Figure 3. nSR100 binds directly upstream of suboptimal 3′ splice sites to promote neural exon inclusion.

(A) nSR100 RNA binding map showing the mean, normalized density of crosslinked sites in 400 nucleotide windows encompassing nSR100-regulated exons (blue) and control non-regulated exons (gray) in 293T cells; ss, splice site.

(B) Top panel, genome browser view of the raw density of nSR100 293T PAR-iCLIP tags surrounding the UGC motif (orange box) upstream of the REST neural exon. Bottom panel, RT-PCR assay monitoring inclusion levels of the REST neural exon upon transfection of wild-type (W) or mutant (M) minigene reporters into 293T cells with and without dox-induced nSR100 expression.

(C) Box plots comparing the 3′ and 5′ splice site strengths of conserved nSR100 target exons (blue) and control, non-regulated exons (white). Asterisks represent significant differences; p values, Wilcoxon rank sum test.

(D) Plots comparing polypyrimidine tract lengths and distances between 3′ splice sites and inferred branch points of nSR100 regulated exons (red), and of PSI-matched control exons (gray) in human; p values, Kolmogorov-Smirnov test.

(E) RT-PCR assays monitoring the effects of mutating UGC motif(s) on inclusion levels of the DAAM1 and MEF2D neural exons. Dox-inducible nSR100-expressing 293T cells were transfected with wild-type (W) minigene reporters (lanes 1-2). 293T cells (not dox-inducible) were transfected with wild-type or mutant (M, M1, M2) reporters, and control (lanes 3, 5, 7) or PTBP1 and PTBP2 siRNAs (lanes 4, 6, 8). PPT, polypyrimidine tract. Asterisk represents a product from usage of an alternative splice site. See also Figure S3.