A CD45 Polymorphism Associated with Multiple Sclerosis Disrupts an Exonic Splicing Silencer

Previous studies have identified a single nucleotide polymorphism that significantly increases the splicing of variable exon 4 in transcripts of the human protein-tyrosine phosphatase CD45. Strikingly, the presence of this polymorphism correlates with susceptibility to the autoimmune disease multiple sclerosis. In this study we investigated the mechanism by which the polymorphism enhances splicing of CD45 exon 4. We found that at least four distinct splicing regulatory elements exist within exon 4 and that the strongest of these elements is an exonic splicing silencer (designated ESS1), which is disrupted by the polymorphism. We show that ESS1 normally functions to repress the weak 5′ splice site (ss) of CD45 exon 4. The ESS1 sequence also suppresses the splicing of a heterologous 5′ss and associates with a specific complex in nuclear extracts. We further demonstrate that ESS1 is juxtaposed to a purine-rich enhancer sequence that activates the use of the 5′ss of exon 4. Thus, proper functioning of the immune system is dependent on a complex interplay of regulatory activities that mediate the appropriate splicing of CD45 exon 4. Previous studies have identified a single nucleotide polymorphism that significantly increases the splicing of variable exon 4 in transcripts of the human protein-tyrosine phosphatase CD45. Strikingly, the presence of this polymorphism correlates with susceptibility to the autoimmune disease multiple sclerosis. In this study we investigated the mechanism by which the polymorphism enhances splicing of CD45 exon 4. We found that at least four distinct splicing regulatory elements exist within exon 4 and that the strongest of these elements is an exonic splicing silencer (designated ESS1), which is disrupted by the polymorphism. We show that ESS1 normally functions to repress the weak 5′ splice site (ss) of CD45 exon 4. The ESS1 sequence also suppresses the splicing of a heterologous 5′ss and associates with a specific complex in nuclear extracts. We further demonstrate that ESS1 is juxtaposed to a purine-rich enhancer sequence that activates the use of the 5′ss of exon 4. Thus, proper functioning of the immune system is dependent on a complex interplay of regulatory activities that mediate the appropriate splicing of CD45 exon 4. ribonuclear particle small nuclear ribonuclear particles nucleotide(s) splice site exonic splicing silencer reverse transcription-polymerase chain reaction wild type exonic splicing enhancer serine-arginine rich human immunodeficiency virus CD45 is a transmembrane protein-tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. In T cells, CD45 functions to maintain the T-cell receptor in a primed state, allowing for activation of the T-cell receptor upon interaction with an antigen-presenting cell (reviewed in Refs. 1Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (662) Google Scholar and 2Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1957) Google Scholar). In mice that contain a knock-out of CD45, the few T cells that survive development are no longer reactive to antigen, and immune function is impaired (3Kishihara K. Penninger J. Wallace V.A. Kundig T.M. Kawai K. Wakenham A. Timms E. Pfeffer K. Ohashi P.S. Thomas M.L. Cell. 1993; 74: 143-156Abstract Full Text PDF PubMed Scopus (459) Google Scholar,4Byth K.F. Conroy L.A. Howlett S. Smith A.J.H. May J. Alexander D.R. Holmes N. J. Exp. Med. 1996; 183: 1707-1718Crossref PubMed Scopus (359) Google Scholar). Similarly, CD45-deficient humans have severe combined immunodeficiency disease (5Cale C.M. Klein N.J. Novelli V. Veys P. Jones A.M. Morgan G. Arch. Dis. Child. 