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• W2154175620 abstract "Alternative RNA processing of the human fibroblast growth factor receptor-1 transcript results in receptor forms that vary in their affinity for fibroblast growth factor. An alternative RNA processing event involving recognition of the α-exon is deregulated during neoplastic transformation of glial cells. We have previously established a splicing reporter/transfection cell culture model system to identify sequences involved in recognition of this exon. In this study, the system was used to identify two sequence elements that differentially function to regulate splicing of this exon. Exclusion of the α-exon in glioblastoma cells specifically required the downstream intron sequence comprising the 5′-splice site. Replacement or mutation of this sequence increasing complementarity to U1 RNA resulted in enhanced exon recognition in SNB-19 glioblastoma cells. Sequences within the exon were found to be required for α-exon inclusion. Deletion and gain-of-function experiments identified a 69-nucleotide exon sequence that was specifically required for α-exon inclusion. These studies indicate that multiple sequences are required for the regulated recognition of the α-exon. Alternative RNA processing of the human fibroblast growth factor receptor-1 transcript results in receptor forms that vary in their affinity for fibroblast growth factor. An alternative RNA processing event involving recognition of the α-exon is deregulated during neoplastic transformation of glial cells. We have previously established a splicing reporter/transfection cell culture model system to identify sequences involved in recognition of this exon. In this study, the system was used to identify two sequence elements that differentially function to regulate splicing of this exon. Exclusion of the α-exon in glioblastoma cells specifically required the downstream intron sequence comprising the 5′-splice site. Replacement or mutation of this sequence increasing complementarity to U1 RNA resulted in enhanced exon recognition in SNB-19 glioblastoma cells. Sequences within the exon were found to be required for α-exon inclusion. Deletion and gain-of-function experiments identified a 69-nucleotide exon sequence that was specifically required for α-exon inclusion. These studies indicate that multiple sequences are required for the regulated recognition of the α-exon. Alternative processing of mRNA precursors provides an important method for creating genetic diversity. In many cases, the process is highly regulated such that unique mRNA forms are found to exist only in specific tissues or at a single developmental stage (1Adams M. Rudner D. Rio D. Curr. Opin. Cell Biol. 1996; 8: 331-339Crossref PubMed Scopus (117) Google Scholar, 2Chabot B. Trends Genet. 1996; 12: 472-478Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 3Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (600) Google Scholar). The mechanisms involved in regulation of alternative RNA processing remain unclear for the most part. However, it has been speculated that different RNAs are likely to share common regulatory factors during alternative splicing. Therefore, deregulation of a single alternative splicing event would be predicted to have widespread cellular effects by disruption of other RNA splicing pathways employing the same regulatory factors.We have continued to examine the mechanisms involved in alternative RNA splicing of transcripts derived from the fibroblast growth factor receptor-1 (FGFR-1) 1The abbreviations used are: FGFR, fibroblast growth factor receptor; FGF, fibroblast growth factor; bp, base pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; snRNA, small nuclear RNA; hMT, human metallothionein; RSV, Rous sarcoma virus. 1The abbreviations used are: FGFR, fibroblast growth factor receptor; FGF, fibroblast growth factor; bp, base pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; snRNA, small nuclear RNA; hMT, human metallothionein; RSV, Rous sarcoma virus. gene. This tyrosine kinase receptor is part of a four-gene family whose members are differentially activated by one or more of at least nine fibroblast growth factor (FGF) peptide ligands (4Green P. Walsh F. Doherty P. Bioessays. 1996; 18: 639-646Crossref PubMed Scopus (129) Google Scholar, 5McKeehan W.L. Kan M. Mol. Reprod. Dev. 1994; 39: 69-81Crossref PubMed Scopus (60) Google Scholar, 6Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1169) Google Scholar). The FGFRs have been implicated in many patterning events, including limb formation, keratinocyte organization, and brain development (7Rubin J. Bottaro D. Chedid M. Miki T. Ron D. Cheon G. Taylor W. Fortney E. Sakata H. Finch P.W. Cell Biol. Int. 1995; 19: 399-411Crossref PubMed Scopus (261) Google Scholar, 8Mason I. Curr. Biol. 1996; 6: 672-675Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 9Webster M.K. Donoghue D.J. Trends Genet. 1997; 13: 178-182Abstract Full Text PDF PubMed Scopus (269) Google Scholar). The great diversity of function is created in part by alternative RNA processing of the FGFRs and ligands. For the FGFR-1 gene, alternative RNA processing of the primary transcript differentially alters ligand affinity, ligand specificity, membrane association, and tyrosine kinase activity (4Green P. Walsh F. Doherty P. Bioessays. 1996; 18: 639-646Crossref PubMed Scopus (129) Google Scholar, 5McKeehan W.L. Kan M. Mol. Reprod. Dev. 1994; 39: 69-81Crossref PubMed Scopus (60) Google Scholar, 6Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1169) Google Scholar, 10Fernig D.G. Gallagher J.T. Prog. Growth Factor Res. 1994; 5: 353-377Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Our interest has been in a regulatory event that affects ligand affinity. The regulated inclusion of a single exon termed “α” results in the production of a receptor with reduced affinity for FGF-1 and FGF-2 (11Wang F. Kan M. Yan G. Xu J. McKeehan W. J. Biol. Chem. 1995; 270: 10231-10235Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). This receptor form (FGFR-1α) contains three extracellular, immunoglobulin-like disulfide loops and is expressed predominantly in the normal adult brain. Recognition of the α-exon is disrupted by glial cell malignancy (12Yamaguchi F. Saya H. Bruner J. Morrison R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 484-488Crossref PubMed Scopus (218) Google Scholar, 13Morrison R. Yamaguchi F. Saya H. Bruner J. Yahanda A. Donehower L. Berger M. J. Neuro-oncol. 