Arianne J. Matlin

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents. Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents. The removal of introns from nascent transcripts by the process of pre-mRNA (precursor to messenger RNA) splicing is an essential step in eukaryotic gene expression. Splicing is mediated by the spliceosome, a highly dynamic, multimegadalton machine composed of five small stable nuclear RNAs (snRNAs) 2The abbreviations used are: snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein; FRT, flp recombinase target; RT, reverse transcription; AdML, adenovirus major late; TPI, triose-phosphate isomerase. 2The abbreviations used are: snRNA, small nuclear RNA; snRNP, small nuclear ribonucleoprotein; FRT, flp recombinase target; RT, reverse transcription; AdML, adenovirus major late; TPI, triose-phosphate isomerase. and more than 100 polypeptides (reviewed in Ref. 1Matlin A.J. Moore M.J. Adv. Exp. Med. Biol. 2007; 623: 14-35Crossref PubMed Scopus (68) Google Scholar). Within the spliceosome, intron excision occurs in two chemical steps: 1) 5′ splice site cleavage accompanied by lariat formation at the branch point adenosine and 2) 3′ splice site cleavage accompanied by exon ligation. Both of these steps are readily observable in in vitro reactions containing crude nuclear extract and ATP as an energy source. In such reactions, spliceosome assembly occurs in a distinctly stepwise fashion. First, the pre-mRNA substrate is coated with a heterogeneous mixture of RNA-binding proteins (referred to as H complex). Interaction of U1 snRNP (U1 snRNA and its associated proteins) with the 5′ splice site and recognition of the branch point adenosine by U2 snRNP generates an early commitment complex (E or CC complex). A subsequent ATP-dependent step stabilizes the U2 snRNP-branch point interaction, resulting in formation of the prespliceosome (A complex). Entry of the U4/U5/U6 tri-snRNP to form B complex is followed by multiple structural rearrangements, which produce the catalytically active C complex, wherein the two chemical steps of splicing occur. Finally, the ligated exon and lariat products are released, and the remaining spliceosome components are disassembled. In the more than two decades since its initial description (2Brody E. Abelson J. Science. 1985; 228: 963-967Crossref PubMed Scopus (222) Google Scholar, 3Grabowski P.J. Seiler S.R. Sharp P.A. Cell. 1985; 42: 345-353Abstract Full Text PDF PubMed Scopus (245) Google Scholar), a wealth of information has been gleaned regarding the parts list of the spliceosome, its gross assembly/disassembly pathway, certain key local structural interactions, and the activities of individual components. However, in comparison with other macromolecular machines, such as the ribosome and RNA polymerase II, our understanding of the spliceosome's inner workings and its detailed structure is still in its infancy. Mechanistic understanding of other macromolecular complexes has been greatly enhanced by the availability of multiple small molecule inhibitors impeding their functional cycles at different points (4Jurica M.S. Nat. Chem. Biol. 2008; 4: 3-6Crossref PubMed Scopus (10) Google Scholar, 5Steitz T.A. Nat. Rev. Mol. Cell. Biol. 2008; 9: 242-253Crossref PubMed Scopus (300) Google Scholar). Such small molecules have been likened to wrenches that can be thrown into the works to freeze cellular machines in specific conformations, making them more amenable to biochemical and structural investigation (4Jurica M.S. Nat. Chem. Biol. 2008; 4: 3-6Crossref PubMed Scopus (10) Google Scholar). Although a set of such wrenches that inhibit splicing in vitro or inhibit specific or alternative splicing events in vivo is being generated (4Jurica M.S. Nat. Chem. Biol. 2008; 4: 3-6Crossref PubMed Scopus (10) Google Scholar, 6Bakkour N. Lin Y.L. Maire S. Ayadi L. Mahuteau-Betzer F. Nguyen C.H. Mettling C. Portales P. Grierson D. Chabot B. Jeanteur P. Branlant C. Corbeau P. Tazi J. PLoS Pathog. 