1997; 76: 163-164Crossref PubMed Scopus (41) Google Scholar, 6Kung C. Pingel J.T. Heikinheimo M. Klemola T. Varkila K. Yoo L.I. Vuopala K. Poyhonen M. Uhari M. Rogers M. Speck S.H. Chatila T. Thomas M.L. Nat. Med. 2000; 6: 343-345Crossref PubMed Scopus (248) Google Scholar). Conversely, T cells that express a constitutively active form of CD45 are hyper-reactive to antigen, and mice containing such cells are prone to develop autoimmune disease (7Majeti R. Xu Z. Parslow T.G. Olson J.L. Daikh D.I. Killeen N. Weiss A. Cell. 2000; 103: 1059-1070Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Thus, both CD45 activity and regulation of this activity are critical for the proper functioning of the immune system. The gene encoding human CD45 encompasses 33 exons. Three of these exons (exons 4, 5, and 6) are alternatively spliced, giving rise to at least five distinct isoforms that differ in their extracellular domain. The alternative splicing of CD45 is highly regulated in both a tissue-specific manner and upon activation of T cells, although the mechanism(s) by which regulation occurs is largely unknown (1Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (662) Google Scholar). The various expressed CD45 isoforms do not differ with regard to their intracellular phosphatase domains. However, it has been proposed that alternative exon usage does indirectly influence phosphatase activity by altering the dimerization of CD45 (7Majeti R. Xu Z. Parslow T.G. Olson J.L. Daikh D.I. Killeen N. Weiss A. Cell. 2000; 103: 1059-1070Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). It has recently been shown that at least some isoforms of CD45 exist as dimers and that in these dimers the phosphatase activity of each molecule is blocked by steric hindrance (7Majeti R. Xu Z. Parslow T.G. Olson J.L. Daikh D.I. Killeen N. Weiss A. Cell. 2000; 103: 1059-1070Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Because the alternate isoforms of CD45 differ in the primary sequence of the extracellular domain, as well as the extent ofO-glycosylation and sialation of this domain, it is likely that they also differ in their ability to dimerize. In particular, exclusion of the variable exons, such as that which occurs upon T cell activation, is predicted to decrease the dimerization of CD45, resulting in a decrease in CD45 function. Thus, the regulation of CD45 splicing may be one mechanism by which an immune response is down-regulated following an initial antigen challenge. Exon 4 is the most tightly regulated of the CD45 variable exons (1Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (662) Google Scholar). In naive T cells, a significant portion of the translated CD45 is encoded by mRNA that includes exon 4. In contrast, the majority of the CD45 protein expressed in activated and memory T cells is encoded by mRNA that lacks exon 4. Several years ago it was shown that a small percentage of the human population expresses aberrantly high levels of exon 4-encoded CD45 isoforms in all cell types (8Schwinzer R. Wonigeit K. J. Exp. Med. 1990; 171: 1803-1808Crossref PubMed Scopus (60) Google Scholar, 9Schwinzer R. Schraven B. Kyas U. Meuer S.C. Wonigeit K. Eur. J. Immunol. 1992; 22: 1095-1098Crossref PubMed Scopus (24) Google Scholar). This misregulation of exon 4 inclusion correlated with the presence of a C to G polymorphism at nucleotide 77 (C77G) of exon 4, which is silent with respect to coding sequence (10Thude H. Hundrieser J. Wonigeit K. Schwinzer R. Eur. J. Immunol. 1995; 25: 2101-2106Crossref PubMed Scopus (63) Google Scholar). Subsequently, we and others demonstrated that engineering the C77G polymorphism into a CD45 minigene is sufficient to confer abnormally high expression of exon 4 in mature mRNA ((11, 12) and see Fig. 1 B). Importantly, consistent with the hypothesis that regulation of CD45 splicing is critical for the appropriate function of the immune system, the C77G polymorphism was recently shown to correlate with development of multiple sclerosis. This conclusion is based on the observation that the prevalence of the C77G polymorphism is significantly greater among multiple sclerosis patients than in healthy controls (13Jacobsen M. Schweer D. Ziegler A. Gaber R. Schock S. Schwinzer R. Wonigeit K. Lindert R.B. Kantarci O. Schaefer-Klein J. Schipper H.I. Oertel W.H. Heidenreich F. Weinshenker B.G. Sommer N. Hemmer B. Nat. Genet. 2000; 26: 495-499Crossref PubMed Scopus (173) Google Scholar). Moreover, this polymorphism co-segregates with disease in at least four independent families (13Jacobsen M. Schweer D. Ziegler A. Gaber R. Schock S. Schwinzer R. Wonigeit K. Lindert R.B. Kantarci O. Schaefer-Klein J. Schipper H.I. Oertel W.H. Heidenreich F. Weinshenker B.G. Sommer N. Hemmer B. Nat. Genet. 