Figure 2.
The hit enhancer of the U2AF2 – RNA complex stalls in vitro pre-mRNA splicing at the H/E-stage. (A) The fluorescence polarization dose-response of U2AF1S34F–U2AF2–SF1–DEK FLRNA titrated with hit enhancer (NSC 194308, chemical structure inset). (B) qRT-PCR of spliced AdML products. The relative amounts of spliced product were normalized to a mock-treated negative control (1% v/v DMSO, gray). The positive control is an SF3B1 inhibitor (PB, 1 µM pladienolide-B). Two-tailed, unpaired t-tests with Welch’s correction compared the indicated NSC 194308 concentration with the mock-treated control: *, p<0.05; **, p<0.005; ***, p<0.0005. (C) Denaturing gel analysis of the radiolabeled pre-mRNA substrate and spliced products from reactions treated with the indicated concentrations of NSC 194308 or a mock-treated control. Spliced products are schematically diagrammed to the left. (D) Native gel analysis of spliceosome assembly at 30 minutes in the presence of the indicated concentrations of NSC 194308, positive control (SSA, 1 µM spliceostatin-A), or mock-treated control. The identities of spliceosome complexes are indicated (left), with assembly occurring in the following order: H/E → A → B → C. NCI DTP, NCI Developmental Therapeutics Program. Data represented in A and B are mean ± SD of three replicates. Data represented in C and D are representative of three replicates. See also Figure S1.