2007; 3: 1530-1539Crossref PubMed Scopus (68) Google Scholar, 7Nottrott S. Hartmuth K. Fabrizio P. Urlaub H. Vidovic I. Ficner R. Luhrmann R. EMBO J. 1999; 18: 6119-6133Crossref PubMed Scopus (164) Google Scholar, 8Soret J. Bakkour N. Maire S. Durand S. Zekri L. Gabut M. Fic W. Divita G. Rivalle C. Dauzonne D. Nguyen C.H. Jeanteur P. Tazi J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8764-8769Crossref PubMed Scopus (98) Google Scholar, 9Stoilov P. Lin C.H. Damoiseaux R. Nikolic J. Black D.L. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 11218-11223Crossref PubMed Scopus (107) Google Scholar, 10Sumanasekera C. Watt D.S. Stamm S. Biochem. Soc. Trans. 2008; 36: 483-490Crossref PubMed Scopus (53) Google Scholar), there is currently a paucity of small molecules that affect general pre-mRNA splicing in vivo. In this paper, we describe a cell-based assay to screen for general splicing inhibitors. This assay takes advantage of the observation that some amount of unspliced pre-mRNA can escape from the nucleus and become available for translation in the cytoplasm (11Legrain P. Rosbash M. Cell. 1989; 57: 573-583Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 12Nasim M.T. Chowdhury H.M. Eperon I.C. Nucleic Acids Res. 2002; 30: e109Crossref PubMed Scopus (36) Google Scholar). By screening for an increase in reporter protein expression from a mammalian pre-mRNA designed such that only the unspliced version generates active protein, we were able to identify a compound that acts as a general inhibitor of splicing both in vivo and in vitro. This inhibitor is the naturally occurring biflavonoid isoginkgetin. In in vitro reactions, isoginkgetin causes accumulation of the prespliceosomal A complex. Like two other compounds recently described as in vivo splicing inhibitors (13Kaida D. Motoyoshi H. Tashiro E. Nojima T. Hagiwara M. Ishigami K. Watanabe H. Kitahara T. Yoshida T. Nakajima H. Tani T. Horinouchi S. Yoshida M. Nat. Chem. Biol. 2007; 3: 576-583Crossref PubMed Scopus (471) Google Scholar, 14Kotake Y. Sagane K. Owa T. Mimori-Kiyosue Y. Shimizu H. Uesugi M. Ishihama Y. Iwata M. Mizui Y. Nat. Chem. Biol. 2007; 3: 570-575Crossref PubMed Scopus (440) Google Scholar), isoginkgetin is a known anti-tumor agent (15Yoon S.O. Shin S. Lee H.J. Chun H.K. Chung A.S. Mol. Cancer Ther. 2006; 5: 2666-2675Crossref PubMed Scopus (92) Google Scholar). Our results suggest that the mechanistic basis of the anti-tumor activity of isoginkgetin is its inhibition of pre-mRNA splicing. Plasmids—Reporter construct I was created by replacing the Renilla luciferase gene in plasmid triose-phosphate isomerase (TPI)/Renilla luciferase 5′ intron (pSHM06T) (16Nott A. Meislin S.H. Moore M.J. RNA. 2003; 9: 607-617Crossref PubMed Scopus (338) Google Scholar) with the firefly luc2 gene from plasmid pGL4.10 (Promega). Construct II is identical to construct I except that site-directed mutagenesis was used to remove an in-frame stop codon in the intron and add a G at position 6 in TPI exon 7. Construct III was generated by site-directed mutagenesis of II to inactivate the 5′ splice site. Constructs I, II, and III were subcloned into vector pcDNA5/FRT for integration into the flp recombinase target (FRT) sequence in HEK293 cells (described below). Cell Culture and Generation of Stable Cell Lines—Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (Invitrogen), as indicated below. Trypan blue (Invitrogen) and Alamar blue (BIOSOURCE) assays were done according to standard protocols. To generate a host cell line containing an integrated FRT site, HEK293 cells (ATCC) were transfected with ScaI-linearized pFRT/lacZeo (Invitrogen) using FuGENE 6 (Roche Applied Science) as per the Flp-In™ system manual (Invitrogen). Stable integrants were selected with 100 μg/ml zeocin. Southern blotting and β-galactosidase assays identified cell clones with integrants expressing intermediate levels of the LacZ-Zeo fusion. Two such clones (293F1 and 293F2) were subsequently co-transfected with one of the three reporter plasmids and pOG44 (Invitrogen), which encodes Flp recombinase. Successful targeting of the reporter plasmid to the FRT site was selected by treatment with 100 μg/ml hygromycin. Luciferase Assays and RNA were grown in or and with or as indicated in the Luciferase