RT-PCR analysis of the in vivo splicing of the U12-dependent P120 intron F from the transiently transfected constructs shown. The region spanning the U12-dependent intron F was amplified using primers in the adjacent exons. The positions of bands corresponding to unspliced and correctly spliced products are shown, as well as the cryptic spliced product. The cryptic spliced band is due to splicing between a pair of U2-dependent splice sites located within the P120 F intron. The constructs used for the various lanes are: (lane 1) empty pCB6 expression vector and (lane 2) wild-type P120 minigene. Lanes 3–10 used the P120 5′ splice site mutant CC5/6GG and the following snRNA expression constructs: (lane 3) none; (lane 4) U6atac GG14/15CC plus U11 GG6/7CC; (lane 5) U6 Mod-1 plus U11 GG6/7CC; (lane 6) U6 Mod-1 plus U11 GG6/7CC plus U4atac Hu U6 Supp.; (lane 7) U6 Mod-1 plus U11 GG6/7CC plus wild-type U4; (lane 8) U6 Mod-2 plus U11 GG6/7CC; (lane 9) U6 Mod-2 plus U11 GG6/7CC plus U4atac Hu U6 Supp.; and (lane 10) U6 Mod-2 plus U11 GG6/7CC plus wild-type U4. Lane M contains molecular size markers.
