Figure 5.

snRNA specificity of reconstituted canonical and U2 Sm cores. (A) Binding of in vitro transcribed snRNAs. ³²P-labeled snRNA from T. brucei were prepared by transcription (20% of U2/U1 and SL/U6/U5 input shown in lanes 1 and 2, respectively). After incubation with recombinant canonical (lanes 3–6) or U2-specific Sm cores (lanes 7–10), co-precipitated RNAs were recovered by His-tag pull-down with Ni-NTA agarose beads and analyzed by denaturing gel electrophoresis (snRNA positions marked on the right; U5 bands denoted by an arrow, U1 bands by asterisks). For each reconstitution reaction, both the pull-down material (P; lanes 3, 5, 7, and 9) and the supernatant fractions (SN; lanes 4, 6, 8, and 10; 20% shown) were analyzed. M, markers (sizes in nucleotides). (B) Binding specificity of Sm site RNA oligonucleotides. ³²P-labeled short RNAs were prepared by T7 transcription, containing the U1 and U2 Sm sites, both as wild-type and mutant versions (U1 WT and U1 UA, U2 WT and U2 UA; RNA sequences shown below, with the Sm sites boxed). Each of them was reconstituted in vitro with recombinant canonical (lanes 5–8) or U2-specific Sm cores (lanes 9–12). Co-precipitated RNAs were recovered by His-tag pull-down with Ni-NTA agarose beads and analyzed by denaturing gel electrophoresis. For comparison, 5% of the input RNAs are shown (lanes 1–4). M, markers (sizes in nucleotides). (C) Sequence requirements for binding canonical and U2-specific Sm cores. ³²P-labeled RNAs derived from the T. brucei U2 snRNA 3′ half (nucleotides 81–148) were in vitro transcribed: WT, wild type; UA, U93A U95A U96A; ΔG, ΔG94 (sequences and secondary structure model shown on the right; 10% of the input RNAs in lanes 1–3). Following reconstitution in vitro with recombinant canonical (lanes 4–6) or U2-specific Sm cores (lanes 7–9), co-precipitated RNAs were recovered by His-tag pull-down with Ni-NTA agarose beads and analyzed by denaturing gel electrophoresis. M, markers (sizes in nucleotides).