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
Autumn
A
Department of Biochemistry, Rm. 2-016, Sanger Hall, Virginia Commonwealth University, 1101 East Marshall St., P.O. Box 980614, Richmond, Virginia 23298-0614, USA.
Roesser
James R
JR
Chalfant
Charles E
CE
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002518:Ceramides / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000235:genetics*; D012322:RNA Precursors; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D020218:Response Elements; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000235:genetics*
D000970:Antineoplastic Agents; D002518:Ceramides; D010750:Phosphoproteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D017413:Ribonucleoprotein, U2 Small Nuclear; C112491:SF3B1 protein, human; D014157:Transcription Factors; D051020:bcl-X Protein; D012313:RNA