Identification of Two RNA cis-Elements That Function to Regulate the 5′ Splice Site Selection of Bcl-x Pre-mRNA in Response to Ceramide

Abstract

Two splice variants derived from the BCL-x gene, proapoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L), are produced via alternative 5′ splice site selection within exon 2 of Bcl-x pre-mRNA. In previous studies, our laboratory demonstrated that ceramide regulated this 5′ splice site selection, inducing the production of Bcl-x(s) mRNA with a concomitant decrease in Bcl-x(L) correlating with sensitization to chemotherapy (Chalfant, C. E., Rathman, K., Pinkerman, R. L., Wood, R. E., Obeid, L. M., Ogretmen, B., and Hannun, Y. A. (2002) J. Biol. Chem. 277, 12587–12595). We have now identified several possible RNA cis-elements within exon 2 of Bcl-x pre-mRNA by sequence analysis. To study the possible roles of these RNA cis-elements in regulating the alternative 5′ splice site selection of Bcl-x pre-mRNA, we developed a BCL-x minigene construct which conferred the same ratio of Bcl-x(L)/Bcl-x(s) mRNA as the endogenous Bcl-x and was responsive to ceramide treatment. Mutagenesis of either a purine-rich splicing enhancer or a pyrimidine tract element within exon 2 induced a change in the ratio of Bcl-x(L)/Bcl-x(s) mRNA from 7 to 1 and 0.23, thereby diminishing the selection of the Bcl-x(L) 5′ splice site with a concomitant increase in Bcl-x(s) 5′ splice site selection. Furthermore, mutagenesis of these cis-elements abolished the ability of ceramide to affect the 5′ splice site selection. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-activating proteins to these regions. SDS-PAGE analysis of cross-linked RNA trans-activating factors with these RNA cis-elements revealed the binding of 215-, 120-, and 30-kDa proteins to the purine-rich element and 120- and 76-kDa proteins to the pyrimidine tract element. In addition, exogenous treatment of A549 cells with ceramide increased the formation of protein complexes with these RNA cis-elements. Therefore, we have identified two ceramide-responsive RNA cis-elements within exon 2 of Bcl-x pre-mRNA, and this is the first report of an RNA cis-element responsive to a bioactive lipid. Two splice variants derived from the BCL-x gene, proapoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L), are produced via alternative 5′ splice site selection within exon 2 of Bcl-x pre-mRNA. In previous studies, our laboratory demonstrated that ceramide regulated this 5′ splice site selection, inducing the production of Bcl-x(s) mRNA with a concomitant decrease in Bcl-x(L) correlating with sensitization to chemotherapy (Chalfant, C. E., Rathman, K., Pinkerman, R. L., Wood, R. E., Obeid, L. M., Ogretmen, B., and Hannun, Y. A. (2002) J. Biol. Chem. 277, 12587–12595). We have now identified several possible RNA cis-elements within exon 2 of Bcl-x pre-mRNA by sequence analysis. To study the possible roles of these RNA cis-elements in regulating the alternative 5′ splice site selection of Bcl-x pre-mRNA, we developed a BCL-x minigene construct which conferred the same ratio of Bcl-x(L)/Bcl-x(s) mRNA as the endogenous Bcl-x and was responsive to ceramide treatment. Mutagenesis of either a purine-rich splicing enhancer or a pyrimidine tract element within exon 2 induced a change in the ratio of Bcl-x(L)/Bcl-x(s) mRNA from 7 to 1 and 0.23, thereby diminishing the selection of the Bcl-x(L) 5′ splice site with a concomitant increase in Bcl-x(s) 5′ splice site selection. Furthermore, mutagenesis of these cis-elements abolished the ability of ceramide to affect the 5′ splice site selection. