Fig. 3.

Accessibility of the 3′ exon to oligo-directed RNase H cleavage. (A) A segment of the ACT1 pre-mRNA is shown; the branchpoint and the 3′ splice site (3′ ss) are marked. DNA oligos complementary to the intron and 3′ exon are shown below the pre-mRNA. (B) ACT1 pre-mRNA was spliced in wild-type extracts in the absence (-) and presence (+) of Prp22-Q804A protein during 20 min at 28°C. Aliquots of the mixtures were then supplemented with the indicated DNA oligo and incubated for 10 min at 22°C. The reaction products were analyzed by denaturing PAGE and the autoradiograms of the dried gels are shown. Lane M: 5′-³²P-labeled Msp1 fragments of pBR322 DNA. (C) In the ACT1-acAC pre-mRNA, the 3′ splice site ag↓AG is changed to ac↓AC. Otherwise, ACT1-acAC is identical to ACT1 pre-mRNA used in (B). ACT1-acAC was spliced in wild-type extracts supplemented with buffer (-), or with dominant-negative Prp22-Q804A protein (+). To aliquots of the reaction mixtures, DNA oligos were added as indicated, and the RNAs were analyzed by denaturing PAGE. The 3′ splice site mutations are marked by * in the symbols for lariat-intermediate and pre-mRNA at the left. A triangle marks a product that migrates at the position of mRNA and is formed in small amounts that vary between experiments. Based on prior studies (Vijayraghavan et al., 1986) showing that the ac↓AC pre-mRNA is spliced accurately in vivo and in vitro, albeit with low efficiency, I surmise that the RNA species marked by the triangle is mRNA.