Figure 5.

 RNase III is required for correct U5 3′-end processing. (A) The rnt1 mutant strain does not produce the long form of U5 in vivo. Total RNAs were extracted from wild-type or rnt1 strains, grown at 26°C or shifted to 37°C for 4 hr. The RNAs were loaded onto a denaturing 6% acrylamide gel, transferred to a nylon membrane, and hybridized to DNA fragments complementary to snRNAs. (B) Extracts from rnt1 cells are deficient in the production of intermediates and U5L. Internally-labeled transcript was incubated in extracts made from wild-type (WT) or mutant rnt1 strains for the times indicated, and products were loaded in a denaturing 6% acrylamide gel. Legends as in Fig. 1B. (C) 3′-end trimming of the short form of U5 in rnt1 extracts. A 5′-labeled transcript was incubated for 5, 15, or 45 min in rnt1 extract and loaded on a 5% sequencing gel, in parallel with a phosphorothioate generated sequence with purines (R) and uridine (U) as in Fig. 3C. Arrowheads on the sequence indicate the 3′ ends after 5 and 45 min of incubation. Note that the trimming must be at the 3′ end, because the 5′ end of the molecule is labeled.