Massiello Autumn, Salas Arelis, Pinkerman Ryan L, Roddy Patrick, Roesser James R, Chalfant Charles E
Department of Biochemistry, Virginia Commonwealth University, Richmond, Virgina 23298, USA.
J Biol Chem. 2004 Apr 16;279(16):15799-804. doi: 10.1074/jbc.M313950200. Epub 2004 Jan 20.
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.
BCL-x基因产生的两种剪接变体，即促凋亡的Bcl-x(s)和抗凋亡的Bcl-x(L)，是通过Bcl-x前体mRNA外显子2内的可变5'剪接位点选择产生的。在先前的研究中，我们实验室证明神经酰胺调节这种5'剪接位点选择，诱导Bcl-x(s) mRNA的产生，同时Bcl-x(L)减少，这与化疗敏感性相关（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）。我们现在通过序列分析在Bcl-x前体mRNA的外显子2内鉴定了几个可能的RNA顺式元件。为了研究这些RNA顺式元件在调节Bcl-x前体mRNA可变5'剪接位点选择中的可能作用，我们构建了一个BCL-x小基因构建体，其产生的Bcl-x(L)/Bcl-x(s) mRNA比例与内源性Bcl-x相同，并且对神经酰胺处理有反应。外显子2内富含嘌呤的剪接增强子或嘧啶序列元件的诱变导致Bcl-x(L)/Bcl-x(s) mRNA比例从7变为1和0.23，从而减少了Bcl-x(L) 5'剪接位点的选择，同时增加了Bcl-x(s) 5'剪接位点的选择。此外，这些顺式元件的诱变消除了神经酰胺影响5'剪接位点选择的能力。体外结合试验和竞争研究表明RNA反式激活蛋白与这些区域有特异性结合。对与这些RNA顺式元件交联的RNA反式激活因子进行SDS-PAGE分析，结果显示215 kDa、120 kDa和30 kDa的蛋白与富含嘌呤的元件结合，120 kDa和76 kDa的蛋白与嘧啶序列元件结合。此外，用神经酰胺对外源A549细胞进行处理增加了与这些RNA顺式元件的蛋白复合物的形成。因此，我们在Bcl-x前体mRNA的外显子2内鉴定了两个对神经酰胺有反应的RNA顺式元件，这是关于对生物活性脂质有反应的RNA顺式元件的首次报道。
