FIG. 1.

Pre-mRNA substrates containing alternative 5′ splice sites require the m⁷GpppG 5′ cap structure for splicing to both sites. (A) Splicing of ApppG- and m⁷GpppG-capped human β-globin IVS-1 substrate psp64 5′Δ16, which contains alternative 5′ splice sites. ApppG-capped RNA is in lanes 1 to 10 and 21 to 23; m⁷GpppG-capped RNA is in lanes 11 to 20 and 24 to 26. Splicing reaction mixtures were incubated for the times indicated (in minutes) above each lane. Lanes 8 to 10 and 18 to 26 were incubated for 120 min. Lanes A, B, and C contain 0, 100, and 1 mM m⁷GpppG cap analogue as a competitor. Lanes D, E, and F contain increasing amounts of SR proteins: 0.75, 1.5, and 3 μg. Cap analogues and SR proteins were preincubated with the extract before the addition of the pre-mRNA and splicing mix. Lane 27 (M) contains size markers. Spliced products and intermediates were resolved on a 6% denaturing polyacrylamide gel. (B) Splicing of m⁷GpppG- and ApppG-capped adenovirus pre-mRNA with a duplicated wild-type 5′ splice site (Ad1WW). (C) Splicing of adenovirus pre-mRNA with duplicated consensus 5′ splice sites (Ad1CC). Splicing reactions were for 40 min (lanes 1 and 3) or 1.5 h (lanes 2 and 4).