assays assay were using a were for using the protein assay RNA was using and according to the protocols. a was to be used both for luciferase activity and for cells were in was with and used for luciferase and the was to of for RNA was using the III (Invitrogen). of RNA was used per and with different of RNA were the being were with at two of RNA to that were the the product an snRNA, an unspliced polymerase III were as a are in screening was at the in with the were in at cell from to the in luciferase activity the cell lines II and were in a using a or a To to for the compound cell lines II and III were in containing to in or for of the cell cell lines II and III were in medium in at a of 100 of compounds or was using a with a to the luciferase activities were using the Luciferase according to the of the cell and assay were to the using a for luciferase were using an was as described S. S. J. M. P. P.A. Nucleic Acids Res. 2008; 36: PubMed Scopus Google Scholar) to and of compound cells were grown in or and with or as described and initial were with isoginkgetin from Isoginkgetin for subsequent was from To their and were to reverse from that activity in cellular assays a that by the In Splicing adenovirus major TPI and 2) pre-mRNA splicing were generated by of plasmids and and as previously described H. E. Moore M.J. EMBO J. PubMed Scopus Google Scholar). cell nuclear extract was as previously described PubMed Scopus Google Scholar, Nucleic Acids Res. PubMed Scopus Google Scholar, Moore M.J. Nucleic Acids Res. PubMed Scopus Google Scholar). Splicing reactions containing nuclear and or isoginkgetin in were for the indicated at C. splicing complexes were as described previously Moore M.J. Nucleic Acids Res. PubMed Scopus Google Scholar, Sharp P.A. Cell. Full Text PDF PubMed Scopus Google Scholar). Splicing were by RNAs To the of was using version for of Cell for in generate for in vivo we created a splicing reporter of the TPI gene exon (16Nott A. Meislin S.H. Moore M.J. RNA. 2003; 9: 607-617Crossref PubMed Scopus (338) Google Scholar) of the firefly luciferase construct In this the codon is in the and active luciferase is generated only from In to generate the screening reporter we in stop from the intron and one at the of the such that the luciferase is of with the codon in the is in the intron is created a reporter identical to the screening reporter except for a 5′ splice site point to splicing to A at position in the three were subcloned of the in plasmid This plasmid an FRT site. Stable cell lines expressing reporter were generated using the recombinase To do we selected HEK293 cell lines transfected with a plasmid containing a FRT site at a position in a active of the as by expression of two of these cell lines (293F1 and 293F2) with the reporter and a plasmid Flp recombinase generated the screening cell lines and and and of RNA from cell line that transcripts from I and II were from construct III were for the lines, with luciferase activity levels were in the cells in the splicing reporter cells and intermediate in the reporter cells the three in both the and the two cell line were used of cell lines containing reporter II or III were used to screen for small molecules that splicing. that such inhibitors increase the luciferase activity from reporter II have or reporter of compound II and III to splicing and other of protein expression cell The the luciferase activities of II and III the screening a for compound cells were grown in in to compounds in or an of for which luciferase activities were luciferase was as the of from the of cells S. S. J. M. P. P.A. Nucleic Acids Res. 2008; 36: PubMed Scopus Google Scholar). A was by the of for line This comparison of two biological that have of this the luciferase activity of cells expressing construct II expressing construct S. S. J. M. P. P.A. Nucleic Acids Res. 2008; 36: PubMed Scopus Google Scholar). compound to have a its in and the for reporter II was the for reporter III compounds were from natural product and some at multiple were known from available and and the were the products of multiple A list of compounds and the in