nuclear localization when compared with FGFR1ß (reviewed in ref. 2 ). FGFR1{alpha} is expressed in normal human pancreatic ductal epithelium and brain; however, the malignant transformation of these tissues results in aberrant production of FGFR1ß (3 , 4) .

The complete mechanism by which alternative RNA processing of FGFR1 is dysregulated during glial cell transformation remains to be elucidated. Alternative RNA splicing decisions frequently require a balance of positive and negative selection, with the serine/arginine (SR) family of proteins having clearly demonstrated roles as mediators of exon inclusion and members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family inhibiting exon inclusion (5) . Whereas these protein families are ubiquitously expressed, altered expression of specific proteins has been associated with aberrant splicing (reviewed in ref. 1 ). Production of FGFR1{alpha} occurs via inclusion of one additional exon. The normal inclusion of this exon requires an exonic splicing enhancer (ESE) sequence (6) . Studies using glioblastoma cells have identified two intronic splicing silencer (ISS) sequences that flank this exon and appear to play a critical role in excluding the exon to produce FGFR1ß (7) . Whether the exon 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{alpha} production to ascertain whether exon inclusion is mediated by SR proteins and whether this process is targeted during glial cell transformation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plasmid Constructs.
The plasmid constructs pFGFR17, {Delta}ESE (pFGFR47), µAVWT-WT ({alpha}-69), and {Delta}ISS (pFGFR104) have been described previously (6 , 7) . The µAVWT 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) . The construct used to generate the wild-type RNA sequence for ultraviolet (UV) cross-linking was generated via insertion of a PCR fragment generated by primers FP103 and FP104 into pGEMTeasy (Promega, Madison, WI) followed by a BstXI/BglII deletion to remove extraneous multilinker sequence. The short hairpin RNA expression plasmids RNA interference (RNAi)-1 and RNAi-2 were generated according to the methodology described by Brummelkamp et al. (11) . A parental expression plasmid, pcDNA3.1/H1A, was created by replacing the cytomegalovirus promoter in pcDNA3.1 (Invitrogen, Carlsbad, CA) with the human H1A promoter. This was accomplished by first removing the cytomegalovirus promoter (BglII-NheI deletion) and then inserting a PCR-generated fragment containing the H1A promoter (product of super-1 and super-2) between the EcoRI and HindIII sites. To generate SRp55-specific short hairpin RNAi (shRNAi) plasmids, two sets of 64-nucleotide–long oligonucleotides (RNAi-1s, RNAi-1as, RNAi-2s, and RNAi-2as) containing BglII and HindIII overhangs were synthesized, purified by PAGE, annealed, and directly inserted into the pcDNA3.1/H1A vector. All of the plasmids were sequenced to confirm their identities. Specific details on the oligonucleotides used to create deletion and mutation plasmids are available on request.

Cell Culture, Transfection, and Reverse Transcription-Polymerase Chain Reaction.
The human glioblastoma cell line SNB19 and human choriocarcinoma cell line JEG3 were maintained as described previously (6) . Transfection experiments were performed in 6-well plates using 2 µg of plasmid DNA (1 µg of each plasmid for cotransfections) per well with GenePORTER 2 transfection reagent (Gene Therapy Systems, San Diego, CA). Forty-eight hours after transfection, total RNA was isolated using the mRNA capture kit (Roche Molecular Biochemicals, Indianapolis, IN), and reverse transcription-PCRs (RT-PCRs) were performed as described previously (6) . The {alpha}-exon inclusion was quantified using a Bio-Rad molecular imager (Model GS-363; Bio-Rad, Hercules, CA) by measuring the incorporation of 32P–end-labeled primer. For detection of endogenous SRp55 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, 32P–end-labeled reverse primers (SRp55-R and GAPDH-2) were used to perform either 18 (SRp55) or 12 (GAPDH) cycles of PCR.

Ultraviolet Cross-Linking and Immunoprecipitation.
UV cross-linking experiments used JEG3 cell nuclear extracts with incubations performed under in vitro splicing conditions as described previously (8) . [32P]UTP-labeled RNA transcripts were generated through SP6-mediated in vitro transcription of BamHI-digested plasmid. Immunoprecipitation of the RNA/protein complexes was performed after UV cross-linking and RNase digestion using the 1H4 antibody (Covance, Berkeley, CA) or {alpha}SC35 antibody (BD Biosciences, San 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'-CCCCGTCCCGGATCCAGTTGATG-3'; 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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
In previous studies, we established that transient transfection of the reporter plasmid pFGFR17 can mimic endogenous FGFR1 {alpha}-exon splicing (6) . Expression of the pFGFR17 minigene in the SNB19 glioblastoma cell line results predominantly in skipping of the {alpha}-exon, whereas the exon is predominantly included (80–90%) in the human choriocarcinoma-derived cell line JEG3 (Fig. 1)Citation . Deletion of a 69-bp fragment within the {alpha}-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.



