Fig. 1.

The influence of 3′ exon length on mRNA release. (A) Experimental outline. (B) Schematic depiction of the ACT1 pre-mRNA. The 5′ exon and intron are depicted as a grey can and a black line, respectively and the sequences of the 3′ exon and the DNA oligos 2-1 and 2–4 are shown. (C) Analysis of the reaction products formed after 20 min of splicing in Δprp16 extract (lane 1), after incubation with oligos (lanes 2 and 3), and after incubation with Prp16 protein (lanes 4 and 5) by denaturing PAGE. Lane M: 5′-³²P-labeled Msp1 fragments of pBR322 DNA, the lengths for several fragments are indicated at right. The symbols at the left indicate the positions of the following nucleic acid species (from top to bottom): lariat-intermediate, lariat-intermediate after RNase H digestion, excised lariat-intron, pre-mRNA, cleaved pre-mRNA, mRNAs and 5′ exon. (D) and (E) Glycerol gradient sedimentation of the reaction mixtures after addition of Prp16. The odd numbered fractions (# 3–29) were analyzed by denaturing PAGE. An aliquot of each reaction mixture before sedimentation was analyzed in the left-most lane (In). The arrows at left mark the positions of spliced products. (D) Analysis of the reaction mixture, in which cleavage of the 3′ exon was directed by DNA oligo 2-1 prior to the addition of Prp16; (E) Analysis of the reaction mixture, in which cleavage of the 3′ exon was targeted by DNA oligo 2–4. Autoradiograms of the dried gels are shown.