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-activating proteins to these regions. SDS-PAGE analysis of cross-linked RNA trans-activating factors with these RNA cis-elements revealed the binding of 215-, 120-, and 30-kDa proteins to the purine-rich element and 120- and 76-kDa proteins to the pyrimidine tract element. In addition, exogenous treatment of A549 cells with ceramide increased the formation of protein complexes with these RNA cis-elements. Therefore, we have identified two ceramide-responsive RNA cis-elements within exon 2 of Bcl-x pre-mRNA, and this is the first report of an RNA cis-element responsive to a bioactive lipid. Ceramide is an important regulator of various stress responses and growth mechanisms, and the formation of ceramide from the hydrolysis of sphingomyelin or from de novo pathways has been observed in response to agonists such as tumor necrosis factor-α, γ-interferon, 1α,25-dihydroxyvitamin D3, interleukin-1, ultraviolet light, heat, chemotherapeutic agents, fatty-acid synthase antigen, and nerve growth factor (1Okazaki T. Bell R.M. 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Also, the addition of exogenous ceramide or the enhancement of cellular levels of ceramide induces cell differentiation, cell cycle arrest, apoptosis, or cell senescence in various cell types (8Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 15823-15831Abstract Full Text PDF PubMed Google Scholar, 9Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Crossref PubMed Scopus (1633) Google Scholar, 10Jayadev S. Liu B. Bielawska A.E. Lee J.Y. Nazaire F. Pushkareva M. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1995; 270: 2047-2052Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). The prominent role of ceramide as a regulator of cellular mechanisms necessitated the identification of target molecules. A family of ceramide-regulated enzymes has been identified, ceramide-activated protein phosphatases, which include the serine/threonine-specific protein phosphatases PP1 1The abbreviations used are: PP1, protein phosphatase 1; CRCE, ceramide-responsive RNA cis-element; EMSA, electromobility shift assay; NIPP1, nuclear inhibitor of PP1; PSF, PTB-associated splicing factor; RT, reverse transcription; snRNP, small nuclear ribonucleoprotein. and PP2A (11Law B. Rossie S. J. Biol. Chem. 1995; 270: 12808-12813Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 12Dobrowsky R.T. Kamibayashi C. Mumby M.C. Hannun Y.A. J. Biol. Chem. 1993; 268: 15523-15530Abstract Full Text PDF PubMed Google Scholar, 13Wolff R.A. Dobrowsky R.T. Bielawska A. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1994; 269: 19605-19609Abstract Full Text PDF PubMed Google Scholar, 14Chalfant C.E. Kishikawa K. Mumby M.C. Kamibayashi C. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 20313-20317Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Interestingly, SR proteins, a family of arginine/serinerich domain containing proteins, have been shown to be specific PP1 substrates and are well established regulators of alternative pre-mRNA processing (15Misteli T. Spector D.L. Mol. Biol. Cell. 1996; 7: 1559-1572Crossref PubMed Scopus (137) Google Scholar, 16Zahler A.M. Lane W.S. Stolk J.A. Roth M.B. Genes Dev. 1992; 6: 837-847Crossref PubMed Scopus (632) Google Scholar, 17Zahler A.M. Neugebauer K.M. Lane W.S. Roth M.B. Science. 1993; 260: 219-222Crossref PubMed Scopus (327) Google Scholar, 18Zahler A.M. Methods Mol. Biol. 1999; 118: 419-432PubMed Google Scholar, 19Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (606) Google Scholar, 20Du C. McGuffin M.E. Dauwalder B. Rabinow L. Mattox W. Mol. Cell. 1998; 2: 741-750Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 21Caceres J.F. Misteli T. Screaton G.R. Spector D.L. Krainer A.R. J. Cell Biol. 1997; 138: 225-238Crossref PubMed Scopus (329) Google Scholar, 22Du K. Peng Y. Greenbaum L.E. Haber B.A. Taub R. Mol. Cell. Biol. 1997; 17: 4096-4104Crossref PubMed Scopus (64) Google Scholar, 23Lin C.H. Patton J.G. RNA (New York). 