FIG. 1.

Rds3p activity is required for pre-mRNA splicing. (A) Northern blot analysis of RNA isolated from yeast strains that express a wild-type RDS3 gene and from yeast strains that express a glucose-repressible GAL1::rds3-1 allele. Samples were taken from yeast grown constitutively on galactose at room temperature (Gal, 23°C) and after shift to glucose-based medium (Glu) for 10 h at 37°C. (B) In vitro assay of splicing conducted in the presence and absence of active Rds3p or Rds3-TAP. The extract used in lanes 4 to 9 and 12 to 18 was preincubated with the TAP-specific calmodulin and IgG affinity resins prior to the assay. In lanes 22 to 27, extract was prepared from the glucose-repressed, heat-inactivated GAL1::rds3-1 mutant. For lanes 7 to 9, 16 to 18, and 25 to 28 the double-affinity-purified Rds3-TAP complex was included in the splicing reaction mixture. The positions of the RPS17A pre-mRNA (P), lariat intermediate (LI), 5′ exon (5′E), mRNA (M), and excised intron (I) are indicated.