FIGURE 6.

Free U4 accumulated in Δ25 extracts after the splicing reaction. (A) Splicing reactions were carried out in wild-type, NTC-depleted, or Δ25 extracts, with or without addition of affinity-purified NTC at a concentration of ∼5 nM. The in vitro synthesized U6 was also added to one reaction in the Δ25 extract (lane 9). RNA was extracted from reaction mixtures and fractionated on a 5% polyacrylamide gel for Northern blotting and probed sequentially with U4, then U6. (B) Splicing reactions were carried out in wild-type or Δ25 extracts, with or without addition of purified NTC. To some reactions carried out in the Δ25 extracts, the in vitro synthesized U6 was also added prior to the reaction (lanes 12,14,16). Reaction mixtures were fractionated on a 4% nondenaturing polyacrylamide gels for Northern blotting and probed sequentially with U4, then U6.