SSA, PB, and HB bind to the same site on SF3B1. (A) Denaturing gel analysis of RNA isolated from splicing reactions incubated with no nuclear extract (NE), DMSO, or the indicated compounds. In lanes 3–8, compounds were added at the specified concentration; in lanes 9–17, splicing reactions contained 1 µM active compound and 1, 10, or 100 µM inactive compound. Identities of bands are schematized to the left as (from top to bottom) lariat intermediate, free lariat, pre-mRNA, mRNA, free intron, and 5′ exon intermediate. Splicing efficiency, shown below each lane, is determined as the percentage of mRNA relative to the sum of pre-mRNA, intermediates, and mRNA. (B) Same as panel A. (C) Columns list the concentration of inactive compounds that restores 50% splicing in the presence of 1 µM active compound (rows). Values are estimated from dosage response plots of the splicing reactions shown in A and B.
