SAP155 Binds to ceramide‐responsive RNA cis‐element 1 and regulates the alternative 5′ splice site selection of Bcl‐x pre‐mRNA

Two splice variants are derived from the BCL‐x gene, proapoptotic Bcl‐x(s) and antiapoptotic Bcl‐x(L), via alternative 5′ splice site selection. In previous studies, our laboratory identified an RNA cis‐element within exon 2 of Bcl‐x pre‐mRNA that is a ceramide responsive termed CRCE 1. In this study, mass spectrometric analysis identified the splicing factor SAP155, as an RNA trans‐acting factor binding to the purine‐rich CRCE 1. The interaction of SAP155 with CRCE 1 was confirmed by the addition of an anti‐SAP155 antibody (Ab) to EMSA decreasing the mobility of a protein:CRCE 1 complex (SuperShift). Furthermore, the down‐regulation of SAP155 in A549 cells by RNA interference (RNAi) technology resulted in the loss of a 155 kDa protein complexed with CRCE 1. Moreover, this down‐regulation of SAP155 induced an increase in the Bcl‐x(s) with a concomitant decrease in the Bcl‐x(L) splice variants and immunoreactive protein levels, thereby decreasing the Bcl‐x(L)/Bcl‐x(s) ratio. Specific down‐regulation of SAP155 also inhibited the ability of exogenous ceramide treatment to further induce the activation of the Bcl‐x(s) 5′ splice site. Additionally, the specific down‐regulation of SAP155 sensitized cells to undergo apoptosis in response to daunorubicin in a manner similar to ceramide. Therefore, we have identified SAP155 as an RNA trans‐acting factor that binds to CRCE 1, functions to regulate the alternative 5′ splice site selection of Bcl‐x pre‐mRNA, and is required for ceramide to induce the activation of the Bcl‐x(s) 5′ splice site. Furthermore, we have demonstrated that activation of the Bcl‐x(s) 5′ splice site can increase the effectiveness of chemotherapeutic drug treatment, thus establishing a role for the alternative splicing mechanism of Bcl‐x in chemotherapeutic sensitivity.—Massiello, A., Roesser, J. R., Chalfant, C. E. SAP155 binds to ceramide‐responsive RNA cis‐element 1 and regulates the alternative 5′ splice site selection of Bcl‐x pre‐mRNA. FASEB J. 20, E921‐E929 (2006)