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  		Fig. 1. Cell-specific recognition of the {alpha}-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 ({Delta}ESE). The graph shows the average percentage of RT-PCR products containing the {alpha}-exon sequence in three independent transfections (±SD).

 
To exclude the potential peripheral effects of other regulatory sequences, we used the heterologous splicing reporter µAVWT, which was originally used to characterize purine-rich (GAR) exonic enhancers (11) . The {alpha}-exon enhancer sequence was able to activate µAVWT 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)Citation . 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 supported 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)Citation . 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.



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  		Fig. 2. Fine mapping of the ESE sequence. The heterologous splicing enhancer reporter plasmid µAVWT 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. 1Citation 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.

 
To confirm a regulatory role for the elements defined using the µAVWT construct, mutations scanning the same regions were introduced into pFGFR17. Construct M1, which introduced mutations immediately upstream of ESE1, had no effect on {alpha}-exon inclusion and therefore defines the 5' boundary of ESE1 (Fig. 3A)Citation . Five constructs (M2–M6) contain mutations targeting the ESE1 region. Construct M2 contains mutations identical to those introduced into µAVWT construct D5 and shows an analogous reduction in splicing (Fig. 3A)Citation . Constructs M4 and M6 define the 3' boundary of ESE1. The M4 construct dramatically reduced {alpha}-exon inclusion, whereas only a partial reduction in {alpha}-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 {alpha}-exon inclusion (Fig. 3A)Citation . The ESS1 and ESE2 regions were targeted with mutations identical to those introduced into µAVWT 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 {alpha}-exon inclusion in transfected JEG3 cells (Fig. 3A)Citation . Furthermore, mutations targeting the ESS1 region were also found to enhance {alpha}-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 µAVWT and strongly support a requirement for the presence of both ESE elements to maintain {alpha}-exon inclusion.



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  		Fig. 3. Definition of the FGFR1 {alpha}-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. 2Citation . 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 {alpha}-exon inclusion indicated below each lane. I, inclusion product; E, exclusion product. B. In vitro transcribed 70-nucleotide labeled RNAs of either wild-type 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.

 
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 {alpha}-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 {alpha}-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)Citation . 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 that whereas the mutations are predicted to create SRp40- and SC35-binding sites, they are clearly not associated with enhanced {alpha}-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. 3CCitation ; 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 {alpha}-exon inclusion, we obtained a SRp55 expression plasmid (13) and created two SRp55 shRNAi expression plasmids. The shRNAi target sites are illustrated in Fig. 4ACitation . Cotransfection of the SRp55 expression plasmid with pFGFR17 did not significantly increase the level of {alpha}-exon inclusion (86% versus 85% using a vector control; Fig. 4BCitation ). 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 {alpha}-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)Citation . JEG3 cells transfected with control shRNAi showed no effect on the level of SRp55 expression or FGFR1 splicing (data not shown).



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  		Fig. 4. SRp55 mediates {alpha}-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 {alpha}-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 ({Delta}ISS) and either a vector, RNAi-2 expression vector, or SRp55 expression vector. The average percentage of {alpha}-exon inclusion in three independent transfections is indicated below each lane. I, inclusion product; E, exclusion product.

 
The observation that reduced SRp55 expression correlates with a loss of {alpha}-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 {alpha}-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 {alpha}-exon inclusion, although the trends were in the appropriate direction (Fig. 4C)Citation . Therefore, either the splicing inhibitory pathway is dominant over the {alpha}-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 ({Delta}ISS) was cotransfected into SNB19 cells. We showed previously (7) that deletion of both ISS elements led to a dramatic increase in {alpha}-exon inclusion in SNB19 cells (Fig. 4CCitation , compare Lanes 1 and 4). A reduction of SRp55 mediated through transfection of RNAi-2 caused a decrease in {alpha}-exon inclusion from 83% to 46%, whereas cotransfection of {Delta}ISS and the SRp55 expression plasmid had no effect on splicing. RNAi-2 was also able to mediate a similar reduction {alpha}-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 {alpha}-exon inclusion and therefore is not targeted for inactivation in the SNB19 cell line. Instead, it appears that the {alpha}-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 {alpha}-exon. The organization of ESE elements in close proximity to or overlapping 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 SRp55-dependent {alpha}-exon inclusion suggests a similar mechanism may exist for FGFR1. However, because {alpha}-exon inclusion was also SRp55 dependent in the {Delta}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 RNAi-mediated 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 {alpha}-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.


   FOOTNOTES
 
Grant support: National Institutes of Health grant CA67946 (to G. Cote).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Gilbert J. Cote, Department of Endocrinology, Unit 435, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-2840; Fax: 713-794-4065; E-mail: gcote{at}mdanderson.org

Received 2/26/04. Revised 8/12/04. Accepted 10/ 3/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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  7. Jin W, Huang ES-C, Bi W, Cote GJ Redundant intronic repressors function to inhibit fibroblast growth factor receptor-1 alpha-exon recognition in glioblastoma cells. J Biol Chem 1999;274:28035-41.[Abstract/Free Full Text]
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