The key structural features of pre-miRs for TRBP-Dicer processing. (A) The predicted secondary structure of chimeric substrates from pre-let-7a, pre-miR-21 representing the effect of cleavage site structure, stem length, and loop size; (red) from pre-miR-21; (blue) from pre-let-7a; (green highlights) indicate where the insertions or mutations were made. HL1 contains the pre-let-7a loop and the pre-miR-21 stem. HL2 has a single nucleotide insertion at the cleavage site of HL1 to create a base pair with the otherwise unpaired nucleotide bulge present in the pre-miR-21 stem. HL3 has five additional base pairs derived from pre-miR-21 inserted onto the end of the HL2 stem. HL4 has a smaller loop than HL2. (B) The effect of stem structure and loop size in Dicer and Dicer–TRBP processing in single turnover conditions ([Dicer] = 400 nM, [Dicer–TRBP] = 50 nM, [RNA] = 2 nM) and in multiple turnover condition ([Dicer], [Dicer–TRBP] = 5 nM, [RNA] = 50 nM; graphs represent data from three experimental replicates). (C) The effect of cleavage site structure for pre-miR-125b processing by Dicer–TRBP complex. Pre-miR-125b and its mutant pre-miR-125b were processed by Dicer and Dicer–TRBP complex ([Dicer], [Dicer–TRBP] = 5 nM, [RNA] = 50 nM; graphs represent data from three experimental replicates).