1994; 18: 207-216Crossref PubMed Scopus (102) Google Scholar). Instead, a receptor form lacking the α-exon (FGFR-1β) and containing only two extracellular, immunoglobulin-like disulfide loops is expressed. There appears to be a strict relationship between the degree of glial cell malignancy and RNA splicing to exclude the α-exon. The result is the predominant expression of a receptor form with higher ligand affinity in glioblastoma cells. This strong association of malignancy with disruption of an RNA processing pathway, as well as the potential involvement of the higher affinity receptor in tumor progression, prompted us to examine the mechanisms involved in greater detail.We previously established a cell culture model to examine the regulatory sequences involved in regulated recognition of the FGFR α-exon (14Cote G.J. Huang E.S.-C. Jin W. Morrison R.S. J. Biol. Chem. 1997; 272: 1054-1060Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). A chimeric minigene containing 4 kilobases of the humanFGFR-1 gene encompassing the α-exon was inserted into a splicing reporter construct. This chimeric gene displayed cell-specific recognition of the α-exon in JEG-3 cells and exclusion in SNB-19 glioblastoma cells. With this model, we narrowed the sequences required to maintain α-exon-regulated recognition to a 375-bp fragment inclusive of the exon. Here we have extended these studies to identify the specific regions of this sequence required for the inclusion and exclusion of the α-exon.DISCUSSIONAlternative processing of the FGFR-1 RNA transcript to include the α-exon is a pathway nearly exclusive to the brain. Therefore, it is curious that deregulation of this splicing event coincides with malignant transformation of glial cells. We had previously established a model system that demonstrated cell-specific recognition of the α-exon (14Cote G.J. Huang E.S.-C. Jin W. Morrison R.S. J. Biol. Chem. 1997; 272: 1054-1060Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). That study identified a minimal sequence unit comprising the α-exon, with 97 bp of upstream sequence and 11 bp of downstream sequence (pFGFR-32), as containing all necessary elements for regulated exon recognition. In this paper, we have attempted to identify the sequences contained within this 375-bp fragment that are responsible for exon skipping in SNB-19 cells and exon inclusion in JEG-3 cells. One consideration in performing this analysis is that our fragment contains the 3′- and 5′-splice sites, which are constitutively required for RNA splicing. Therefore, it is not possible to perform a simple deletion of intron sequence flanking the α-exon, as it would remove these required elements. To avoid this problem, we performed substitutions. Exon 4 of the FGFR-1gene is included in all cell types shown to express the gene (5McKeehan W.L. Kan M. Mol. Reprod. Dev. 1994; 39: 69-81Crossref PubMed Scopus (60) Google Scholar). When this exon and analogous flanking sequence were placed in an identical context (pFGFR-33), the exon did not display cell-specific regulation. The exon was skipped at a constant level, suggesting that recognition was weakened by the context of our splicing reporter. As weakly recognized splice sites are a prerequisite for alternative splicing, these sequences provided an ideal candidate for our substitution experiments.We first turned our attention to the question of why the α-exon is skipped in SNB-19 cells. The possibilities to consider are the presence of an inhibitory element, the absence of an enhancer, or both. The first mechanism would function through a unique silencer element, whereas an enhancer-based mechanism requires that the exon be weakly recognized through its splice sites. Sequence substitutions between the α-exon and exon 4 (Fig. 2) implicated the downstream intron sequence. When this region was replaced or mutated, a significant enhancement of the α-exon was observed. Consistent with this observation, when the α-exon 3′-intron was placed downstream of exon 4, it was skipped (Fig. 2 B). Furthermore, there is a direct correlation between the level of α-exon inclusion and predicted U1 snRNA base pairing. The constructs pFGFR-36 and pFGFR-40, which have the same number of U1 snRNA base pairs, show similar levels of α-exon inclusion in SNB-19 cells. The construct pFGFR-39, which has the lowest level of exon inclusion, also contains the least number of predicted U1 snRNA base pairs (Fig. 2 A). Combined, these data support a role for a weak 5′-splice site in the α-exon skipping in SNB-19 cells. However, while there is good correlation with exon inclusion and base pairing, we cannot rule out that this effect is coincidental and that these changes are interfering with a negative regulatory domain.While the 5′-splice site clearly plays an important role in α-exon skipping, it is not the sole element involved. Several observations suggest the presence of additional factors. First, while substitution or mutation of the 5′-splice site enhanced α-exon inclusion in SNB-19 cells, the same construct expressed in JEG-3 cells consistently showed higher inclusion. This may simply represent overall enhanced splicing ability of the JEG-3 cells or the presence of additional inhibitory factors in the SNB-19 cells. The gain-of-function experiments using a constitutive enhancer (GAR4) (Fig. 4) provide support for the former hypothesis. However, the presence of additional FGFR-1flanking intron sequence clearly affects the level of α-exon inclusion. In SNB-19 cells, there is a significant and reproducible increase in the level of α-exon inclusion for pFGFR-32 compared with pFGFR-17 (37% versus 20%) (Fig. 1). An even greater difference in the level of α-exon inclusion can be attributed to flanking intron sequence when comparing pFGFR-45 (65% inclusion) (Fig. 3) with pFGFR-47 (25% inclusion) (Fig. 5). In support of these findings, we have recently identified a sequence within the upstream intron that appears to be required for maximum repression of α-exon inclusion in SNB-19 cells. 2W. Jin, E. S.