Figure 5.

Secondary structure determination of RevCen. (A) Secondary structure probing of in vitro transcribed, [γ-³²P]ATP 5′-end-labelled RevCen RNA analysed on polyacrylamide gels. Partial RNA cleavages were performed as described in Materials and methods section. An OH ladder and a T1 ladder were used to assess cleavage positions. The positions of G residues are marked on the right. (B) Refined prediction of the RevCen secondary structure using the Mfold software using constraints from structural probing data in (A). The siRNAs VII and VIII are indicated in light and dark orange. The grey shade indicates the part of RevCen, which could not be resolved. Nucleotides are highlighted with different colours to show probe-dependent cleavage, as indicated in the colour key. (C) Cleavage of RevCen by human Dicer in vitro. RNA fragments ranging from 21–33 nt were formed when treating RevCen with recombinant human Dicer. RNA was labeled after incubation. The increasing dose of Dicer is indicated. Incubation times were as follows: 5 min for lanes 1 and 4; 30 min for lanes 2 and 5; 2 h for lanes 3, 6 and 7. Concentrations of Dicer: lane 7: 0 units; lanes 1–3: 0.1 units and lanes 4–6: 1.0 units.