U4 degradation and consequent splicing inhibition require active splicing conditions. (A) Secondary structure of U4 (light gray) base-paired to U6 (dark gray) (Brow and Guthrie 1988) and as a free particle (Myslinski and Branlant 1991), with the targeting oligonucleotide binding site indicated (black line). (B) Extent of U4 degradation increases with actin concentration. Northern blot of U4 in splicing extract in the absence or presence of targeting oligonucleotide and increasing amounts of unlabeled actin pre-mRNA. (C) U4 degradation does not affect other snRNAs. Denaturing Northern blot of untreated splicing extract (SE, lane 1), mock-depleted extract (lane 2), and U4 depleted extract (DSE, lane 3), probed for all five yeast snRNAs as indicated on the side. U5L indicates long U5; U5S, short U5; and U4-RNaseH, U4 cleavage product. (D) Extent of splicing inhibition increases with the concentration of cold actin in the U4 degradation step. Denaturing gel showing the generation of splicing intermediates and products after incubation of radioactively labeled actin pre-mRNA in mock-depleted (lane 3) or U4-depleted (lanes 4–8) extract preincubated with increasing amounts of unlabeled actin pre-mRNA. Lanes 1 and 2 are standard splicing controls incubated for 0 or 30 min, respectively. Lanes 3–8 are the same reactions as in panel B. Bands corresponding to splicing substrate, intermediates, and products are indicated to the left of the gel (black and white boxes, 5′ and 3′ exons; black line, intron). The absolute splicing efficiency of each reaction is indicated below the gel.
