2088 Nucleic Acids Research, 2005, Vol. 33, No. 7 proteins participate in the regulation of pre-mRNA splicing is currently unknown. Of interest, A030 with the exogenous NLS activates N30 splicing as efﬁciently as F011 in the pre-mRNAs derived from minigenes J and H, which contain the shorter intron, whereas this isoform poorly activates N30 splicing in the pre-mRNA from minigene G, which contains the full- length intron. This observation implies that the interactions of A030 with different factors are required in the different pre- mRNA contexts. Therefore, the isoforms of Fxh and A2BP1 described here may have different effects on other UGCAUG- regulated alternative splicing. The involvement of the hexanucleotide UGCAUG in regu- lated alternative splicing has been experimentally demon- strated in a number of neural cell-speciﬁc, as well as other tissue-speciﬁc, model systems (13–21). This hexanucleotide element plays a role, in most cases, as an enhancer in regu- lating alternative splicing of cassette-type exons as well as mutually exclusive exons. Furthermore, computational ana- lysis has revealed that UGCAUG is over-represented in the introns in which splicing is regulated, compared with the con- stitutively spliced introns (43). This analysis also pointed to the UGCAUG element as playing a role in the regulation of tissue-speciﬁc alternative splicing in a wide range of tissues, but not in speciﬁc tissues. To date, KH-type splicing regulatory protein (KSRP) (44), A2BP1 (21) and Fxh (this study) are known to be capable of binding to UGCAUG. KSRP is expressed ubiquitously and tissue-speciﬁc variants of this gene have not been described so far. In this study, we have described the existence of tissue- dependent isoforms of Fxh and A2BP1, which, while not identical in some areas of the molecule, may contain the same RRM. The physiological relevance of these isoforms is that they have different splicing activities and different subcellular localizations. The brain isoforms promote N30 inclusion more efﬁciently than the muscle isoforms of both Fxh and A2BP1. The isoforms lacking the RRM are normally expressed to a signiﬁcant extent in skeletal muscles. This isoform is incapable of activating N30 splicing and, moreover, can inhibit N30 inclusion. The properties of these isoforms are consistent with Fxh and A2BP1 acting as regulators for N30 splicing, since N30 is included in neuronal cells, but exclu- ded in muscles. Therefore, despite the tissue-independent occurrence of UGCAUG as a regulatory element, given the tissue-dependent isoforms of the UGCAUG-binding proteins (Fxh and A2BP1) with different activities, the hexanucleotide UGCAUG could confer tissue speciﬁcity on regulated spli- cing. One of the major problems in understanding the mech- anisms responsible for alternative pre-mRNA splicing is the manner in which tissue speciﬁcity is determined. In verteb- rates, to date, only a few tissue-speciﬁc proteins have been identiﬁed as splicing regulators (8–12). Here, we have shown that the tissue-dependent isoforms of the sequence-speciﬁc RNA-binding proteins, which themselves are generated by alternative splicing, have different activities in tissue- speciﬁc alternative splicing of target pre-mRNA. Therefore, these isoforms play a role in the determination of tissue spe- ciﬁcity of target pre-mRNA splicing. The discovery of these tissue-dependent isoforms of the UGCAUG-binding proteins with different splicing activities adds an important new dimen- sion to the molecular mechanisms responsible for regulating tissue-dependent alternative splicing mediated via UGCAUG. ACKNOWLEDGEMENTS We are grateful to Dr Robert S. Adelstein (NHLBI) for con- tinued encouragement, helpful discussions and critical reading of the manuscript. We acknowledge the professional skills and advice of Dr Christina A. Combs (Light Microscopy Core Facility, NHLBI, NIH) and the excellent editorial assis- tance of Catherine S. Magruder. Funding to pay the Open Access publication charges for this article was provided by Division of Intramural Research, NHLBI, NIH, USA. Conflict of interest statement. None declared. REFERENCES 1. Smith,C.W. and Valcarcel,J. (2000) Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem. Sci., 25, 381–388. 2. Black,D.L. (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem., 72, 291–336. 3. Cartegni,L., Chew,S.L. and Krainer,A.R. (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nature Rev. Genet., 3, 285–298. 4. Stoilov,P., Meshorer,E., Gencheva,M., Glick,D., Soreq,H. and Stamm,S. (2002) Defects in pre-mRNA processing as causes of and predisposition to diseases. DNA Cell Biol., 21, 803–818. 5. Faustino,N.A. and Cooper,T.A. (2003) Pre-mRNA splicing and human disease. Genes Dev., 17, 419–437. 6. Graveley,B.R. (2000) Sorting out the complexity of SR protein functions. RNA, 6, 1197–1211. 7. Wagner,E.J. and Garcia-Blanco,M.A. (2001) Polypyrimidine tract binding protein antagonizes exon definition. Mol. Cell. Biol., 21, 3281–3288. 8. Jensen,K.B., Dredge,B.K., Stefani,G., Zhong,R., Buckanovich,R.J., Okano,H.J., Yang,Y.Y. and Darnell,R.B. (2000) Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. Neuron, 25, 359–371. 9. Polydorides,A.D., Okano,H.J., Yang,Y.Y., Stefani,G. and Darnell,R.B. (2000) A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing. Proc. Natl Acad. Sci. USA, 97, 6350–6355. 10. Markovtsov,V., Mikolic,J.M., Goldman,J.A., Turck,C.W., Chou,M.Y. and Black,D.L. (2000) Cooperative assembly of an hnRNP complex induced by a tissue-specific homology of polypyrimidine tract binding protein. Mol. Cell. Biol., 20, 7463–7479. 11. Ladd,A.N., Charlet,N. and Cooper,T.A. (2001) The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol. Cell. Biol., 21, 1285–1296. 12. Zhang,W., Liu,H., Han,K. and Grabowski,P.J. (2002) Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR. RNA, 8, 671–685. 13. Huh,G.S. and Hynes,R.O. (1994) Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element. Genes Dev., 8, 1561–1574. 14. Kawamoto,S. (1996) Neuron-specific alternative splicing of nonmuscle myosin II heavy chain-B pre-mRNA requires a cis-acting intron sequence. J. Biol. Chem., 271, 17613–17616. 15. Del Gatto,F., Plet,A., Gesnel,M.C., Fort,C. and Breathnach,R. (1997) Multiple interdependent sequence elements control splicing of a fibroblast growth factor receptor 2 alternative exon. Mol. Cell. Biol., 17, 5106–5116. 16. Hedjran,F., Yeakley,J.M., Huh,G.S., Hynes,R.O. and Rosenfeld,M.G. (1997) Control of alternative pre-mRNA splicing by distributed penta- meric repeats. Proc. Natl Acad. Sci. USA, 94, 12343–12347. 17. Modafferi,E.F. and Black,D.L. (1997) A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon. Mol. Cell. Biol., 17, 6537–6545. 18. Lim,L.P. and Sharp,P.A. (1998) Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats. Mol. Cell. Biol., 18, 3900–3906. 19. Deguillien,M., Huang,S.C., Moriniere,M., Dreumont,N., Benz,E.J.,Jr and Baklouti,F. (2001) Multiple cis elements regulate an alternative