2000; 26: 495-499Crossref PubMed Scopus (173) Google Scholar). Therefore, we were interested in understanding how the regulation of CD45 splicing is altered by the C77G change within exon 4. In general, the splicing of any given exon is primarily determined by the sequences at the 3′ and 5′ exon-intron boundaries (splice sites). These splice site sequences function as binding sites for small nuclear ribonuclear particles (snRNPs),1 which form the core of the spliceosome and direct RNA cleavage and ligation (for review see Ref. 14Reed R. Palandjian L. Krainer A.R. Eukaryotic mRNA Processing.in: Oxford University Press, Oxford, UK1997: 103-129Google Scholar). However, in mammals the sequences at the 3′ and 5′ splice sites are poorly conserved and are often insufficient for optimal recognition by the snRNPs (15Black D.L. RNA ( N. Y. ). 1995; 1: 763-771PubMed Google Scholar, 16Reed R. Curr. Opin. Genet. Dev. 1996; 6: 215-220Crossref PubMed Scopus (251) Google Scholar). Therefore, the splicing of many mammalian exons is dependent on the presence of additional sequences within the exon itself or within the flanking intron. Numerous splicing enhancers have been identified that promote exon splicing by helping to recruit snRNPs to the splice sites and/or by promoting polyadenylation (reviewed in Refs. 17Wang J. Manley J.L. Curr. Opin. Genet. Dev. 1997; 7: 205-211Crossref PubMed Scopus (99) Google Scholar, 18Blencowe B.J. Trends Biochem. Sci. 2000; 25: 106-110Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar, 19Smith C.W.J. Valcarcel J. Trends Biochem. Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). In addition, other sequences have been shown to inhibit splicing of a given exon via mechanisms that include the steric hindrance of closely neighboring splice sites, sequestration of snRNPs, and sequestration of splice sites (17Wang J. Manley J.L. Curr. Opin. Genet. Dev. 1997; 7: 205-211Crossref PubMed Scopus (99) Google Scholar, 19Smith C.W.J. Valcarcel J. Trends Biochem. Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). The activity of such enhancing and inhibitory sequence elements may be constitutive or may be regulated by specific trans-acting factors, thereby allowing for regulated splice site use (19Smith C.W.J. Valcarcel J. Trends Biochem. Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (758) Google Scholar). In fact, it has recently been proposed that the splicing of most, if not all, mammalian exons are influenced by at least some constitutive enhancer or inhibitor elements in addition to the canonical splice sites (16Reed R. Curr. Opin. Genet. Dev. 1996; 6: 215-220Crossref PubMed Scopus (251) Google Scholar, 20Schaal T.D. Maniatis T. Mol. Cell. Biol. 1999; 19: 261-273Crossref PubMed Scopus (165) Google Scholar). In this study, we found that indeed multiple, opposing, splicing regulatory elements exist within exon 4 of CD45. Moreover, we show that the multiple sclerosis-associated C77G mutation disrupts the strongest of these elements, namely an exonic silencer element that functions by inhibiting the use of the 5′splice sites (ss) of exon 4. The minigene constructs used in this study were all derived from the previously described minigene MG4, which is subcloned into the MluI-ClaI sites of the stable expression vector pAWneo3 (12Lynch K.W. Weiss A. Mol. Cell. Biol. 2000; 20: 70-80Crossref PubMed Scopus (115) Google Scholar). The two-intron substitution constructs, QC1–QC10 and C77G, were all synthesized using the QuikChange Kit (Stratagene). The resultant mutant minigenes were fully sequenced and reinserted into a fresh pAWneo3 vector to ensure that no extraneous mutations existed that might alter the splicing of the minigene. The single-intron constructs encompassing exons 3 and 4 or 4 and 7 were synthesized using PCR to truncate exon 4. For exon 3–4 constructs a PCR primer was used that terminates exon 4 at nucleotide 181 followed by a ClaI restriction site. For exon 4–7 constructs a PCR primer was used that contains an MluI restriction site followed immediately by exon 4 beginning at nucleotide 18. The C77G, QC2, QC3, QC4, and QC5 mutations were then introduced into the truncated minigenes as described above for the three-exon minigene. The 