Fig. 5. U4 RNA remains bound to U6 RNA in snu114ΔN heat-inactivated spliceosomes. Wild-type (wt; lanes 4–6) and snu114ΔN (lanes 7–9) spliceosomes were assembled at non-permissive temperature (30°C) using a biotinylated actin pre-mRNA and were affinity purified on streptavidin beads. Spliceosomal RNAs were fractionated on an SDS gel. RNAs were analysed by northern blotting after probing the membrane with U4 (A) and U6 (B) probes. Lane 3 is a control reaction using non-biotinylated substrate. The U4/U6 duplex (lane 1) was obtained after phenol–chloroform extraction at 4°C of the reaction shown in lane 6, before affinity purification. In lane 2, the U4/U6 duplex was dissociated by heating the sample for 5 min at 95°C. (The free U6 RNA is only weakly visible.)