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. 2009 Sep;15(9):1729–1739. doi: 10.1261/rna.1625309

FIGURE 6.

FIGURE 6.

Ntc90 is not required for spliceosome activation. (A) The U5, U6 stability assay. Splicing reactions were carried out in mock-depleted (lanes 4–6), NTC-depleted (lanes 7–9), or Ntc90-depleted extracts (lanes 1–3) using biotinylated Ac/Cla pre-mRNA as the substrate, and the spliceosome was precipitated with streptavidin Sepharose. After washing off unbound materials, the pellet was separated into two fractions: one was for total precipitate (T, lanes 1,4,7), and the other had splicing buffer added and was incubated at room temperature for 20 min. After separating supernatant (S) and pellet (P) fractions, RNA was extracted and analyzed by Northern blotting. (B) UV cross-linking of the U6 snRNA with pre-mRNA. Splicing reactions were carried out in wild-type (lane 1), Ntc90-depleted (lane 2), 30Δ20Δ (lane 3), mock-depleted (lane 4), or NTC-depleted extracts (lane 5), using Ac/Cla pre-mRNA as the substrate. The reaction mixtures were precipitated with the anti-Smd1 antibody followed by UV irradiation. The cross-linked products X1, X2a, and X2b are as described by Chan et al. (2003) and Chan and Cheng (2005).