-C. Huang, W. Bi, and G. J. Cote, unpublished observation. These observations indicate that the additional repressor sequences may be required to enforce α-exon exclusion in the presence of reduced enhancer activity.The weak 5′-splice site of the α-exon identifies the requirement for an enhancer element to promote inclusion in JEG-3 cells. Two regions were implicated by the substitution constructs. Replacement of the α-exon (pFGFR-35) reduced exon inclusion, whereas the α-exon 3′-intron was capable of enhancing recognition of a heterologous exon 4 (pFGFR-37) (Fig. 2). The inverse swaps did not support these findings. The α-exon was not included at a higher level than exon 4 (compare pFGFR-33 with pFGFR-38), and replacement of the α-exon 3′-intron did not cause a significant reduction in exon inclusion (compare pFGFR-34 with pFGFR-32) (Fig. 2). These contradictory results only serve to emphasize the complex relationship of splicing elements and splice sites. Exon enhancers typically function to facilitate 3′-splice site recognition when one or both splice sites are weak (15Xu R. Teng J. Cooper T. Mol. Cell. Biol. 1993; 13: 3660-3674Crossref PubMed Scopus (178) Google Scholar, 17Inoue K. Ohno M. Shimura Y. Gene Expr. 1995; 4: 177-182PubMed Google Scholar, 18Watakabe A. Tanaka K. Shimura Y. Genes Dev. 1993; 7: 407-418Crossref PubMed Scopus (307) Google Scholar). Also, the need for these enhancers can be overcome by increasing the U1 snRNA complementarity to the downstream 5′-splice site, consistent with serine-arginine-rich our findings in both cell lines.Exon enhancer elements are believed to function through the binding of serine-arginine-rich proteins, which then act to stabilize interaction between the factors associated with splice sites flanking the exon (3Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (600) Google Scholar,19Fu X. RNA. 1995; 1: 663-680PubMed Google Scholar, 21Hertel K. Lynch K. Maniatis T. Curr. Opin. Cell Biol. 1997; 9: 350-357Crossref PubMed Scopus (120) Google Scholar). Two major classes of nonspecific exon enhancer elements have been identified, purine-rich and A/C-rich sequences (19Fu X. RNA. 1995; 1: 663-680PubMed Google Scholar, 21Hertel K. Lynch K. Maniatis T. Curr. Opin. Cell Biol. 1997; 9: 350-357Crossref PubMed Scopus (120) Google Scholar, 22Coulter L. Landree M. Cooper T. Mol. Cell. Biol. 1997; 17: 2143-2150Crossref PubMed Scopus (206) Google Scholar). Examination of the α-exon sequence revealed several potential enhancer sequences of both types. Deletion of α-exon sequence confirmed the presence of enhancer elements within this exon. A 165-base pair internal deletion of the α-exon showed a dramatic reduction in exon inclusion in JEG-3 cells. Further deletion analysis failed to localize this effect to a single region, suggesting a redundancy of enhancer elements, although we cannot rule out an effect of exon size. Inclusion of the α-exon could be restored by the addition of as few as 24 bp, but the reciprocal deletion experiments did not result in exon exclusion (compare pFGFR-43 and pFGFR-44) (Fig. 3). A similar result was observed when the 69-bp sequence was deleted in pFGFR-45 (Fig. 3). To directly address the possibility of multiple enhancer elements, we examined the ability of the individual sequences to provide enhancer function in a previously characterized heterologous splicing reporter (μAVWT.BSC) (15Xu R. Teng J. Cooper T. Mol. Cell. Biol. 1993; 13: 3660-3674Crossref PubMed Scopus (178) Google Scholar). These gain-of-function experiments identified two regions as capable of enhancer function, a 69-nucleotide sequence and a 72-nucleotide sequence (Fig. 4). While deletion experiments suggest that the 72-nucleotide sequence is not required for α-exon recognition, this sequence clearly functions as a potent enhancer. This enhancer activity is not surprising as we identified five purine-rich elements within the sequence. It is intriguing, however, that in the absence of the 69-nucleotide element and in the presence of flanking intron sequence (pFGFR-47) (Fig. 5), this sequence does not function as an enhancer. The converse deletion of the 72-nucleotide sequence also had no affect on α-exon splicing. Therefore, it would appear that the 72-nucleotide enhancer is dispensable and that the 69-nucleotide enhancer is critical for α-exon inclusion.The 69-nucleotide sequence does not contain purine- or A/C-rich sequences. Data base searches failed to identify any extensive sequence homologies for the 69-nucleotide region outside of the FGFRs. This is not unexpected given the relatively small size of most reported regulatory sequences. Whether the 69-nucleotide region contains sequences representing a new class of constitutive enhancer elements or is indeed a cell- or gene-specific splicing enhancer remains to be addressed. The gain-of-function experiments clearly suggest that the JEG-3 cell line has greater enhancer activity compared with the SNB-19 cell line. However, the deletion experiments performed in the pFGFR-17 construct indicate that intron sequences may modulate some of the enhancer effects. It will be interesting to determine if theFGFR-1 intron has similar effects in the context of the heterologous splicing reporter (μAVWT). It is also unclear if the 69-nucleotide sequence is the only element required for α-exon inclusion. There clearly are multiple sequences within the α-exon sequence that function as enhancers. This is not an uncommon theme among enhancer-regulated genes. The best characterized example is the female-specific inclusion of exon 4 of the Drosophila doublesex gene (23Lynch K.W. Maniatis T. Genes Dev. 1996; 10: 2089-2101Crossref PubMed Scopus (196) Google Scholar, 24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This exon contains six repeats that are recognized by the splicing factor TRA-2 and a single purine-rich enhancer that is thought to stabilize interactions through the recruitment of serine-arginine-rich proteins (23Lynch K.W. Maniatis T. Genes Dev. 1996; 10: 2089-2101Crossref PubMed Scopus (196) Google Scholar, 24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). While maximal inclusion of exon 4 requires the presence of all enhancer sequences, this effect is additive, and only a single enhancer is required to maintain exon recognition (24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A similar complex relationship may exist for tissue-specific recognition of the α-exon.In this study, we used a reporter/transfection model system to identifycis-elements involved in the “cassette-type” recognition of the FGFR-1 α-exon. The exon skipping phenotype in SNB-19 glioblastoma cells requires a “weak” 5′-splice site; enhancers within the α-exon facilitate inclusion in JEG-3 cells. Both elements are required, but are not sufficient for cell-specific recognition of the α-exon. Clearly, no single sequence directly mediates the regulation of α-exon splicing. However, we feel justified to suggest a mechanism in which a reduction of enhancer function in glioblastoma cells results in α-exon skipping. Both the constitutive (purine-rich) and the α-exon (α-69) enhancer activities were found to be significantly reduced in SNB-19 cells (Fig. 4). The exclusion of α-exon in SNB-19 cells is likely to be enforced by repressor regulatory sequences acting in addition to the suboptimal 5′-splice site. A complex mechanism involving multiple intron enhancer and repressor elements has been proposed to explain the regulated inclusion of the exons that alternatively code for the third Ig domain of the FGFR-2 gene (20Carstens R.P. McKeehan W.L Garcia-Blanco M.A. Mol. Cell. Biol. 1998; 18: 2205-2217Crossref PubMed Scopus (85) Google Scholar, 25Del Gatto F. Plet A. Gesnel M.