our assay can be the The of compounds the as by treatment or the line by II and III compounds such as the inhibitor and the translation inhibitor luciferase activity of both II and for A of compounds a for one reporter a the other this one compound as a very luciferase activity in cells expressing construct II in cells expressing construct III This compound is the biflavonoid a natural product in a of with II and is the for screening compounds the is a the of from the and of is as the of the the vector and the line by from to is the for screening compounds the The is a the of from the and of is as the of the the vector and the line by from to in a new and of Isoginkgetin as an in Splicing the isoginkgetin results from the cells containing I, II, and III as as the cell line a reporter were with isoginkgetin screening in or with Luciferase activity of reporter II with isoginkgetin that of reporter III only in the the reporter III was with an inhibition of a in luciferase activity was isoginkgetin treatment of cells expressing reporter I. To that the in luciferase activity were to inhibition of we of RNA from and This that to isoginkgetin was to the generated from I and II from to unspliced pre-mRNA that this accumulation of unspliced pre-mRNA was of isoginkgetin with luciferase activity from reporter I to and that from reporter II to increase a similar The of isoginkgetin treatment was since treatment with 100 isoginkgetin to a increase in pre-mRNA accumulation than treatment with or isoginkgetin To the of isoginkgetin splicing was specific to our reporter system or more we TPI, and of these transcripts pre-mRNA accumulation treatment with isoginkgetin A and and intron a known substrate for the minor spliceosome Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). for other isoginkgetin treatment to an increase in the amount of unspliced intron these results that isoginkgetin is a general inhibitor of splicing that both the major and minor spliceosomes. for a general inhibitor of an essential step in gene treatment of cells with isoginkgetin at for splicing in Trypan blue and Alamar blue that this was accompanied by a of cell with removal of isoginkgetin both expression of luciferase from reporter I and cellular B and Thus, isoginkgetin appears to be a inhibitor of pre-mRNA splicing. Isoginkgetin Splicing in isoginkgetin inhibit splicing in we splicing assays in cell nuclear extract using multiple RNA these isoginkgetin was in which affect splicing at of were for in standard reactions supplemented with isoginkgetin Splicing of AdML, and TPI to the in vivo splicing transcripts was by isoginkgetin in a three splicing was by with an of This in vitro splicing inhibition was with different nuclear extract and of the with the compound was for inhibition Thus, as in isoginkgetin is a general inhibitor of splicing in vitro. Isoginkgetin and in the at which splicing is splicing reactions containing pre-mRNA isoginkgetin were to In the of and C complexes was readily a ATP a complex, a than A complex Sharp P.A. Cell. Full Text PDF PubMed Scopus Google Scholar, R. R. RNA. 1999; 5: PubMed Scopus Google Scholar). This complex was observable in reactions containing isoginkgetin ATP In reactions containing both isoginkgetin and or complex with B or C be these reactions a or complex with identical to that of A complex and Thus, appears that isoginkgetin the A to B resulting in of the pre-mRNA in a In comparison with other steps in gene there is currently a paucity of small molecule of pre-mRNA Such their is can be useful tools for both cell biological and biochemical Here we describe the development of a set of stable mammalian cell lines that we in a screen to identify a new general inhibitor of pre-mRNA splicing. Our was that inhibition of splicing to a readily in our an increase in luciferase This is similar to one previously in where a in to expression of of one 5′ splice site C. PubMed Google Scholar). This system has highly for other of pre-mRNA as Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, F. Rosbash M. EMBO J. PubMed Scopus Google Scholar). In to a of other reporter for general or alternative splicing in mammalian cells a protein activity have been described P. Lin C.H. Damoiseaux R. Nikolic J. Black D.L. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 11218-11223Crossref PubMed Scopus (107) Google Scholar, 12Nasim M.T. Chowdhury H.M. Eperon I.C. Nucleic Acids Res. 2002; 30: e109Crossref PubMed Scopus (36) Google Scholar, N. R. A. S. J. P. RNA. 2006; PubMed Scopus Google Scholar, C. EMBO J. PubMed Scopus Google Scholar). M.T. Chowdhury H.M. Eperon I.C. Nucleic Acids Res. 2002; 30: e109Crossref PubMed Scopus (36) Google Scholar) a reporter system in which a transfected plasmid encodes β-galactosidase in the exon and firefly luciferase in the The two are by an intron containing multiple in-frame stop Thus, β-galactosidase is from both pre-mRNA and luciferase is only from the In this specific inhibition of splicing is by a in luciferase activity a in β-galactosidase reporter was by and N. R. A. S. J. P. RNA. 2006; PubMed Scopus Google Scholar) to exon of exon In their expression of the expression of of the initial an increase in by of a in splicing into a in protein expression. This system was used to identify two small molecules that exon N. R. A. S. J. P. RNA. 2006; PubMed Scopus Google Scholar). recently an alternative splicing reporter expressing protein or protein in an alternative in a small screen P. Lin C.H. Damoiseaux R. Nikolic J. Black D.L. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 11218-11223Crossref PubMed Scopus (107) Google Scholar). particular was the of this cell-based assay by the of two alternative splicing their P. Lin C.H. Damoiseaux R. Nikolic J. Black D.L. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 11218-11223Crossref PubMed Scopus (107) Google Scholar) identified a of known such as as the splicing of exon their initial reporter Although some of these compounds were in our have in our This is with the that are splicing In to the our cell-based assay was designed to identify a general inhibitor of pre-mRNA splicing. It is luciferase as a and three identical By using the system to generate cell lines transfected at identical we at the of to different By the expression of two that by only one at the RNA were identical at the protein we of compounds RNA or protein RNA or protein Here we used reporter cell lines II and III to identify a pre-mRNA splicing However, other of these cell lines be used to screen for compounds a different step in a compound of unspliced RNA from the nucleus splicing be to increase the luciferase from construct III only from I and that compounds this particular set of were identified in our screen and may be of screening a small of compounds we were able to identify a splicing the natural product which was by our Isoginkgetin has previously been described as one of from G. of which have been as activities Lee Med. Chem. 2005; PubMed Scopus Google Scholar, Chun PubMed Scopus Google Scholar, Sci. 57: PubMed Scopus Google Scholar) (reviewed in Ref. H. Res. 2008; PubMed Scopus Google Scholar). the of isoginkgetin is as an inhibitor of cell isoginkgetin similar to we of splicing both in vivo and in (15Yoon S.O. Shin S. Lee H.J. Chun H.K. Chung A.S. Mol. Cancer Ther. 2006; 5: 2666-2675Crossref PubMed Scopus (92) Google Scholar) that cell was in the levels of proteins in cell gene expression were to from inhibition of the pathway, the target of isoginkgetin was identified in that two other of compounds have as general pre-mRNA splicing and a of (13Kaida D. Motoyoshi H. Tashiro E. Nojima T. Hagiwara M. Ishigami K. Watanabe H. Kitahara T. Yoshida T. Nakajima H. Tani T. Horinouchi S. Yoshida M. Nat. Chem. Biol. 2007; 3: 576-583Crossref PubMed Scopus (471) Google Scholar, 14Kotake Y. Sagane K. Owa T. Mimori-Kiyosue Y. Shimizu H. Uesugi M. Ishihama Y. Iwata M. Mizui Y. Nat. Chem. Biol. 2007; 3: 570-575Crossref PubMed Scopus (440) Google Scholar, H. Y. H. M. T. S. K. J. PubMed Scopus Google Scholar). Although these natural products were from different and have different both target the complex. which of the three proteins and is a of U2 and for early spliceosome assembly and branch point adenosine to A complex J. B. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). known as the A complex the ATP-dependent of spliceosome and U2 snRNP associated with the branch site. with being the target of we recently that which is of more than A. T. K. J. Chem. Soc. 2007; PubMed Scopus Google Scholar), A complex formation in vitro. J. K. and K. In isoginkgetin appears to act by a different since its the accumulation of A complex in in vitro splicing Although we have identified the target of be components of both the major and minor for the A to B complex of such a is the which stable with the U4/U5/U6 tri-snRNP and to B complex R. Hartmuth K. S. Urlaub H. Ficner R. Luhrmann R. Nat. Mol. Biol. 2008; PubMed Scopus Google Scholar). of its isoginkgetin a useful for the accumulation of for mechanistic and structural studies. Like and its have been reported to have was their as anti-tumor that the to the identification of as the cellular target of and A have both anti-tumor and activity inhibit splicing by two different of A complex formation the A to B complex that their anti-tumor activity is a of to some for cell to general splicing In for of the general splicing to a cell to splicing of The for of at the the cell to R. S. M. T.A. M. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). A similar may be at with small molecule inhibitors of splicing. The may be the of an to in certain proteins in Thus, small molecule inhibitors of general pre-mRNA splicing represent an new avenue for the development of novel anti-cancer agents. for and and for of the of for and results to with

PTB (polypyrimidine tract-binding protein) is a repressive regulator of alternative splicing. We have investigated the role of PTB in three model alternative splicing systems. In the α-actinin gene, PTB represses the SM (smooth muscle) exon by binding to key sites in the polypyrimidine tract. Repressive binding to these sites is assisted by co-operative binding to additional downstream sites. SM exon splicing can be activated by CELF proteins, which also bind co-operatively to interspersed sites and displace PTB from the pyrimidine tract. Exon 11 of PTB pre-mRNA is repressed by PTB in an autoregulatory feedback loop. Exon 11-skipped RNA gets degraded through nonsense-mediated decay. Less than 1% of steady-state PTB mRNA is represented by this isoform, but inhibition of nonsense-mediated decay by RNA interference against Upf1 shows that at least 20% of PTB RNA is consumed by this pathway. This represents a widespread but under-appreciated role of alternative splicing in the quantitative regulation of gene expression, an important addition to its role as a generator of protein isoform diversity. Repression of α-tropomyosin exon 3 is an exceptional example of PTB regulation, because repression only occurs at high levels in SM cells, despite the fact that PTB is widely expressed. In this case, a PTB-interacting cofactor, raver1, appears to play an important role. By the use of ‘tethering’ assays, we have identified discrete domains within both PTB and raver1 that mediate their repressive activities on this splicing event.

The alpha-actinin gene has a pair of alternatively spliced exons. The smooth muscle (SM) exon is repressed in most cell types by polypyrimidine tract binding protein (PTB). CELF (CUG-BP and ETR3-like factors) family proteins, splicing regulators whose activities are altered in myotonic dystrophy, were found to coordinately regulate selection of the two alpha-actinin exons. CUG-BP and ETR3 activated the SM exon, and along with CELF4 they were also able to repress splicing of the NM (nonmuscle) exon both in vivo and in vitro. Activation of SM exon splicing was associated with displacement of PTB from the polypyrimidine tract by binding of CUG-BP at adjacent sites. Our data provides direct evidence for the activity of CELF proteins as both activators and repressors of splicing within a single-model system of alternative splicing, and suggests a model whereby alpha-actinin alternative splicing is regulated by synergistic and antagonistic interactions between members of the CELF and PTB families.