5′ss UP mutations were also introduced into the exon 3–4–7 and 4–7 constructs using the QuikChange mutagenesis method. For the CD45 exon 4-globin exon 2 chimera, the CD45 portion contains exon 4 (beginning at nucleotide 18) and ∼200 nucleotides (nt) of normal flanking downstream intron (up to a naturally occurringBglII site) were ligated to an engineered BglII site within the normal β-globin intron ∼60 nucleotides upstream of exon 2. For the ESS1 insertion into the β-globin transcript, complementary oligonucleotides were synthesized that contain the entire ESS1 sequence (nt 29–88) with or without the C77G change, flanked byNcoI sites. The oligos were annealed and inserted into the natural NcoI site located 92 nucleotides upstream of the 5′ss of globin exon 1 (20Schaal T.D. Maniatis T. Mol. Cell. Biol. 1999; 19: 261-273Crossref PubMed Scopus (165) Google Scholar). The plasmid templates for RNA transcription were generated as follows. Complementary oligos corresponding to nucleotides 40–90 of CD45 exon 4 were annealed and inserted into the SmaI site of a variant of pSP64 (Promega), which has a T7 promoter immediately adjacent to theSmaI site, to give the plasmid pT7-ESS1. pT7-ESS1-C77G was created in the same way except that the synthesized oligos contain the C77G polymorphism. The plasmid Rx2 was described previously (21Lynch K.W. Maniatis T. Genes Dev. 1996; 10: 2089-2101Crossref PubMed Scopus (199) Google Scholar). All RT-PCR assays were done exactly as detailed previously (12Lynch K.W. Weiss A. Mol. Cell. Biol. 2000; 20: 70-80Crossref PubMed Scopus (115) Google Scholar). The conditions were determined empirically to result in a signal that was linear with respect to input RNA. RT-PCR reactions were resolved on 5% denaturing polyacrylamide gels and quantitated using a phosphorimaging device (Fuji). Nuclear extract was prepared from JSL1 cells using a standard protocol (22Eperon, I. C., Krainer, A. R., RNA Processing: A Practical Approach, Higgins, S. J., Hames, B. D., 1, 1994, 57, 102, Oxford University Press, New York.Google Scholar). The competitor RNAs, WT, C77G, and Dsx, were synthesized from the plasmids pT7-ESS1, pT7-ESS1-C77G, and Rx2, respectively, by transcription with T7 RNA polymerase (Promega). For the radiolabeled ESS1 RNA probe, [32P]CTP was added during transcription. Following transcription, all RNAs were gel-purified. To assay binding, ESS1 probe RNA was incubated with the JSL1 nuclear extract under standard splicing conditions (22Eperon, I. C., Krainer, A. R., RNA Processing: A Practical Approach, Higgins, S. J., Hames, B. D., 1, 1994, 57, 102, Oxford University Press, New York.Google Scholar) for 15 min at 30 °C in the presence or absence of cold competitor RNA. The reactions were then placed on ice, and heparin was added to a final concentration of 0.5 mg/ml. Reactions were loaded on a prerun 4.5% polyacrylamide 0.5× TBE gel and run at 150 V for 1.5 h at room temperature. Gels were dried and analyzed by autoradiography. The effect of the C77G polymorphism on the splicing of CD45 exon 4 can be explained by one of two general models. Either the polymorphism disrupts a sequence that normally represses the recognition of exon 4, or alternatively the change from C to G at position 77 creates a novel splicing enhancer element. The former possibility is supported by data from a linker scanning mutagenesis of CD45 exon 4, which was designed to determine the sequences involved in regulating the exclusion of this exon from transcripts expressed in thymocytes (23Streuli M. Saito H. EMBO J. 1989; 8: 787-796Crossref PubMed Scopus (88) Google Scholar). That study identified three regions corresponding to nt 8–11, 40–92, and 126–137 that, when mutated, resulted in the aberrant inclusion of exon 4. Although the identification of nt 40–92 as repressing sequences is consistent with a model in which the C77G polymorphism disrupts a splicing repressor element, there are two important caveats to this original study. First, in the linker scanning substitutions there was some modification of exon length and no indication of whether the substituting sequences had any inherent effect on splicing (23Streuli M. Saito H. EMBO J. 1989; 8: 787-796Crossref PubMed Scopus (88) Google