Figure 4

Mismatched flanking 5′ splice site strengths leads to alternative skipping of exon 7.

(A) Schematic of the RT-PCR assay construct. Assays were performed using oligonucleotide primers positioned upstream and downstream of the rabbit exons. (B) Exon skipping as detected in minigene RT-PCR assay. Lane labels specify transfected construct and reagents in U2OS cells. First line: −: untransfected control, P: empty plasmid (pXJ41), M: USP4-E7 minigene construct (pDG467). Second line: +: oligo dT-primed cDNA reaction, *: random-primed cDNA reaction, −: random primers, no reverse transcriptase (RT). A 200 base pair (bp) product of is visible after primed reactions of pXJ41-transfected cells. Exon-retained and -skipped products are visible as 350 and 200 bp bands, respectively, in pDG467-transfected cells. Products of approximately 800 and 1500 bp were generated pXJ41- and pDG467-transfected conditions, respectively, in the absence of RT and likely arose from amplification of DNA rather than cDNA. (C) Schematic of site-directed mutations. The wild type (WT) sequences and the boundaries of E₇ (yellow box) are shown. In the branch point (BP) mutant a YURAY sequence (indicated in pink) was inserted 32 residues upstream of E₇. In the splice site (SS) mutants the downstream exon-intron boundary sequence were optimized (substituted residues indicated in green). The combination mutant (BP + SS) incorporated both mutations. (D) RT-PCR analysis of site-directed mutants. Lane labels correspond to transfected mutant constructs as per Figure 4C. Exon-skipped products are absent from SS and BP + SS mutants.