-C. Fort C. Breathnach R. Mol. Cell. Biol. 1997; 17: 5106-5116Crossref PubMed Scopus (82) Google Scholar). Whether a similar complexity is required for inclusion of the α-exon remains to be elucidated. Identification of the exon enhancer sequences, however, provides us with key information to begin to address this possibility. Alternative processing of mRNA precursors provides an important method for creating genetic diversity. In many cases, the process is highly regulated such that unique mRNA forms are found to exist only in specific tissues or at a single developmental stage (1Adams M. Rudner D. Rio D. Curr. Opin. Cell Biol. 1996; 8: 331-339Crossref PubMed Scopus (117) Google Scholar, 2Chabot B. Trends Genet. 1996; 12: 472-478Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 3Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (600) Google Scholar). The mechanisms involved in regulation of alternative RNA processing remain unclear for the most part. However, it has been speculated that different RNAs are likely to share common regulatory factors during alternative splicing. Therefore, deregulation of a single alternative splicing event would be predicted to have widespread cellular effects by disruption of other RNA splicing pathways employing the same regulatory factors. We have continued to examine the mechanisms involved in alternative RNA splicing of transcripts derived from the fibroblast growth factor receptor-1 (FGFR-1) 1The abbreviations used are: FGFR, fibroblast growth factor receptor; FGF, fibroblast growth factor; bp, base pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; snRNA, small nuclear RNA; hMT, human metallothionein; RSV, Rous sarcoma virus. 1The abbreviations used are: FGFR, fibroblast growth factor receptor; FGF, fibroblast growth factor; bp, base pair(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; snRNA, small nuclear RNA; hMT, human metallothionein; RSV, Rous sarcoma virus. gene. This tyrosine kinase receptor is part of a four-gene family whose members are differentially activated by one or more of at least nine fibroblast growth factor (FGF) peptide ligands (4Green P. Walsh F. Doherty P. Bioessays. 1996; 18: 639-646Crossref PubMed Scopus (129) Google Scholar, 5McKeehan W.L. Kan M. Mol. Reprod. Dev. 1994; 39: 69-81Crossref PubMed Scopus (60) Google Scholar, 6Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1169) Google Scholar). The FGFRs have been implicated in many patterning events, including limb formation, keratinocyte organization, and brain development (7Rubin J. Bottaro D. Chedid M. Miki T. Ron D. Cheon G. Taylor W. Fortney E. Sakata H. Finch P.W. Cell Biol. Int. 1995; 19: 399-411Crossref PubMed Scopus (261) Google Scholar, 8Mason I. Curr. Biol. 1996; 6: 672-675Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 9Webster M.K. Donoghue D.J. Trends Genet. 1997; 13: 178-182Abstract Full Text PDF PubMed Scopus (269) Google Scholar). The great diversity of function is created in part by alternative RNA processing of the FGFRs and ligands. For the FGFR-1 gene, alternative RNA processing of the primary transcript differentially alters ligand affinity, ligand specificity, membrane association, and tyrosine kinase activity (4Green P. Walsh F. Doherty P. Bioessays. 1996; 18: 639-646Crossref PubMed Scopus (129) Google Scholar, 5McKeehan W.L. Kan M. Mol. Reprod. Dev. 1994; 39: 69-81Crossref PubMed Scopus (60) Google Scholar, 6Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1169) Google Scholar, 10Fernig D.G. Gallagher J.T. Prog. Growth Factor Res. 1994; 5: 353-377Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Our interest has been in a regulatory event that affects ligand affinity. The regulated inclusion of a single exon termed “α” results in the production of a receptor with reduced affinity for FGF-1 and FGF-2 (11Wang F. Kan M. Yan G. Xu J. McKeehan W. J. Biol. Chem. 1995; 270: 10231-10235Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). This receptor form (FGFR-1α) contains three extracellular, immunoglobulin-like disulfide loops and is expressed predominantly in the normal adult brain. Recognition of the α-exon is disrupted by glial cell malignancy (12Yamaguchi F. Saya H. Bruner J. Morrison R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 484-488Crossref PubMed Scopus (218) Google Scholar, 13Morrison R. Yamaguchi F. Saya H. Bruner J. Yahanda A. Donehower L. Berger M. J. Neuro-oncol. 1994; 18: 207-216Crossref PubMed Scopus (102) Google Scholar). Instead, a receptor form lacking the α-exon (FGFR-1β) and containing only two extracellular, immunoglobulin-like disulfide loops is expressed. There appears to be a strict relationship between the degree of glial cell malignancy and RNA splicing to exclude the α-exon. The result is the predominant expression of a receptor form with higher ligand affinity in glioblastoma cells. This strong association of malignancy with disruption of an RNA processing pathway, as well as the potential involvement of the higher affinity receptor in tumor progression, prompted us to examine the mechanisms involved in greater detail. We previously established a cell culture model to examine the regulatory sequences involved in regulated recognition of the FGFR α-exon (14Cote G.J. Huang E.S.