Scholar). Secondly, that study was done prior to the discovery of the C77G polymorphism, so a direct comparison has not been done between the linker scanning results and the effect of the C77G mutation. We have previously developed and characterized a cell line JSL1, which faithfully reproduces the pattern of CD45 splicing that is observed in mature, naive T cells (12Lynch K.W. Weiss A. Mol. Cell. Biol. 2000; 20: 70-80Crossref PubMed Scopus (115) Google Scholar). In addition, we have identified a CD45 minigene consisting of variable exon 4 flanked by constitutive exons 3 and 7 that, when stably expressed in JSL1 cells, shows a pattern of exon 4 inclusion consistent with that seen in the endogenous CD45 gene (12Lynch K.W. Weiss A. Mol. Cell. Biol. 2000; 20: 70-80Crossref PubMed Scopus (115) Google Scholar). Importantly, when the C77G polymorphism is engineered into our CD45 minigene, it results in a dramatic increase in the inclusion of exon 4 ((12) and Fig. 1 B). Therefore, our minigene and cell line provide a good model system to study in more detail the effect of the C77G polymorphism on the splicing of CD45 exon 4. To understand why the C77G polymorphism increases the inclusion of exon 4, we first wanted to map potential exonic regulatory elements within exon 4 in the context of our minigene. To do this we systematically changed blocks of 20 nt within exon 4 to a heterologous 20-mer sequence (Fig. 1 A). The heterologous sequence we used for these substitutions is a sequence from the IgM gene that was previously shown to lack intrinsic splicing enhancer activity (24Watakabe A. Tanaka K. Shimura Y. Genes Dev. 1993; 7: 407-418Crossref PubMed Scopus (307) Google Scholar). The resultant mutant minigenes, as well as the wild type counterpart, were transfected into the JSL1 cells, and individual clones that stably expressed the desired minigene were isolated and expanded. To determine the relative splicing of exon 4 in these various minigenes, we analyzed total cellular RNA by an RT-PCR assay, which we have described previously (12Lynch K.W. Weiss A. Mol. Cell. Biol. 2000; 20: 70-80Crossref PubMed Scopus (115) Google Scholar). To ensure that any differences seen were not the result of random clonal variation, we analyzed four to six independent clones for each minigene. In each case we detected no significant variation among the independent clones; however, for simplicity, in this study we only show two to three representative clones for each minigene. Consistent with our previous results, 40–50% of transcripts from the wild type minigene contain exon 4 ((12) and Fig. 1, B andC). Strikingly, the splicing of exon 4 is dramatically stimulated when nucleotides 29–48, 49–68, or 69–88 are substituted with the heterologous sequence, similar to the effect of the C77G polymorphism (compare Fig. 1 C, QC2,QC3, and QC4, and Fig. 1 B). The stimulation of exon 4 splicing observed in constructs QC2–QC4 is unlikely to be a consequence of the insertion of the heterologous sequence because the substitution of this sequence at other locations within exon 4 had either the opposite effect (QC5 andQC8) or no effect (QC1, QC7, andQC10) on the inclusion of exon 4 (Fig. 1 C). In addition, the results shown here with QC2–QC4 are very similar to those obtained by Streuli and Saito (23Streuli M. Saito H. EMBO J. 1989; 8: 787-796Crossref PubMed Scopus (88) Google Scholar) upon the substitution of nucleotides 40–92 with a sequence unrelated to that used in this study. Thus, we conclude that the region of exon 4 encompassing nucleotides 29–88 most likely represents a strong exonic splicing silencer (ESS), which represses the inclusion of this exon. In addition to the strong ESS at nucleotides 29–88 (ESS1), our mutational analysis reveals the presence of multiple additional elements within exon 4 that influence its splicing, albeit more weakly than ESS1 (Fig. 1 C). Substitution of nucleotides 169–188 results in the increased inclusion of exon 4. In contrast, the inclusion of exon 4 is decreased significantly upon substitution of nucleotides 89–108 and 149–168, indicating that these sequences may normally function to enhance exon 4 splicing. Therefore, multiple, competing splicing regulatory elements