FIG. 2.

Splicing defects associated with slt mutations. (A) In vitro splicing defects associated with the slt11, slt22, and slt17 mutations. Splicing reactions with ³²P-labeled actin pre-mRNA substrate were performed at 25 and 33°C for 20 min with whole-cell extracts prepared from wt, slt11, slt22, and slt17 cells. Precursor, intermediates (free 5′ exon and lariat intron–3′ exon), and final products (5′ exon–3′ exon and lariat intron) of the splicing reaction are indicated between the gels. Arrows in lanes 12 and 14 indicate preferential accumulation of lariat-intron–3′-exon intermediate in slt17 extract. (B) Primer-extension analyses of inhibition of splicing in vivo. The left gels show reduced levels of mature actin RNA in slt15 cells. The right gels show inhibition of pre-U3 splicing in slt16 cells. The two upper bands labeled A and B correspond to pre-U3A and pre-U3B, respectively. Cells were grown at 25°C for several generations and then were shifted to 37°C. Total yeast RNA was isolated before (grown at 25°C) and following a shift to 37°C for the times indicated. Levels of spliced and unspliced RNAs were measured by primer extension with labeled oligonucleotide complementary to the second exon. Precursor and mature RNAs are indicated.