-C. Jin W. Morrison R.S. J. Biol. Chem. 1997; 272: 1054-1060Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). A chimeric minigene containing 4 kilobases of the humanFGFR-1 gene encompassing the α-exon was inserted into a splicing reporter construct. This chimeric gene displayed cell-specific recognition of the α-exon in JEG-3 cells and exclusion in SNB-19 glioblastoma cells. With this model, we narrowed the sequences required to maintain α-exon-regulated recognition to a 375-bp fragment inclusive of the exon. Here we have extended these studies to identify the specific regions of this sequence required for the inclusion and exclusion of the α-exon. DISCUSSIONAlternative processing of the FGFR-1 RNA transcript to include the α-exon is a pathway nearly exclusive to the brain. Therefore, it is curious that deregulation of this splicing event coincides with malignant transformation of glial cells. We had previously established a model system that demonstrated cell-specific recognition of the α-exon (14Cote G.J. Huang E.S.-C. Jin W. Morrison R.S. J. Biol. Chem. 1997; 272: 1054-1060Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). That study identified a minimal sequence unit comprising the α-exon, with 97 bp of upstream sequence and 11 bp of downstream sequence (pFGFR-32), as containing all necessary elements for regulated exon recognition. In this paper, we have attempted to identify the sequences contained within this 375-bp fragment that are responsible for exon skipping in SNB-19 cells and exon inclusion in JEG-3 cells. One consideration in performing this analysis is that our fragment contains the 3′- and 5′-splice sites, which are constitutively required for RNA splicing. Therefore, it is not possible to perform a simple deletion of intron sequence flanking the α-exon, as it would remove these required elements. To avoid this problem, we performed substitutions. Exon 4 of the FGFR-1gene is included in all cell types shown to express the gene (5McKeehan W.L. Kan M. Mol. Reprod. Dev. 1994; 39: 69-81Crossref PubMed Scopus (60) Google Scholar). When this exon and analogous flanking sequence were placed in an identical context (pFGFR-33), the exon did not display cell-specific regulation. The exon was skipped at a constant level, suggesting that recognition was weakened by the context of our splicing reporter. As weakly recognized splice sites are a prerequisite for alternative splicing, these sequences provided an ideal candidate for our substitution experiments.We first turned our attention to the question of why the α-exon is skipped in SNB-19 cells. The possibilities to consider are the presence of an inhibitory element, the absence of an enhancer, or both. The first mechanism would function through a unique silencer element, whereas an enhancer-based mechanism requires that the exon be weakly recognized through its splice sites. Sequence substitutions between the α-exon and exon 4 (Fig. 2) implicated the downstream intron sequence. When this region was replaced or mutated, a significant enhancement of the α-exon was observed. Consistent with this observation, when the α-exon 3′-intron was placed downstream of exon 4, it was skipped (Fig. 2 B). Furthermore, there is a direct correlation between the level of α-exon inclusion and predicted U1 snRNA base pairing. The constructs pFGFR-36 and pFGFR-40, which have the same number of U1 snRNA base pairs, show similar levels of α-exon inclusion in SNB-19 cells. The construct pFGFR-39, which has the lowest level of exon inclusion, also contains the least number of predicted U1 snRNA base pairs (Fig. 2 A). Combined, these data support a role for a weak 5′-splice site in the α-exon skipping in SNB-19 cells. However, while there is good correlation with exon inclusion and base pairing, we cannot rule out that this effect is coincidental and that these changes are interfering with a negative regulatory domain.While the 5′-splice site clearly plays an important role in α-exon skipping, it is not the sole element involved. Several observations suggest the presence of additional factors. First, while substitution or mutation of the 5′-splice site enhanced α-exon inclusion in SNB-19 cells, the same construct expressed in JEG-3 cells consistently showed higher inclusion. This may simply represent overall enhanced splicing ability of the JEG-3 cells or the presence of additional inhibitory factors in the SNB-19 cells. The gain-of-function experiments using a constitutive enhancer (GAR4) (Fig. 4) provide support for the former hypothesis. However, the presence of additional FGFR-1flanking intron sequence clearly affects the level of α-exon inclusion. In SNB-19 cells, there is a significant and reproducible increase in the level of α-exon inclusion for pFGFR-32 compared with pFGFR-17 (37% versus 20%) (Fig. 1). An even greater difference in the level of α-exon inclusion can be attributed to flanking intron sequence when comparing pFGFR-45 (65% inclusion) (Fig. 3) with pFGFR-47 (25% inclusion) (Fig. 5). In support of these findings, we have recently identified a sequence within the upstream intron that appears to be required for maximum repression of α-exon inclusion in SNB-19 cells. 2W. Jin, E. S.-C. Huang, W. Bi, and G. J. Cote, unpublished observation. These observations indicate that the additional repressor sequences may be required to enforce α-exon exclusion in the presence of reduced enhancer activity.The weak 5′-splice site of the α-exon identifies the requirement for an enhancer element to promote inclusion in JEG-3 cells. Two regions were implicated by the substitution constructs. Replacement of the α-exon (pFGFR-35) reduced exon inclusion, whereas the α-exon 3′-intron was capable of enhancing recognition of a heterologous exon 4 (pFGFR-37) (Fig. 2). The inverse swaps did not support these findings. The α-exon was not included at a higher level than exon 4 (compare pFGFR-33 with pFGFR-38), and replacement of the α-exon 3′-intron did not cause a significant reduction in exon inclusion (compare pFGFR-34 with pFGFR-32) (Fig. 2). These contradictory results only serve to emphasize the complex relationship of splicing elements and splice sites. Exon enhancers typically function to facilitate 3′-splice site recognition when one or both splice sites are weak (15Xu R. Teng J. Cooper T. Mol. Cell. Biol. 1993; 13: 3660-3674Crossref PubMed Scopus (178) Google Scholar, 17Inoue K. Ohno M. Shimura Y. Gene Expr. 1995; 4: 177-182PubMed Google Scholar, 18Watakabe A. Tanaka K. Shimura Y. Genes Dev. 1993; 7: 407-418Crossref PubMed Scopus (307) Google Scholar). Also, the need for these enhancers can be overcome by increasing the U1 snRNA