Splicing and splice factor SRp55 participate in the response to DNA damage by changing isoform ratios of target genes

Abstract

Alternative splicing is an important source of protein diversity, and is an established but not yet fully understood mechanism for gene regulation in higher eukaryotes. Its regulation is governed by a variety of mechanisms, including variation in the expression levels of splicing factors engaged in spliceosome formation. SRp55 is one of the most ubiquitous splicing factors and one that can be up-regulated by DNA damage in the absence of p53, and we had previously found that depletion of its activity increased resistance to DNA damage in p53-dependant manner. To assess its influence on the splicing patterns of genes involved in apoptosis, we performed splice-specific microarray analysis of cells treated with siRNA specific for this gene. This analysis, backed by RT-PCR verification, identified three genes, KSR1, ZAK and mda7/IL24, which are sensitive to SRp55 depletion. We also analyzed the splice patterns of apoptosis-related genes in p53-deficient U2OS cells following treatment with the genotoxic drug mitomycin C. This analysis revealed that DNA damage resulted in changes in splicing activity that modified the splicing pattern of Fas, a key pro-apoptotic, p53-inducible death receptor. Interestingly, this modification led to an enrichment of the anti-apoptotic soluble Fas isoform, and this secreted isoform was detected in the media surrounding cells subjected to DNA damage. These findings show that modulation of splicing activity in p53-deficient cells during the early response to sub-lethal DNA damage results in a change in the splicing of target genes, thus modifying the cellular response to genotoxic agents.

Figure 1. Characterization of RNA samples subjected to SpliceArray™ analysis and effect of SRp55 silencing on cell survival

A. Left panel: Level of ATF3, CUTL, SFRS6, and CFL1 transcripts in U2OSE64b cells treated with 2 µg/ml mitomycin C for 5 and 10 hrs as determined by RT-PCR analysis. Right panel: The SRp55 protein encoded by the SFRS6 gene has elevated expression levels in U2OSE64b cells after incubation with mitomycin C (2 µg/ml) for 5 hrs, but not in the control U2OSAS cells (right panel). Lanes 1 and 3 show lysates of untreated U2OSAS and U2OSE64b cells, and lanes 2 and 4 show lysates of U2OSAS and U2OSE64b cells treated with 2 µg/ml mitomycin C for 5 hrs, respectively. The levels of splicing factors were determined by immunoblotting with monoclonal antibody 1H4 (ATCC, Manassas, VA). β-Actin antibodies were used for re-blotting to verify uniformity of sample loading. B. Left panel: Depletion of doxycycline (−Dox) in the medium effectively decreases p53 levels in the Tet-Off U2OSE6tet24 cell line but not in control U2OSAS cells. U2OSAS or Tet-Off U2OSE6 cells, grown in the presence or absence of 10 ng/ml Dox, were lysed and the lysates analyzed for p53 by ELISA. Right panel: Sensitivity of SRp55-depleted cells to mitomycin C treatment depends on the level of p53. U2OStetE624 cells, maintained in the presence of three concentrations of doxycycline, were transfected with SRp55 or scrambled siRNAs, then subjected to DNA damage by treatment with 4 µg/ml mitomycin C for 24 hrs. Viable cells were quantified using the MTT assay, and the differences between SRp55 silenced and control cells were compared by subtracting the number of viable cells in control scrambled siRNA samples from the number of surviving cells transfected with SRp55 siRNA. Means from triplicate measurements are shown, with the error bars representing the standard deviation. C. Left panel: Treatment of U2OS cells with SFRS6-specific siRNA effectively decreases the SFRS6 mRNA level. The level of SFRS6 mRNA was determined by RT-PCR and normalized against expression levels of the CFL1 gene. Right panel: Inhibition by siRNA leads to a decrease in the level of the SRp55 protein. Immunoblot of protein lysates isolated from control U2OS cells (CNTRL) and U2OS cells transfected with SFRS6 siRNA (siRNA). The β-actin antibodies were used to verify uniformity of sample loading.

