Fig. 4: Regulation of compound-induced splicing for HTT-C2 and SMN-C3.

a 5′ splice sites (ss) sequence at regions (−4 to −1 and +1 to +6) were studied for the enrichment of Inc (included) vs. NC (no change) exons. Significance scores are shown. Wide boxes represent exons. b Schematic of 5′ss sequence logo in the three exon groups: annotated NC, annotated (inclusion) Inc and psiExons Inc. c Diagram illustrating the design of huntingtin (HTT) minigene constructs for studying compound-induced splicing. d Polymerase chain reaction (PCR) analysis of RNA extracts from HEK293 cells transfected with wild-type (wt) human HTT minigene or constructs with point mutations in the −2 to +3 region of the 5′ss; cells were treated with dimethyl sulphoxide (DMSO) or HTT-C2 (0.010–1 µM). The data are from a single transfection experiment with multiple concentrations tested for a given construct; the “WT” control construct has been used multiple times (n > 3). e Sequence of the 20-nucleotide region upstream of the 5′ss of HTT stop-codon psiExon49a showing partial deletions and mutations performed on this region, and their effects on HTT-C2 induced splicing (lower panel). Through partial deletion or mutation of the nucleotides CAGGA at positions −38 to −34, this region was shown to be important in regulating splicing events. The data are from a single transfection experiment with multiple concentrations tested for a given construct; the “WT” control construct has been used multiple times (n > 3).