1995; 1: 234-245PubMed Google Scholar, 24Lopato S. Mayeda A. Krainer A.R. Barta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3074-3079Crossref PubMed Scopus (78) Google Scholar, 25Mermoud J.E. Cohen P.T. Lamond A.I. EMBO J. 1994; 13: 5679-5688Crossref PubMed Scopus (279) Google Scholar). Furthermore, endogenous ceramide has been found recently to modulate the phosphorylation status of SR proteins in a PP1-dependent manner (26Chalfant C.E. Ogretmen B. Galadari S. Kroesen B.J. Pettus B.J. Hannun Y.A. J. Biol. Chem. 2001; 276: 44848-44855Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). PP1 also associates with the splice factor, PTB-associated splicing factor (PSF), and the PP1 regulatory subunit, nuclear inhibitor of PP1 (NIPP1), has been demonstrated to co-localize with splicing factors (15Misteli T. Spector D.L. Mol. Biol. Cell. 1996; 7: 1559-1572Crossref PubMed Scopus (137) Google Scholar, 25Mermoud J.E. Cohen P.T. Lamond A.I. EMBO J. 1994; 13: 5679-5688Crossref PubMed Scopus (279) Google Scholar, 27Boudrez A. Beullens M. Groenen P. Van Eynde A. Vulsteke V. Jagiello I. Murray M. Krainer A.R. Stalmans W. Bollen M. J. Biol. Chem. 2000; 275: 25411-25417Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 28Hirano K. Erdodi F. Patton J.G. Hartshorne D.J. FEBS Lett. 1996; 389: 191-194Crossref PubMed Scopus (59) Google Scholar). Finally, dephosphorylation of SR proteins stimulated by PP1 induces alternative 5′ splice site selection in adeno pre-mRNA in vitro, demonstrating that pre-mRNA splicing events are regulated in a phosphorylation-dependent manner (29Cardinali B. Cohen P.T. Lamond A.I. FEBS Lett. 1994; 352: 276-280Crossref PubMed Scopus (45) Google Scholar). Thus, PP1 plays an important role in regulating mRNA processing. To this end, our laboratory recently described a pathway linking the generation of ceramide and activation of PP1 to the regulation of the alternative 5′ splice site selection of Bcl-x pre-mRNA. Ceramide treatment resulted in a decrease in Bcl-x(L) mRNA with concomitant increase in Bcl-x(s) mRNA in A549 cells. This effect required the generation of endogenous ceramide through the de novo pathway, and more importantly, inhibitors of protein phosphatase-1 abolished the ability of ceramide to affect Bcl-x alternative splicing (30Chalfant C.E. Rathman K. Pinkerman R.L. Wood R.E. Obeid L.M. Ogretmen B. Hannun Y.A. J. Biol. Chem. 2002; 277: 12587-12595Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Thus, both the phospho-state of SR proteins and Bcl-x 5′ splice site selection are regulated by the generation of de novo ceramide and subsequent PP1 activation. The involvement of PP1 and endogenous ceramide in the dephosphorylation of SR proteins and the effects on Bcl-x alternative splicing suggested ceramide regulated the protein-RNA complexes that mediate the 5′ splice site selection of Bcl-x pre-mRNA. In this study, we identified a purine-rich RNA cis-element that corresponds to an SRp30a-binding site, as well as a polypyrimidine tract site known to bind RNA splice factors, such as PSF, within exon 2 of Bcl-x mRNA. Furthermore, both RNA cis-elements regulated Bcl-x 5′ splice site selection and were found to be responsive to ceramide. Cell Culture—A549 adenocarcinoma cells were grown in 50% RPMI 1640 (Invitrogen) and 50% Dulbecco's modified Eagle's medium (Invitrogen) supplemented with l-glutamine, 10% (v/v) fetal bovine serum (Sigma), 100 units/ml penicillin G sodium (Invitrogen), and 100 μg/ml streptomycin sulfate (Invitrogen). Cells were maintained at less than 80% confluency under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C). For treatments with d-erythro-C6 ceramide (Matreya) or long chain ceramides (Brain) (Sigma), A549 cells were plated at 4 × 105 cells/35-mm plate in the same media. BCL-x Minigene Construct—PCR amplification of Bcl-x genomic sequence was accomplished using Human Genomic DNA (Promega) as template and two separate amplification reactions. The first PCR amplified a 1443-bp sequence of the BCL-x gene containing exon 1, intron 1, exon 2, and the flanking region of intron 2 and utilized the following primers: sense, 5′-GCATTCCTCGAGGGCGGATTTGAATGTAGGT-3′; antisense, 5′-GCATTCGCGGCCGCGAGAGAAGCCCAGGATAGGAC-3′ (Integrated DNA Technologies, Inc.). The amplification reaction was carried out for 30 