likely exist within CD45 exon 4. Because of our interest in the C77G polymorphism, this study focuses on understanding the role of the ESS1 element and its effect on the flanking splice sites and juxtaposed regulatory element (nt 89–108). The first step in the splicing of an exon involves the recognition of the 3′- and 5′ss (14Reed R. Palandjian L. Krainer A.R. Eukaryotic mRNA Processing.in: Oxford University Press, Oxford, UK1997: 103-129Google Scholar). Although the recognition of one splice site often facilitates the use of the other through a process known as exon definition, the two splice sites can also be recognized independently and are both potential sites of regulation (16Reed R. Curr. Opin. Genet. Dev. 1996; 6: 215-220Crossref PubMed Scopus (251) Google Scholar, 25Graveley B. RNA ( N. Y. ). 2000; 6: 1197-1211Crossref PubMed Scopus (883) Google Scholar). To determine which of the splice sites of exon 4 is regulated by the C77G polymorphism and by ESS1, we made single-intron minigene constructs in which exon 4 was truncated either just upstream of the 5′ss or just downstream of the 3′ss (see Fig. 2, A andB, respectively). Strikingly, RT-PCR analysis of multiple clones that stably express these minigenes indicates that the 3′ss of exon 4 is recognized constitutively (Fig. 2 A,WT), whereas splicing of the 5′ss of exon 4 in the absence of the 3′ss is very inefficient (Fig. 2 B, WT). Moreover, in the presence of the C77G polymorphism, the use of the 5′ss is dramatically increased (Fig. 2 B), whereas this polymorphism has no apparent effect on 3′ss utilization (Fig.2 A). In all cases the RT-PCR assays were done under conditions in which no signal was detectable when reverse transcriptase was omitted, confirming that the slower migrating band is indeed unspliced RNA and not contaminating genomic DNA (data not shown). To confirm that the 5′ss of exon 4 and not the 3′ss of exon 7 is the sequence that is regulated by the C77G polymorphism, we constructed a minigene chimera consisting of CD45 exon 4 fused to a heterologous exon. The heterologous exon we chose to use is the 3′ss and complete exon 2 of the human β-globin gene, which is spliced constitutively (20Schaal T.D. Maniatis T. Mol. Cell. Biol. 1999; 19: 261-273Crossref PubMed Scopus (165) Google Scholar). The splicing of the resultant CD45 exon 4-globin exon 2 chimera is inefficient because of the weak 5′ss of CD45 exon 4 (Fig.2 C, WT). However, as predicted, the substitution of the C77G polymorphism into the chimera strongly enhances the splicing of the 5′ss of exon 4 to the heterologous β-globin exon (Fig. 2 C, C77G). Finally, to determine whether the C77G polymorphism functions similarly to the other ESS1 mutations, we constructed the QC2, QC3, and QC4 substitutions in the context of the CD45 exon 4-exon 7 minigene. As shown in Fig. 2 D, the QC2-QC4 substitutions indeed strongly promote use of the 5′ss of exon 4, indicating that these substitutions are mechanistically similar to the C77G mutation. Taken together, these data indicate that ESS1 inhibits use of the 5′ss of exon 4 and further indicate that the C77G polymorphism allows for inclusion of exon 4 by disrupting the activity of this silencer. The canonical sequence of a mammalian 5′ss is AG/GUAAGU in which / indicates the point of cleavage (14Reed R. Palandjian L. Krainer A.R. Eukaryotic mRNA Processing.in: Oxford University Press, Oxford, UK1997: 103-129Google Scholar). Although the 5′ss of CD45 exon 4 contains the most critical of these nucleotides (the GU at positions +1 and +2 relative to the cleavage site and a G at position +5), it differs from the consensus at the other positions in the intron (Fig.3, A and B). The differences between the consensus 5′ss sequence and that present at the 5′ss of exon 4 suggest that the 5′ss of exon 4 might be inherently weak. To test this possibility and investigate the influence of 5′ss strength on exon 4 splicing, we constructed minigenes in which the 5′ss of exon 4 was mutated to exactly match the canonical mammalian 5′ss. As shown in Fig. 3, A and B, respectively, this 5′ss UP mutation results in a significant increase in the splicing of a two-exon single-intron construct and results in the complete inclusion of exon 