Fig. 5.

A change in accessibility of the 3′ exon is not unique to ACT1 mRNA and it is a natural intermediate in the pathway. (A) Schematic drawings of pre-mRNA and mRNA, indicating the approximate positions of DNA oligos that were used. (B) ³²P-lableled transcripts, RPS17A pre-mRNA and U3 precursor were spliced in wild-type extract in the presence and absence of added Prp22-Q804A protein. Then, aliquots of the mixtures were supplemented with the respective DNA oligos a or b to allow for cleavage by endogenous RNase H. Oligos: RPS17A (a) 5′-TGGTTCTAACTCTACC, (b) 5′-CTTAGAAGCACGCTTGA; U3 snoRNA (a) 5′-CCTATAGAAATGATCC, (b) 5′-GTGACGATTCCTAT. The reaction products were analyzed by denaturing PAGE. (C) Outline of the experimental strategy. The ACT1 pre-mRNA used in this experiment consisted of a 5′ exon that was 98 nt in length, the normal 302-nt intron and a 3′ exon of 195 nt. ACT1 pre-mRNA was spliced in Δprp22 extract to form lariat-intermediates and 5′ exon. The mixture was then depleted of ATP by endogenous hexokinase upon addition of glucose. Aliquots of the reaction mixture were supplemented with wild-type Prp22 protein and ATP as indicated by (+) or buffer (-). DNA oligos were then added to aliquots of each mixture and the RNA products were analyzed upon further incubation. An autoradiogram of the dried gel is shown. ³²P-labeled Msp1 fragments of pBR322 DNA were separated in the left-most lane (M). The symbols at the left indicate the positions of the RNA species generated by splicing.