Free U6-II-Sm but not U6-II co-IPs with the SmD1 protein. (A) Extracts from SmD1-3xFLAG strains or a control lacking the 3xFLAG epitope tag were immunoprecipitated using anti-FLAG antibodies. RNAs from the immunoprecipitate, supernatant, or total extract were extracted using phenol, precipitated, and analyzed by primer extension. (B) Averages of the ratios of U6 to U4 in anti-FLAG IPs relative to WT samples from the experiment in panel A (n = 3). (C) Analysis of U6 base-pairing interactions with U4 snRNA. RNAs from the immunoprecipitate, supernatant, or total extract were isolated under conditions which maintain intact base-pairing. RNAs were then analyzed via hybridization with radiolabeled DNA probes complementary to U6 and U1 snRNAs. (D) Average efficiencies of free U6 IP relative to WT U6 and after normalization to U1 from the experiment in panel C (n = 3).
