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.
Highlights
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-␣, ␥-interferon, 1␣,25-dihydroxyvitamin D3, interleukin-1, ultraviolet light, heat, chemotherapeutic agents, fatty-acid synthase antigen, and nerve growth factor [1,2,3,4,5,6,7]
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 [30]
Identification of RNA cis-Elements in Exon 2 of the BCL-x Gene—Previously, our laboratory reported that the alternative 5Ј splice site selection of Bcl-x was regulated by ceramide, correlating with the phosphorylation status of SR proteins [26, 30]
Summary
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-␣, ␥-interferon, 1␣,25-dihydroxyvitamin D3, interleukin-1, ultraviolet light, heat, chemotherapeutic agents, fatty-acid synthase antigen, and nerve growth factor [1,2,3,4,5,6,7]. 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 5Ј splice site selection of Bcl-x pre-mRNA. Ceramide treatment resulted in a decrease in Bclx(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 [30]. 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 Both RNA cis-elements regulated Bcl-x 5Ј splice site selection and were found to be responsive to ceramide
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
