Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

Plad-B blocks splicing during ATP-dependent prespliceosome formation in extracts from humanized yeast. (A) In vitro splicing assays of a ³²P-radiolabeled actin pre-mRNA. Indicated extracts were incubated at 23°C for 20 min with either H₂O or 2 mM ATP, and in the presence of DMSO or the indicated concentration of Plad-B. RNA was extracted from the reactions, resolved on a 6% acrylamide/8 M urea gel and visualized by autoradiography. Lariat-3′ exon, lariat, pre-mRNA, and mature mRNA are shown. Lane “m” is a 50 bp marker, and “no n.e.” is a no extract control lane. (B) Half of the in vitro splicing reactions in panel A were also directly loaded on nondenaturing gels to visualize splicing-complex formation. Prespliceosomes/spliceosomes (psp/sp) and commitment complexes (cc) are indicated. (C) Quantification of splicing reactions in panel A measuring relative splicing efficiency in the presence of increasing concentration of Plad-B. (D) Quantification of splicing reaction assay in panel B measuring relative ATP-dependent complex formation in the presence of an increasing concentration of Plad-B.