Figure 2.

Screening splicing factors that regulate Cdh23 exon68 inclusion. (a) Schematic drawing of the screening strategy. The Cdh23 minigene reporter was constructed by fusing mouse Cdh23 genomic sequence ranging from exon 67 to exon 69 in frame with a mCherry-encoding sequence. A single base pair insertion in exon 68 and a single base pair deletion in exon 69 introduced in the minigene are indicated in red. When exon 68 is included in the mature mRNA, a mCherry fusion protein is expressed. In contrast, when exon 68 is not included in the mature mRNA, the single base pair deletion in exon 69 will cause a frameshift and mCherry fusion protein will not be expressed. (b) The Cdh23 minigene and various splicing factors were overexpressed in COS7 cells, and the resultant fluorescence was examined using a confocal microscope. Scale bar, 200 μm. (c) The relative mCherry fluorescence intensity from (b) was analyzed using ImageJ software. (d) The Cdh23 minigene and various splicing factors were overexpressed in COS7 cells, and RT-PCR was performed to examine the inclusion of exon 68. (e, f) The Cdh23 minigene and either Rbm24 or control siRNAs were transfected into COS7 cells, and the expression level of Rbm24 and Cdh23(+68) was examined by performing RT-PCR (e) and qPCR (f). β-Actin was used as internal control. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.