complementarity to the downstream 5′-splice site, consistent with serine-arginine-rich our findings in both cell lines.Exon enhancer elements are believed to function through the binding of serine-arginine-rich proteins, which then act to stabilize interaction between the factors associated with splice sites flanking the exon (3Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (600) Google Scholar,19Fu X. RNA. 1995; 1: 663-680PubMed Google Scholar, 21Hertel K. Lynch K. Maniatis T. Curr. Opin. Cell Biol. 1997; 9: 350-357Crossref PubMed Scopus (120) Google Scholar). Two major classes of nonspecific exon enhancer elements have been identified, purine-rich and A/C-rich sequences (19Fu X. RNA. 1995; 1: 663-680PubMed Google Scholar, 21Hertel K. Lynch K. Maniatis T. Curr. Opin. Cell Biol. 1997; 9: 350-357Crossref PubMed Scopus (120) Google Scholar, 22Coulter L. Landree M. Cooper T. Mol. Cell. Biol. 1997; 17: 2143-2150Crossref PubMed Scopus (206) Google Scholar). Examination of the α-exon sequence revealed several potential enhancer sequences of both types. Deletion of α-exon sequence confirmed the presence of enhancer elements within this exon. A 165-base pair internal deletion of the α-exon showed a dramatic reduction in exon inclusion in JEG-3 cells. Further deletion analysis failed to localize this effect to a single region, suggesting a redundancy of enhancer elements, although we cannot rule out an effect of exon size. Inclusion of the α-exon could be restored by the addition of as few as 24 bp, but the reciprocal deletion experiments did not result in exon exclusion (compare pFGFR-43 and pFGFR-44) (Fig. 3). A similar result was observed when the 69-bp sequence was deleted in pFGFR-45 (Fig. 3). To directly address the possibility of multiple enhancer elements, we examined the ability of the individual sequences to provide enhancer function in a previously characterized heterologous splicing reporter (μAVWT.BSC) (15Xu R. Teng J. Cooper T. Mol. Cell. Biol. 1993; 13: 3660-3674Crossref PubMed Scopus (178) Google Scholar). These gain-of-function experiments identified two regions as capable of enhancer function, a 69-nucleotide sequence and a 72-nucleotide sequence (Fig. 4). While deletion experiments suggest that the 72-nucleotide sequence is not required for α-exon recognition, this sequence clearly functions as a potent enhancer. This enhancer activity is not surprising as we identified five purine-rich elements within the sequence. It is intriguing, however, that in the absence of the 69-nucleotide element and in the presence of flanking intron sequence (pFGFR-47) (Fig. 5), this sequence does not function as an enhancer. The converse deletion of the 72-nucleotide sequence also had no affect on α-exon splicing. Therefore, it would appear that the 72-nucleotide enhancer is dispensable and that the 69-nucleotide enhancer is critical for α-exon inclusion.The 69-nucleotide sequence does not contain purine- or A/C-rich sequences. Data base searches failed to identify any extensive sequence homologies for the 69-nucleotide region outside of the FGFRs. This is not unexpected given the relatively small size of most reported regulatory sequences. Whether the 69-nucleotide region contains sequences representing a new class of constitutive enhancer elements or is indeed a cell- or gene-specific splicing enhancer remains to be addressed. The gain-of-function experiments clearly suggest that the JEG-3 cell line has greater enhancer activity compared with the SNB-19 cell line. However, the deletion experiments performed in the pFGFR-17 construct indicate that intron sequences may modulate some of the enhancer effects. It will be interesting to determine if theFGFR-1 intron has similar effects in the context of the heterologous splicing reporter (μAVWT). It is also unclear if the 69-nucleotide sequence is the only element required for α-exon inclusion. There clearly are multiple sequences within the α-exon sequence that function as enhancers. This is not an uncommon theme among enhancer-regulated genes. The best characterized example is the female-specific inclusion of exon 4 of the Drosophila doublesex gene (23Lynch K.W. Maniatis T. Genes Dev. 1996; 10: 2089-2101Crossref PubMed Scopus (196) Google Scholar, 24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This exon contains six repeats that are recognized by the splicing factor TRA-2 and a single purine-rich enhancer that is thought to stabilize interactions through the recruitment of serine-arginine-rich proteins (23Lynch K.W. Maniatis T. Genes Dev. 1996; 10: 2089-2101Crossref PubMed Scopus (196) Google Scholar, 24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). While maximal inclusion of exon 4 requires the presence of all enhancer sequences, this effect is additive, and only a single enhancer is required to maintain exon recognition (24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A similar complex relationship may exist for tissue-specific recognition of the α-exon.In this study, we used a reporter/transfection model system to identifycis-elements involved in the “cassette-type” recognition of the FGFR-1 α-exon. The exon skipping phenotype in SNB-19 glioblastoma cells requires a “weak” 5′-splice site; enhancers within the α-exon facilitate inclusion in JEG-3 cells. Both elements are required, but are not sufficient for cell-specific recognition of the α-exon. Clearly, no single sequence directly mediates the regulation of α-exon splicing. However, we feel justified to suggest a mechanism in which a reduction of enhancer function in glioblastoma cells results in α-exon skipping. Both the constitutive (purine-rich) and the α-exon (α-69) enhancer activities were found to be significantly reduced in SNB-19 cells (Fig. 4). The exclusion of α-exon in SNB-19 cells is likely to be enforced by repressor regulatory sequences acting in addition to the suboptimal 5′-splice site. A complex mechanism involving multiple intron enhancer and repressor elements has been proposed to explain the regulated inclusion of the exons that alternatively code for the third Ig domain of the FGFR-2 gene (20Carstens R.P. McKeehan W.L Garcia-Blanco M.A. Mol. Cell. Biol. 1998; 18: 2205-2217Crossref PubMed Scopus (85) Google Scholar, 25Del Gatto F. Plet A. Gesnel M.