Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

Figure 3

A noncanonical 5′ splice site represents an unusual feature of E24a, and transplantation of this splice site renders a constitutive exon susceptible to splicing silencing. (a) Graph illustrates relative splice site scores determined using the MAXENT scoring algorithm for E18, E19, and E24a. Sequences used for scoring are shown. (b) Splicing reporter, DIPwt, contains constitutive exon, E3, as the middle exon with 12 bases of flanking intron sequence. E3 and adjacent intron sequences are derived from the mouse DIP13beta (APPL2) gene. Sequences of the 5′ splice sites of DIPwt and DIP5pss are shown at right (5′ to 3′); vertical lines illustrate potential base pairing to U1 snRNA (3′ to 5′). The 5′ splice site of CASK E24a was transferred into DIP5pss by constructing two-point mutations (underscored). (c) The gel panel shows results of splicing assays performed after transfecting DIPwt and DIP5pss reporters into N18TG2 cells. Transfections and RT-PCR amplification were carried out in triplicate. Numbers below gel panels represent average % exon inclusion value ± standard deviation.