Figure 5. Nova2 regulates alternative splicing of Dab1 in a context dependent manner.

(A) Scheme of Dab1 protein, which consists of PTB domain and tyrosine phospholylation sites, and amino acids encoded by Dab1 7bc exons. PTB: phospho-tyrosine binding. (B) Nova2 CLIP tags from E14.5 cortex (blue/cyan/purple) and P7 brains (red/orange/pink) by each three individual CLIP experiments, map to Nova2 regulating Dab1 transcripts. The alternatively spliced region is highlighted. Two red boxes in highlight indicate Dab1 7bc exons. (C) Alternative splicing analysis of Dab1 in wild type and Nova2 KO using 5 time points: E10.5 brain, E14.5 cortex (Ctx), E16.5 Ctx, P0 Ctx and P10 Ctx total RNA. Each corresponding band was confirmed by sequencing. (D) Quantitation of Dab1 vs Dab1.7bc mRNA expression data in (C); each point represent the average of three biological replicates (see detailed data in Figure S5C contains the ratio of Dab1.7b/c and steady state level of total Dab1 between embryo and postnatal cortex in Figure S5B). (E–G) Two upstream intronic sequences of Dab1 7b and 7c exons are necessary for the regulation of alternative splicing of Dab1.7bc exons as a pair by Nova2. (E) Schematic representation of pGloDab1.7bc and its derivative minigenes containing the mouse intronic regions surrounding and including exon 7b and 7c between human globin constitutive exon1 and 3. Asterisks indicate site of point mutations in Nova binding YCAY clusters (see Figure S5D, F for details). (F) total RNA was isolated from 293T cells transiently transfected with WT or mutant pGloDab1.7bc minigenes (0.25 μg)and pNova2 (0.5 μg) as indicated, and spliced products analyzed by RT-PCR. Three biological replicates were used in each analysis. (G) Model of Nova2-mediated Dab1.7bc exon repression. .