-C. Fort C. Breathnach R. Mol. Cell. Biol. 1997; 17: 5106-5116Crossref PubMed Scopus (82) Google Scholar). Whether a similar complexity is required for inclusion of the α-exon remains to be elucidated. Identification of the exon enhancer sequences, however, provides us with key information to begin to address this possibility. Alternative processing of the FGFR-1 RNA transcript to include the α-exon is a pathway nearly exclusive to the brain. Therefore, it is curious that deregulation of this splicing event coincides with malignant transformation of glial cells. We had previously established a model system that demonstrated cell-specific recognition of the α-exon (14Cote G.J. Huang E.S.-C. Jin W. Morrison R.S. J. Biol. Chem. 1997; 272: 1054-1060Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). That study identified a minimal sequence unit comprising the α-exon, with 97 bp of upstream sequence and 11 bp of downstream sequence (pFGFR-32), as containing all necessary elements for regulated exon recognition. In this paper, we have attempted to identify the sequences contained within this 375-bp fragment that are responsible for exon skipping in SNB-19 cells and exon inclusion in JEG-3 cells. One consideration in performing this analysis is that our fragment contains the 3′- and 5′-splice sites, which are constitutively required for RNA splicing. Therefore, it is not possible to perform a simple deletion of intron sequence flanking the α-exon, as it would remove these required elements. To avoid this problem, we performed substitutions. Exon 4 of the FGFR-1gene is included in all cell types shown to express the gene (5McKeehan W.L. Kan M. Mol. Reprod. Dev. 1994; 39: 69-81Crossref PubMed Scopus (60) Google Scholar). When this exon and analogous flanking sequence were placed in an identical context (pFGFR-33), the exon did not display cell-specific regulation. The exon was skipped at a constant level, suggesting that recognition was weakened by the context of our splicing reporter. As weakly recognized splice sites are a prerequisite for alternative splicing, these sequences provided an ideal candidate for our substitution experiments. We first turned our attention to the question of why the α-exon is skipped in SNB-19 cells. The possibilities to consider are the presence of an inhibitory element, the absence of an enhancer, or both. The first mechanism would function through a unique silencer element, whereas an enhancer-based mechanism requires that the exon be weakly recognized through its splice sites. Sequence substitutions between the α-exon and exon 4 (Fig. 2) implicated the downstream intron sequence. When this region was replaced or mutated, a significant enhancement of the α-exon was observed. Consistent with this observation, when the α-exon 3′-intron was placed downstream of exon 4, it was skipped (Fig. 2 B). Furthermore, there is a direct correlation between the level of α-exon inclusion and predicted U1 snRNA base pairing. The constructs pFGFR-36 and pFGFR-40, which have the same number of U1 snRNA base pairs, show similar levels of α-exon inclusion in SNB-19 cells. The construct pFGFR-39, which has the lowest level of exon inclusion, also contains the least number of predicted U1 snRNA base pairs (Fig. 2 A). Combined, these data support a role for a weak 5′-splice site in the α-exon skipping in SNB-19 cells. However, while there is good correlation with exon inclusion and base pairing, we cannot rule out that this effect is coincidental and that these changes are interfering with a negative regulatory domain. While the 5′-splice site clearly plays an important role in α-exon skipping, it is not the sole element involved. Several observations suggest the presence of additional factors. First, while substitution or mutation of the 5′-splice site enhanced α-exon inclusion in SNB-19 cells, the same construct expressed in JEG-3 cells consistently showed higher inclusion. This may simply represent overall enhanced splicing ability of the JEG-3 cells or the presence of additional inhibitory factors in the SNB-19 cells. The gain-of-function experiments using a constitutive enhancer (GAR4) (Fig. 4) provide support for the former hypothesis. However, the presence of additional FGFR-1flanking intron sequence clearly affects the level of α-exon inclusion. In SNB-19 cells, there is a significant and reproducible increase in the level of α-exon inclusion for pFGFR-32 compared with pFGFR-17 (37% versus 20%) (Fig. 1). An even greater difference in the level of α-exon inclusion can be attributed to flanking intron sequence when comparing pFGFR-45 (65% inclusion) (Fig. 3) with pFGFR-47 (25% inclusion) (Fig. 5). In support of these findings, we have recently identified a sequence within the upstream intron that appears to be required for maximum repression of α-exon inclusion in SNB-19 cells. 2W. Jin, E. S.-C. Huang, W. Bi, and G. J. Cote, unpublished observation. These observations indicate that the additional repressor sequences may be required to enforce α-exon exclusion in the presence of reduced enhancer activity. The weak 5′-splice site of the α-exon identifies the requirement for an enhancer element to promote inclusion in JEG-3 cells. Two regions were implicated by the substitution constructs. Replacement of the α-exon (pFGFR-35) reduced exon inclusion, whereas the α-exon 3′-intron was capable of enhancing recognition of a heterologous exon 4 (pFGFR-37) (Fig. 2). The inverse swaps did not support these findings. The α-exon was not included at a higher level than exon 4 (compare pFGFR-33 with pFGFR-38), and replacement of the α-exon 3′-intron did not cause a significant reduction in exon inclusion (compare pFGFR-34 with pFGFR-32) (Fig. 2). These contradictory results only serve to emphasize the complex relationship of splicing elements and splice sites. Exon enhancers typically function to facilitate 3′-splice site recognition when one or both splice sites are weak (15Xu R. Teng J. Cooper T. Mol. Cell. Biol. 1993; 13: 3660-3674Crossref PubMed Scopus (178) Google Scholar, 17Inoue K. Ohno M. Shimura Y. Gene Expr. 1995; 4: 177-182PubMed Google Scholar, 18Watakabe A. Tanaka K. Shimura Y. Genes Dev. 1993; 7: 407-418Crossref PubMed Scopus (307) Google Scholar). Also, the need for these enhancers can be overcome by increasing the U1 snRNA complementarity to the downstream 5′-splice site, consistent with serine-arginine-rich our findings in both cell lines. Exon enhancer elements are believed to function through the binding of serine-arginine-rich proteins, which then act to stabilize interaction between the factors