cycles (94 °C, 30 s; 68 °C, 1.5 min). The second PCR amplified a 477-bp sequence of the BCL-x gene containing the flanking region of intron 2 and partial exon 3 used the following primers: sense, 5′-GCATTCGCGGCCGCCCTGACCCTGCCCATCTCGTT-3′; antisense, 5′-GCATTCAAGCTTACCAGCGGTTGAAGCGTTCCT-3′ (Integrated DNA Technologies, Inc.). The PCR was carried out for 30 cycles (94 °C, 30 s; 56 °C, 30 s; 72 °C, 40 s). The BCL-x minigene was then in the (Invitrogen), by a In 1, the 477-bp was the by using enzymes and In 2, the 1443-bp was the by using enzymes and For both and were by sequence analysis. The construct a minigene of cells were with the BCL-x minigene construct using following the A549 cells were plated at confluency in to Cells were with 2 of with of for 4 the cells were with d-erythro-C6 ceramide for and for minigene by reverse RNA from A549 cells was with (Invitrogen). 1 of A549 RNA was using and the (Invitrogen). 1 of the reverse reaction was amplified using a specific for the sequence (Integrated DNA Technologies, a specific to Bcl-x exon 2 (Integrated DNA Technologies, and (Invitrogen). cycles (94 °C, 30 and 68 °C, 1 the PCR was by cis-elements within exon 2 of the BCL-x minigene construct were to using the mutagenesis were to and for in at the were from both and A549 adenocarcinoma cells to the of R.M. Res. PubMed Scopus Google Scholar). were by a of the using the protein following were used for RNA 1, and 2, RNA binding were in a of containing of 1 or 2 of nuclear protein 40 of and of in of 1 3 and 5% The reaction were at 4 for by of were on a 5% for of complexes for 1.5 at at 4 The was then using with a SDS-PAGE the same for the binding described for electromobility shift were in for and then on a for of the of the studies were using of either specific or The following competitor were and of RNA in 2 of the BCL-x our laboratory that the alternative 5′ splice site selection of Bcl-x was regulated by correlating with the phosphorylation status of SR proteins (26Chalfant C.E. Ogretmen B. Galadari S. Kroesen B.J. Pettus B.J. Hannun Y.A. J. Biol. Chem. 2001; 276: 44848-44855Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, C.E. Rathman K. Pinkerman R.L. Wood R.E. Obeid L.M. Ogretmen B. Hannun Y.A. J. Biol. Chem. 2002; 277: 12587-12595Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Furthermore, the phosphorylation status of SR proteins has been shown to 5′ splice site selection (29Cardinali B. Cohen P.T. Lamond A.I. FEBS Lett. 1994; 352: 276-280Crossref PubMed Scopus (45) Google Scholar). Therefore, exon 2 of the BCL-x gene was for known to bind specific SR proteins as well as for possible RNA cis-elements M. Krainer A.R. Genes Dev. 1998; PubMed Scopus Google Scholar). possible SRp30a-binding site 1, from Bcl-x intron two possible 2 and and from Bcl-x intron a purine-rich exon splicing enhancer from Bcl-x intron and pyrimidine tract site from Bcl-x intron were found within exon 2 1, To these RNA cis-elements a role in regulating the alternative 5′ splice site selection of Bcl-x pre-mRNA, a minigene was by of two DNA of the BCL-x gene containing exon 1, intron 1, exon 2 of intron 2, and of exon 3 1, possible RNA cis-elements were modified in the minigene construct by mutagenesis 1, using this the were the splice within exon 2, and the of of the RNA cis-element RNA cis-element 1, the ratio of Bcl-x(L)/Bcl-x(s) mRNA from 7 to of the pyrimidine tract the Bcl-x(s) and Bcl-x(L) 5′ splice the production of minigene the ratio from 7 to Furthermore, ceramide affect the alternative splicing of both these Bcl-x 1, of the cis-elements within exon 2 effect on the and RNA processing of Bcl-x pre-mRNA Therefore, two regulatory for Bcl-x 5′ splice site selection were identified within exon 2, and these as either possible of the Bcl-x(L) 5′ splice site or of the Bcl-x(s) 5′ splice RNA the the were identified, the ability of RNA trans-activating factors to bind these RNA cis-elements was assays using nuclear from A549 and cells and a