Figure 6.
PRMT6 regulates alternative splicing of endogenous VEGF and Syk. (A) Schematic representation of the major spliced products of the VEGF gene (not to scale). (B) Effect of PRMT6 knockdown on alternative splicing of VEGF. MCF-7 cells were transfected either with control siRNA (Ctrl-siRNA) or siRNA targeting PRMT6 (P6-siRNA-1) and treated either with or without 10−9 M E2 for 12 h as indicated. RNA was harvested from the cells and the relative expression level of each spliced isoform was determined by Q-RT-PCR analysis as detailed in ‘Materials and Methods’ section. (C) The relative VEGF 189:VEGF 165 ratio was obtained by dividing the relative VEGF 189 cDNA level by the relative VEGF 165 cDNA level for each experimental condition. (D) Schematic representation of the major alternatively spliced products of the endogenous Syk gene (not to scale). (E) Representation of the RHCglo-Syk-Exon 7 minigene construct, not to scale. (F) Effect of PRMT6 knockdown on splicing of endogenous Syk. MCF-7 cells were transfected either with control siRNA (Ctrl-siRNA) or siRNA targeting PRMT6 (P6-siRNA-1) and treated either with or without 10–9 M E2 for 12 h as indicated. RNA was harvested from the cells and Syk splicing was determined by reverse transcriptase-PCR. The relative Syk[L]:Syk[S] ratio was obtained by dividing the relative Syk[L] cDNA level by the relative Syk[S] cDNA level for each experimental condition. An autoradiograph of the radiolabeled Syk and β-2-microglobulin reverse transcriptase-PCR products from a representative experiment is shown. Lane 1; radiolabelled 100-bp DNA marker; lane 2; Ctrl-siRNA without 10−9 M E2; lane 3; Ctrl-siRNA with 10−9 M E2, lane 4; P6-siRNA-1 without 10−9 M E2; lane 5; P6-siRNA-1 with 10−9 M E2. (G) Effect of PRMT6 on the splicing of an RHCglo-Syk-Exon 7 minigene. HeLa cells were transiently co-transfected with a RHCglo-Syk-Exon 7 minigene along with expression vectors for PRMT6, the PRMT6 V86K/D88A mutant or CARM1. RNA was harvested from the cells and Syk splicing determined by reversetranscriptase-PCR. The relative RHCglo-Syk-Exon 7 inclusion (Syk[I]): RHCglo-Syk-Exon 7 exclusion (Syk[E]) ratio was obtained by dividing the relative Syk[I] cDNA level by the relative Syk[E] cDNA level for each experimental condition. RT-PCR without reverse transcriptase did not produce any detectable PCR products (data not shown). A representative autoradiograph of the radiolabelled RHCglo-Syk-Exon 7 minigene products is shown. For all experiments, each data point represents the mean and SD of results from four transfected cultures (or six transfected cultures for the RHCglo-Syk-Exon 7 minigene experiment). Results shown are from a single experiment, which is representative of two independent experiments. *P < 0.005, #P < 0.05.