The central domain of U4 functions during U4/U6 formation and splicing. (A) Secondary structure of U4 (gray) with targeting oligonucleotide binding site indicated (black line). (B) U4 requires its central domain to re-associate with U6. Non-denaturing solution hybridization gel showing the base-pairing status of U6 in mock-depleted extract (lane 1) or extract depleted of U4 in the absence (lane 2) or presence (lane 3) of added unlabeled actin pre-mRNA. The positions of base-paired wild-type (WT) U4/U6, U6 base-paired to the truncated 5′ portion of U4 (U4*/U6), and free U6 are indicated to the left of the gel. The fraction of U6 base-paired to U4 in each reaction is indicated below the gel. (C) Degraded U4 supports splicing when associated with U6. Denaturing gel showing splicing activity in mock-depleted extract (lane 1) or extract depleted of U4 in the absence (lane 2) or presence (lane 3) of pre-mRNA added during the U4 degradation step. Splicing substrate, intermediates, and products are indicated to the left of the gel, as in Figure 1. Absolute splicing efficiency of each reaction is indicated below the gel. (D) Deletion of U4's central domain increases steady-state levels of U4/U6 di-snRNP. U4/U6 analysis as in B of mock-depleted (lane 1) or depleted (lanes 2–5) extracts reconstituted with no U4 (lane 2) or U4 variants as shown. Note that U4/U6 migrates faster with the mutant U4s due to their smaller size. (E) U4's central domain is necessary for splicing. Splicing analysis of the reactions in panel D.
