FIG. 3.

U2 snRNA interactions of U2-40K, U2B″, and the U2-specific Sm proteins. (A through D) Analysis of U2B″ and U2-40K protein binding to U2-3′half RNA. Increasing amounts of GST-U2B″ (50 to 1,010 nM, as indicated below) (A), or GST-U2-40K protein (50 to 1,010 nM) (B), or His-FLAG U2B″ (50 to 2,000 nM) (C) were combined with ³²P-labeled U2-3′half snRNA (5 nM). The reactions shown in panel C also contained a constant amount of GST-U2-40K (300 nM). (D) In addition, to test for the role of the 3′-terminal loop of U2 snRNA, the formation of the RNP complex of GST-U2-40K (300 nM), His-FLAG-U2B″ (2,000 nM), and ³²P-labeled TbU2-3′half wild-type RNA (2 nM) was analyzed in the presence of unlabeled competitor RNA [TbU2-3′half wild type or TbU2-3′half hul4 RNA at a 1- to 10-fold molar excess, as indicated below]. RNP complexes were separated from free RNA by non-denaturing gel electrophoresis. In each panel, free RNA was analyzed as well (input lanes). (E and F) snRNA binding specificity of the U2-40K/B″ complex. ³²P-labeled wild-type and ΔG94 mutant (ΔG) T. brucei U2-3′half RNAs (nucleotides 67 through 148 of U2 snRNA) were reconstituted in vitro with recombinant canonical (lanes 8 through 12) or U2-specific (lanes 2 through 6) Sm core proteins, followed by the addition of GST-U2B″, GST-U2-40K, His-FLAG-U2B″/GST-U2-40K, or GST alone (as indicated) and further incubation for 30 min at 30°C. The respective RNA inputs are analyzed in lanes 1 and 7 (5% of each) (E). To control for efficient Sm core assembly, His-tag pulldown assays were performed (lanes 2 and 8). For the other reconstitutions, coprecipitated RNAs were recovered by GST pulldown and analyzed by denaturing gel electrophoresis (E). (F) The efficiencies of the reconstitutions were quantitated and diagrammed, using as a measure the percentages of precipitated RNAs to their respective inputs with standard deviations (n = 3). Gray bars, wild-type U2-3′half RNA; black bars, ΔG mutant RNA. The lane numbers refer to the gel shown in panel E.