Protein kinase C-dependent control of Bcl-x alternative splicing

Abstract

The alternative splicing of Bcl-x generates the proapoptotic Bcl-x(S) protein and the antiapoptotic isoform Bcl-x(L). Bcl-x splicing is coupled to signal transduction, since ceramide, hormones, and growth factors alter the ratio of the Bcl-x isoforms in different cell lines. Here we report that the protein kinase C (PKC) inhibitor and apoptotic inducer staurosporine switches the production of Bcl-x towards the x(S) mRNA isoform in 293 cells. The increase in Bcl-x(S) elicited by staurosporine likely involves signaling events that affect splicing decisions, because it requires active transcription and no new protein synthesis and is independent of caspase activation. Moreover, the increase in Bcl-x(S) is reproduced with more specific inhibitors of PKC. Alternative splicing of the receptor tyrosine kinase gene Axl is similarly affected by staurosporine in 293 cells. In contrast to the case for 293 cells, PKC inhibitors do not influence the alternative splicing of Bcl-x and Axl in cancer cell lines, suggesting that these cells have sustained alterations that uncouple splicing decisions from PKC-dependent signaling. Using minigenes, we show that an exonic region located upstream of the Bcl-x(S) 5' splice site is important to mediate the staurosporine shift in Bcl-x splicing. When transplanted to other alternative splicing units, portions of this region confer splicing modulation and responsiveness to staurosporine, suggesting the existence of factors that couple splicing decisions with PKC signaling.

FIG. 1.

Alternative splicing of the Bcl-x pre-mRNA. (A) The Bcl-x pre-mRNA is alternatively spliced to produce two major isoforms, Bcl-x_L and Bcl-x_S. The positions of the primers used in RT-PCR assays are shown. (B) Sequences and factors documented to affect the alternative splicing of Bcl-x. The CRCE1 and CRCE2 elements modulate the response of A549 cells to ceramide that activates the use of the 5′ splice site of Bcl-x_S (57). CRCE1 is bound by the U2 snRNP protein SAP155, and its knockdown increases the production of Bcl-x_S (56). B2G is bound by hnRNP H and stimulates the production of Bcl-x_S (23). B3 enhances the use of the Bcl-x_L 5′ splice site (23). The intron regulatory element (IRE) has been implicated in mediating the activity of growth factors and TPA on Bcl-x splicing in various cell lines (50). The SB1 element was previously shown to decrease the production of the Bcl-x_S isoform (23), and its position is indicated. Sam68 and hnRNP A1 also contribute to Bcl-x splicing, but the cis-acting elements mediating these effects have not yet been mapped (63).

FIG. 2.

Staurosporine affects Bcl-x pre-mRNA alternative splicing in 293 cells. (A) RT-PCR assays were performed on total RNA extracted from different human cell lines treated or not with staurosporine (25 nM). (B to E) RT-PCR assays were also carried out to amplify portions of endogenous Bcl-x mRNAs when 293 cells were treated with increasing concentrations of staurosporine (B), when they were treated for different incubation times with 25 nM of staurosporine (C), or when the indicated concentrations of DRB (D) or cycloheximide (CHX) (E) were added 1 h prior to addition of 50 nM of staurosporine. A size ladder is shown on the left for all gels. The sizes of the different PCR products corresponding to the Bcl-x splice isoforms are indicated in panel A, and the Bcl-x_L/Bcl-x_S ratios are shown below the lane numbers.

FIG. 3.

Caspase-independent and PKC-dependent shifts in Bcl-x splicing. (A) Western blotting performed to detect PARP cleavage induced by the different drugs in the absence or the presence of z-VAD-fmk. The position of the cleaved PARP product obtained with vinorelbine is shown. (B) RT-PCR assays were performed on total RNA from 293 cells treated for 18 h with the caspase inhibitor z-VAD-fmk and/or staurosporine. The ratios of Bcl-x_L and Bcl-x_S products are indicated. (C) Bcl-x splicing profiles for different cell lines treated with the indicated concentrations of staurosporine (ST) and the specific PKC inhibitor Gö6976 (Gö). The graphs plot the Bcl-x_L/Bcl-x_S ratio. (D) Impact of the PKC inhibitor calphostin C on the endogenous Bcl-x splicing profile in 293 cells. Error bars indicate standard deviations.

FIG. 4.

PKC-dependent alternative splicing of Axl. (A) An RT-PCR assay was carried out to amplify endogenous adenovirus E1a products in 293 cells treated or not with 50 nM of staurosporine. (B) RT-PCR assays were designed to amplify splicing products from Bcl-x and Axl in mock- or staurosporine-treated 293 cells. The sizes of the PCR products are indicated, as well as the structures of the Bcl-x and Axl alternative splicing units. (C) Different cell lines were treated with staurosporine or Gö6976. RT-PCR assays were performed to amplify Axl splicing products. The inclusion/exclusion ratios are shown below lane numbers.

FIG. 5.

The SB1 element represses the Bcl-x_S 5′ splice site, and its activity is neutralized by staurosporine. (A) Diagram representing the SB1 element on the Bcl-x gene and the structures of minigenes X2 and X2.13. Derivatives of X2 and X2.13 carrying mutations (two GGUs mutated to CCAs) that improve Bcl-x_L production were also used (X2-W and X2.13-W). The positions of the primers used to amplify mRNA products from these minigenes are indicated. (B) The Bcl-x splicing products were monitored by RT-PCR amplification from total RNA isolated from mock- or staurosporine-treated 293 cells transfected with minigenes. The analysis of amplified products was done on an automated microfluidic station, and electropherograms are shown. The sizes of the amplified Bcl-x products are indicated, as well as the x_L/x_S ratios. The graphs on the right represent the quantitated x_L/x_S average ratios from another set of experiments performed with the X2.13, X2, X2.13-W, and X2-W minigenes. Standard deviations are provided. (C) Graph representing the x_L/x_S ratios obtained following transfections with the X2 and X2.13 minigenes in HeLa cells.

FIG. 6.

Portions of SB1 can modulate splicing in heterologous pre-mRNAs. (A) Diagrams of the Bcl-x X2, the globin-derived Dup51, and the adenovirus E1a pre-mRNAs, indicating the portions of SB1 that were inserted into Dup51 and E1a. (B) RT-PCR assays were performed to amplify products derived from the minigenes transfected in 293 cells that were treated or not with staurosporine. Values for the exclusion/inclusion ratio are shown below lane numbers. (C) RT-PCR assays were carried out to amplify products derived from the E1a minigene transfected in 293 cells that were treated or not with staurosporine. The positions and structures of the various splicing products are indicated.