FIG. 2.

RNase H protection of consensus 5′ splice sites. The substrate pre-mRNA was m⁷GpppG-capped C174C, derived from β-globin IVS-2, which contained two alternative consensus 5′ splice sites. Pre-mRNA was incubated in HeLa nuclear extract that had been depleted of CBC or had been mock depleted. Oligonucleotides that direct RNase H cleavage to the consensus 5′ splice sites were added either just before the pre-mRNA (0) or 25 min after the pre-mRNA (25). No MgCl₂ or ATP was added to the reaction mixtures. No oligonucleotide was added to the reaction mixtures in lanes 2, 3, 10, and 11; an oligonucleotide directed against the upstream site was added in lanes 4, 5, 12, and 13; an oligonucleotide directed against the downstream site was added in lanes 6, 7, 14, and 15; both were added to the reaction mixtures in lanes 8, 9, 16, and 17. The reaction mixtures were resolved on a 6% polyacrylamide gel. The products of RNase H cleavage are indicated with bars above the diagrams of the pre-mRNA on the left-hand side.