Alternative Splicing of Cdh23 Exon 68 Is Regulated by RBM24, RBM38, and PTBP1

Figure 4

An ESS is required for PTBP1-mediated inhibition of Cdh23 exon 68 inclusion. (a) Schematic drawing of the location of the potential PTBP1-binding ESS and the donor splicing site of intron 68. (b) Cdh23 minigene was expressed in COS7 cells together with the splicing regulators as indicated, and the inclusion of Cdh23 exon 68 was examined by performing RT-PCR. (c) Wild-type Cdh23 minigene or minigene with donor site mutation was expressed in COS7 cells together with the splicing regulators as indicated, and the inclusion of Cdh23 exon 68 was examined by performing RT-PCR. (d) Cdh23 minigene was expressed in COS7 cells together with constant amount of RBM24 and various amount of PTBP1, and the inclusion of Cdh23 exon 68 was examined by performing RT-PCR. The expression level of RBM24 and PTBP1 was confirmed by performing western blot. (e) Cdh23 minigene with donor site mutation or donor site/ESS double mutations was expressed in COS7 cells together with PTBP1, and the inclusion of Cdh23 exon 68 was examined by performing RT-PCR. (f, g) Wild-type Cdh23 minigene or mutant with deletion of the potential PTBP1-binding ESS was expressed in COS7 cells with or without PTBP1. Binding of PTBP1 with Cdh23 pre-mRNA was examined by performing native RIP followed by RT-PCR (f) or qPCR (g). Error bars represent from triplicate experiments. ; .

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