sequence for 1 or 2 were cross-linked and by an nuclear the of the several protein-RNA complexes by The of these in A549 nuclear was by the of specific or competitor and analysis. The of the specific to 1 were to be and 30 and binding of these proteins to 1 was the addition of a specific competitor 2, analysis of 2 revealed specific complexes of and as with 1, were the addition of a specific competitor 2, that RNA trans-activating factors with the were using nuclear from cells Ceramide the of RNA to the of A549 cells to d-erythro-C6 ceramide in induces the production of endogenous ceramide To the binding of RNA trans-activating factors to the is responsive to A549 cells were with exogenous ceramide at a and that induces de novo ceramide generation and alternative 5′ splice site selection of the Bcl-x exon 2 (26Chalfant C.E. Ogretmen B. Galadari S. Kroesen B.J. Pettus B.J. Hannun Y.A. J. Biol. Chem. 2001; 276: 44848-44855Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, C.E. Rathman K. Pinkerman R.L. Wood R.E. Obeid L.M. Ogretmen B. Hannun Y.A. J. Biol. Chem. 2002; 277: 12587-12595Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, B. Pettus B.J. Wood R. J. Bielawska A. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). from A549 cells with d-erythro-C6 ceramide for and 30 demonstrated an increase in formation for 1 and 2 as by increase in formation was also observed for the 215-, 120-, and 30-kDa complexes to 1 at both and 30 of ceramide treatment an increase in formation at both and 30 of ceramide treatment was also observed for 2 to of and Thus, exogenous ceramide at treatment that the activation of the Bcl-x(s) 5′ splice site and the production of endogenous long chain ceramides also the formation of protein complexes with the the formation of protein complexes to the with the of chain ceramides to long chain we the exogenous addition of long chain ceramides induces a more formation of treatment of A549 cells with ceramides induced a increase in the 1 and 2 complexes as as 30 A and Thus, long chain ceramides a increase in the formation of proteins to the our laboratory that ceramide via alternative the of the splice with a concomitant in the anti-apoptotic splice Bcl-x(L) (30Chalfant C.E. Rathman K. Pinkerman R.L. Wood R.E. Obeid L.M. Ogretmen B. Hannun Y.A. J. Biol. Chem. 2002; 277: 12587-12595Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). In this study, two RNA cis-elements have been identified that the alternative 5′ splice site selection of Bcl-x pre-mRNA. Furthermore, the protein-RNA of these RNA cis-elements was regulated by and we the RNA as ceramide-responsive RNA cis-elements are important for several a of the regulation of Bcl-x alternative a SR proteins, and the regulation of Bcl-x RNA processing has now been Finally, this is the first identification of responsive to a bioactive lipid. in the that the of the cell be by the ratio of anti-apoptotic Bcl-x(L) to and of Bcl-x(s) cells to chemotherapeutic J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, 1999; 17: PubMed Scopus Google Scholar, J.A. R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). This has been demonstrated by the alternative 5′ splice site selection of Bcl-x to increase the of Bcl-x(s) at the of the levels of Bcl-x(L) 1999; 17: PubMed Scopus Google Scholar, J.A. R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). In studies, Bcl-x alternative splicing was using an specific for Bcl-x pre-mRNA at a site the Bcl-x(L) 5′ splice site 1999; 17: PubMed Scopus Google Scholar, J.A. R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of this to Bcl-x pre-mRNA induced an increase in the of Bcl-x(s) and a decrease in the of Bcl-x(L) in sensitization of the cells to chemotherapeutic and inducing long 1999; 17: PubMed Scopus Google Scholar, J.A. R. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). the study by 1999; 17: PubMed Scopus Google demonstrated that of the Bcl-x(L) 5′ splice site to the activation of the Bcl-x(s) splice on the of Krainer and Mayeda A. J.F. Krainer A.R. Mol. Cell. Biol. 2000; PubMed Scopus Google this that the Bcl-x(L) 5′ splice site the with This binding of the is with of the with RNA trans-activating factors, such as SR are with these previous We demonstrated that the mutagenesis of 1 or 2 induces a decrease in Bcl-x(L) mRNA with concomitant increase in This that both as binding for RNA trans-activating factors that the binding of the to the Bcl-x(L) 5′ splice with the of 1999; 17: PubMed Scopus Google this that the Bcl-x(s) 5′ splice site is binding of the to the Bcl-x(L) 5′ splice site is This study also the by which ceramide the 5′ splice site selection of Bcl-x pre-mRNA. This is to the that a purine-rich RNA cis-element a 30-kDa and ceramide the of this protein with The 30-kDa is well known to bind purine-rich RNA cis-elements splicing and alternative 5′ splice selection. Therefore, the that 1 a with an protein is with our previous that the dephosphorylation of SR proteins in response to ceramide with the regulation of alternative 5′ splice site selection of Bcl-x pre-mRNA. Thus, a the generation of PP1 formation to 1, and subsequent Bcl-x alternative splicing be that the pyrimidine tract 2, is also responsive to ceramide for formation well with this This is to the that 2 to an that to the polypyrimidine splicing factor an binding site for PP1, a ceramide-activated protein phosphatase and the target of ceramide for SR proteins K. Erdodi F. Patton J.G. Hartshorne D.J. FEBS Lett. 1996; 389: 191-194Crossref PubMed Scopus (59) Google Scholar). Furthermore, a study by and Y. Cohen M. S. B. Patton J.G. J. Mol. Biol. Cell. 2001; PubMed Scopus Google demonstrated that the of change protein binding to with protein and SR proteins Thus, that a to 2 with SR proteins to 1, and this be regulated by the of PP1 in response to ceramide. The that a is observed for both 1 and 2 this Furthermore, the regulation of Bcl-x 5′ splice site selection by ceramide is PP1-dependent (30Chalfant C.E. Rathman K. Pinkerman R.L. Wood R.E. Obeid L.M. Ogretmen B. Hannun Y.A. J. Biol. Chem. 2002; 277: 12587-12595Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Thus, this study a possible regulatory role for SR proteins and a in the alternative splicing of the ceramide the formation of protein-RNA complexes at 1 and studies in the K. Peng Y. Greenbaum L.E. Haber B.A. Taub R. Mol. Cell. Biol. 1997; 17: 4096-4104Crossref PubMed Scopus (64) Google Scholar, G.R. J.F. Mayeda A. Bell M. Bell Krainer A.R. EMBO J. 1995; PubMed Scopus Google Scholar, K. Lee Haber B.A. Taub R. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google that in the of of SR proteins in response to growth factors and cellular also the alternative splicing of specific For Taub and K. Lee Haber B.A. Taub R. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google first the SR using a in a of responsive to that this increase in the of in response to was required for the of exon of the gene K. Peng Y. Greenbaum L.E. Haber B.A. Taub R. Mol. Cell. Biol. 1997; 17: 4096-4104Crossref PubMed Scopus (64) Google Scholar). Krainer and G.R. J.F. Mayeda A. Bell M. Bell Krainer A.R. EMBO J. 1995; PubMed Scopus Google demonstrated that increased levels of activation with in the alternative splicing of and pre-mRNA. the effect of long chain ceramides on the formation at 1 and 2 a via the activation of PP1, studies are in the effect of ceramide on the 5′ splice site selection of Bcl-x pre-mRNA is through an In these the identification of two ceramide-responsive RNA cis-elements that to Bcl-x alternative splicing in response to and thereby increase the of a cell to This is the first report of a RNA and these also a role for ceramide in the regulation of formation of RNA trans-activating factors to these RNA cis-elements. This of regulation have to and chemotherapeutic specific regulation of the alternative splicing of Bcl-x is to the sensitization of cells to to a target for We and for of the