The 3′ stem–loop of U4 is required for efficient base-pairing to U6. (A) Secondary structure of U4 snRNA with the 3′ ends of the truncation mutants indicated. (B) U4 truncations lacking the 3′ stem–loop do not support reconstitution of splicing. Denaturing gel analysis of splicing in mock-depleted (lane 1) or U4-depleted (lanes 2–6) extract reconstituted using wild-type (WT) or mutant IVT U4. Splicing substrate, intermediates, and products are indicated to the left of the gel, as in Figure 1. (C) The 3′ stem–loop of U4 is required for base-pairing to U6 at 283 nM U4. Non-denaturing solution hybridization gel analysis of the base-pairing status of U6 in mock-depleted (lane 1) or U4-depleted (lanes 2–6) extract reconstituted using WT or mutant IVT U4. (D) U4 truncations can be driven into complexes with U6 by increasing their concentration above 283 nM. Non-denaturing solution hybridization analysis of extent of U4/U6 formation as in panel C. (Lane 1) 283 nM full-length IVT U4. Increasing concentrations of truncated U4 mutants: 283, 566, 1132, and 2830 nM (1–142 only to 1132 nM).
