Reconstitution of splicing activity in U4-depleted extract by IVT U4 is not limited by the extent of base-paired U4/U6 formation. (A) Maximum splicing reconstitution is obtained with addition of 283 nM IVT U4. Denaturing splicing gel as in Figure 1D. (Lane 1) Mock-depleted extract. (Lanes 2–9) Depleted extract with IVT U4 added as indicated above lanes. Absolute splicing efficiency of each reaction is indicated below gel. (B) Extent of base-pairing with U6 snRNA increases with added IVT U4. Non-denaturing solution hybridization gel probed using a radioactively labeled oligonucleotide complementary to U6, showing the base-pairing status of U6. (Lane 1) Untreated splicing extract. (Lane 2) Mock-depleted extract. (Lanes 3–10) U4-depleted splicing extract with IVT U4 added as indicated. The percentage of U6 in base-paired complex is shown below the gel.