Figure 2. Splicing patterns of KSR1, ZAK and mda7/IL24 genes are sensitive to SRp55 expression

A. Schemes of the regions of the KSR1 and ZAK gene structures involved in the tested AS events. Arrows F, R1 and R2 show the primers used for RT-PCR analyses for each of these genes. Rectangles A5, B, C, and E show localization of the SpliceArray™ probes. B. Left panel: Increased expression of the KSR1 isoform with truncated exon 21 (KSR1-2) in SRp55-depleted U2OS cells. The graph shows quantification of the ratio between KSR1 isoforms, as recognized by primers F+R1 (KSR1-1) and F+R2 (KSR1-2) in U2OS cells with different levels of SRp55 expression. Right panel: Changes in the splicing of the ZAK gene in SRp55-depleted U2OS cells. The graph shows quantification of the ratio between the two ZAK isoforms, as recognized by primers F+R1 (β isoform) and F+R2 (α isoform) in U2OS cells with different levels of SRp55 expression. Two lower panels show RT-PCR analysis of SFRS6 expression in these RNA samples isolated from U2OS cells treated with scrambled siRNA (Control), 0.25 µg or 0.5 µg SRFS6 specific siRNA (si1 and si2, respectively). C. Silencing of SRp55 expression by specific siRNA up-regulates the abundance of the isoform represented by a 319 bp band in RT-PCR analysis using the primers designated by arrows F and R. D. The amount of the SRp55-sensitive isoform is proportional to the levels of silencing of SRp55. U2OS cells were treated with scrambled siRNA (CNTRL), 0.25 µg or 0.5 µg SRFS6 specific siRNA (si1 and si2, respectively) to decrease SRp55 expression gradually. Isolated RNA was subjected to RT-PCR analysis.

Figure 3. Mitomycin C treatment induces up-regulation of Fas gene expression in U2OSE6AS cells and changes its splicing pattern in U2OSE64b cells

A. Scheme of alternative splicing of the Fas gene. Skipping exon 6, which codes for the transmembrane domain of the receptor, leads to expression of the soluble isoform (S), while retention of the exon produces membrane bound receptor (R). Two arrows (F and R) show the primers used to distinguish between these isoforms. B. Time course of Fas gene expression during mitomycin C (2 µg/ml) treatment by RT-PCR analysis. The CFL1 gene was used as a control. The ratio of the RT-PCR Fas R isoform bands to control (0 hrs of drug treatment) is shown between the panels, with the density of the S isoform for the U2OSE64b cells after 5 hrs of treatment noted separately. C. Detection of soluble Fas in cell culture media. The concentration of soluble Fas protein secreted into the media was estimated by ELISA in media collected from U2OSE6AS and U2OSE64b cells untreated and treated with mitomycin C (MMC) (2 µg/ml) for 5 hrs. Means from triplicate measurements are shown, with the error bars representing the standard deviation. D. Immunoprecipitation analysis of media collected from treated and untreated U2OSE64b cell cultures. Lane 1 – Fas immunoprecipitation of medium from untreated U2OSE64b cells. Lane 2 - Fas immunoprecipitation of medium from U2OSE64b cells treated with 2 µg/ml mitomycin C for 5 hrs. Lane 3 – U2OSE64b lysate.

Figure 4. Regulation of Fas expression differs in p53-deficient cells during the early response to DNA damage

A. A scheme of the p53-dependent response to DNA damage. p53 induces expression of the Fas gene, leading to an increased amount of Fas receptor molecules on the cellular surface, thus making the cell more sensitive to FasL-mediated apoptosis. B. A scheme of the early response to DNA damage in the absence of p53. In p53-deficient cells, the total expression of the Fas gene does not change and activation of AS leads to increased expression of the soluble isoform of the receptor. This results in a decreased amount of Fas receptor on the cellular surface, accompanied by the appearance of the soluble anti-apoptotic isoform in the intercellular space, and resulting in protection of the cell from FasL-mediated apoptosis.