U1A binding between the two GU-rich regions inhibits CstF64K binding. (A) CstF64K UV cross-linking assays. Uniformly radiolabeled RNA was incubated with recombinant GSTCstF64KRBD (2.5 μM) and increasing concentrations of U1A (as indicated), also expressed as a molar ratio of U1A to GSTCstF64KRBD. Products were cross-linked with UV light, subjected to RNase A digestion, and run on SDS-12% PAGE. wt, wild type. (B) Phosphorimager quantitation of panel A. CstF64K cross-link, results expressed as a percentage of CstF64K binding without added U1A; U1A cross-linking, results expressed a percentage of 500 nM U1A binding the wild-type substrate (lane 4, U1A cross-link). Solid circles, wild type; solid squares, IgM 1790-2085 mut 248; open circles, IgM 1790-2085 mut ds12; open squares, IgM 1790-2085 mut 248 ds12. Data are means of triplicates from three separate cross-linkings ± SE.