associated with splice sites flanking the exon (3Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (600) Google Scholar,19Fu X. RNA. 1995; 1: 663-680PubMed Google Scholar, 21Hertel K. Lynch K. Maniatis T. Curr. Opin. Cell Biol. 1997; 9: 350-357Crossref PubMed Scopus (120) Google Scholar). Two major classes of nonspecific exon enhancer elements have been identified, purine-rich and A/C-rich sequences (19Fu X. RNA. 1995; 1: 663-680PubMed Google Scholar, 21Hertel K. Lynch K. Maniatis T. Curr. Opin. Cell Biol. 1997; 9: 350-357Crossref PubMed Scopus (120) Google Scholar, 22Coulter L. Landree M. Cooper T. Mol. Cell. Biol. 1997; 17: 2143-2150Crossref PubMed Scopus (206) Google Scholar). Examination of the α-exon sequence revealed several potential enhancer sequences of both types. Deletion of α-exon sequence confirmed the presence of enhancer elements within this exon. A 165-base pair internal deletion of the α-exon showed a dramatic reduction in exon inclusion in JEG-3 cells. Further deletion analysis failed to localize this effect to a single region, suggesting a redundancy of enhancer elements, although we cannot rule out an effect of exon size. Inclusion of the α-exon could be restored by the addition of as few as 24 bp, but the reciprocal deletion experiments did not result in exon exclusion (compare pFGFR-43 and pFGFR-44) (Fig. 3). A similar result was observed when the 69-bp sequence was deleted in pFGFR-45 (Fig. 3). To directly address the possibility of multiple enhancer elements, we examined the ability of the individual sequences to provide enhancer function in a previously characterized heterologous splicing reporter (μAVWT.BSC) (15Xu R. Teng J. Cooper T. Mol. Cell. Biol. 1993; 13: 3660-3674Crossref PubMed Scopus (178) Google Scholar). These gain-of-function experiments identified two regions as capable of enhancer function, a 69-nucleotide sequence and a 72-nucleotide sequence (Fig. 4). While deletion experiments suggest that the 72-nucleotide sequence is not required for α-exon recognition, this sequence clearly functions as a potent enhancer. This enhancer activity is not surprising as we identified five purine-rich elements within the sequence. It is intriguing, however, that in the absence of the 69-nucleotide element and in the presence of flanking intron sequence (pFGFR-47) (Fig. 5), this sequence does not function as an enhancer. The converse deletion of the 72-nucleotide sequence also had no affect on α-exon splicing. Therefore, it would appear that the 72-nucleotide enhancer is dispensable and that the 69-nucleotide enhancer is critical for α-exon inclusion. The 69-nucleotide sequence does not contain purine- or A/C-rich sequences. Data base searches failed to identify any extensive sequence homologies for the 69-nucleotide region outside of the FGFRs. This is not unexpected given the relatively small size of most reported regulatory sequences. Whether the 69-nucleotide region contains sequences representing a new class of constitutive enhancer elements or is indeed a cell- or gene-specific splicing enhancer remains to be addressed. The gain-of-function experiments clearly suggest that the JEG-3 cell line has greater enhancer activity compared with the SNB-19 cell line. However, the deletion experiments performed in the pFGFR-17 construct indicate that intron sequences may modulate some of the enhancer effects. It will be interesting to determine if theFGFR-1 intron has similar effects in the context of the heterologous splicing reporter (μAVWT). It is also unclear if the 69-nucleotide sequence is the only element required for α-exon inclusion. There clearly are multiple sequences within the α-exon sequence that function as enhancers. This is not an uncommon theme among enhancer-regulated genes. The best characterized example is the female-specific inclusion of exon 4 of the Drosophila doublesex gene (23Lynch K.W. Maniatis T. Genes Dev. 1996; 10: 2089-2101Crossref PubMed Scopus (196) Google Scholar, 24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This exon contains six repeats that are recognized by the splicing factor TRA-2 and a single purine-rich enhancer that is thought to stabilize interactions through the recruitment of serine-arginine-rich proteins (23Lynch K.W. Maniatis T. Genes Dev. 1996; 10: 2089-2101Crossref PubMed Scopus (196) Google Scholar, 24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). While maximal inclusion of exon 4 requires the presence of all enhancer sequences, this effect is additive, and only a single enhancer is required to maintain exon recognition (24Hertel K.J. Maniatis T. Mol. Cell. 1998; 1: 449-455Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A similar complex relationship may exist for tissue-specific recognition of the α-exon. In this study, we used a reporter/transfection model system to identifycis-elements involved in the “cassette-type” recognition of the FGFR-1 α-exon. The exon skipping phenotype in SNB-19 glioblastoma cells requires a “weak” 5′-splice site; enhancers within the α-exon facilitate inclusion in JEG-3 cells. Both elements are required, but are not sufficient for cell-specific recognition of the α-exon. Clearly, no single sequence directly mediates the regulation of α-exon splicing. However, we feel justified to suggest a mechanism in which a reduction of enhancer function in glioblastoma cells results in α-exon skipping. Both the constitutive (purine-rich) and the α-exon (α-69) enhancer activities were found to be significantly reduced in SNB-19 cells (Fig. 4). The exclusion of α-exon in SNB-19 cells is likely to be enforced by repressor regulatory sequences acting in addition to the suboptimal 5′-splice site. A complex mechanism involving multiple intron enhancer and repressor elements has been proposed to explain the regulated inclusion of the exons that alternatively code for the third Ig domain of the FGFR-2 gene (20Carstens R.P. McKeehan W.L Garcia-Blanco M.A. Mol. Cell. Biol. 1998; 18: 2205-2217Crossref PubMed Scopus (85) Google Scholar, 25Del Gatto F. Plet A. Gesnel M.-C. Fort C. Breathnach R. Mol. Cell. Biol. 1997; 17: 5106-5116Crossref PubMed Scopus (82) Google Scholar). Whether a similar complexity is required for inclusion of the α-exon remains to be elucidated. Identification of the exon enhancer sequences, however, provides us with key information to begin to address this possibility." @default.
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