4 in a three-exon construct. Thus, the 5′ss of exon 4 is inherently weak, and this characteristic is important for its incomplete inclusion and susceptibility to regulation. However, we do note that in the single-intron construct in which the UP mutation does not result in complete recognition of the 5′ss, the presence of the C77G polymorphism does allow for further use of the 5′ss (Fig.3 A, 90% versus 96% splicing). Therefore, the ESS1 can suppress, albeit weakly, the utilization of an optimized 5′ss. As mentioned above, the ESS1 is only one of several exonic elements that appear to influence the splicing of exon 4. We were particularly interested in the presence of a putative exonic splicing enhancer (ESE) positioned immediately downstream of ESS1 (nt 89–108). Previous studies have identified similar cases of a juxtaposed splicing enhancer and inhibitor that function to counterbalance each other (26Kan J.L.C. Green M.R. Genes Dev. 1999; 13: 462-471Crossref PubMed Scopus (122) Google Scholar). The sequence of the ESS1 proximal enhancer element, which we term ESE1, closely resembles that of a canonical purine-rich splicing enhancer. Most well characterized purine-rich enhancers have been shown to enhance the use of a weak 3′ss (24Watakabe A. Tanaka K. Shimura Y. Genes Dev. 1993; 7: 407-418Crossref PubMed Scopus (307) Google Scholar, 27Tanaka K. Watakabe A. Shimura Y. Mol. Cell. Biol. 1994; 14: 1347-1354Crossref PubMed Scopus (206) Google Scholar, 28Lavigueur H. Branche L. Kornblitt A.R. Chabot B. Genes Dev. 1993; 7: 2405-2417Crossref PubMed Scopus (274) Google Scholar, 29Sun Q. Mayeda A. Hampson R.K. Krainer A.R. Rottman F.M. Genes Dev. 1993; 7: 2598-2608Crossref PubMed Scopus (246) Google Scholar). However, there is at least one example of enhancement of a 5′ss by a purine-rich enhancer (30Bourgeois C.F. Popeilarz M. Stevenin J. Mol. Cell. Biol. 1999; 19: 7347-7356Crossref PubMed Scopus (61) Google Scholar) as well as an example of 5′ss enhancement by a non-purine-rich element (31Ryner L.C. Goodwin S.F. Castrillon D.H. Anand A. Villella A. Baker B.S. Hall J.C. Taylor B.J. Wasserman S.A. Cell. 1996; 87: 1079-1089Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Therefore, we wanted to determine whether ESE1 stimulates the use of exon 4 splicing by enhancing 3′ss recognition or alternatively, whether ESE1 functions to counter repression of the 5′ss by the ESS1. As shown in Fig.4 A, splicing to the 3′ss of exon 4 is equally efficient in the presence or absence of ESE1. In contrast, disruption of ESE1 by the QC5 substitution further decreases the inefficient recognition of the 5′ss by 2-fold (Fig. 4 B). Thus, we conclude that the ESE1 element activates exon 4 inclusion by enhancing 5′ss use. Given that the ESS1 and ESE1 appear to influence the same step in exon 4 recognition, we next wanted to determine whether the activities of ESS1 and ESE1 were inter-related or separable. For example, if the ESS1 represses 5′ss use by directly suppressing the enhancer activity of ESE1, then when the ESE1 element is absent, disruption of the ESS1 should have no effect. As shown in Fig. 4 C, this prediction is clearly not met. Instead, we observe that disruption of ESS1 by the C77G polymorphism in the background of the ESE1-eliminating substitution QC5 results in a significant increase in exon inclusion (from 14 to 58%). Similarly, elimination of ESE1 in the background of the C77G polymorphism decreases the splicing of exon 4 from 95 to 58%. Therefore, we conclude that the ESS1 and ESE1 function independently of each other to influence utilization of the 5′ss of exon 4. To further determine whether the ESS1 truly functions as a separable splicing silencer element, we tested whether the presence of the ESS1 could confer repression on a heterologous splicing construct. When stably expressed in JSL1 cells, exons 1 and 2 of a human β-globin minigene are spliced together with relatively high efficiency (Fig.5, Glo). However, this efficiency is significantly decreased when the ESS1 is inserted ∼80 nt upstream of the 5′ss of exon 1 (Fig. 5, GloESS) such that the relative percentage of unspliced transcript increases by more than 3-fold. Importantly, at least half of the ESS1 repression of β-g