Nuclease protection assays to probe hDicer interactions with pre-miRNAs. (A) RNase V1 protection assays. (Lanes 1) 5′-end 32P-labeled RNA substrates without any treatment. (Lanes 2) RNAs treated with hDicer for 3 min at room temperature. (Lanes 3) RNAs treated with RNase V1 for 1 min at room temperature. (Lanes 4) RNAs treated with RNase V1 in the presence of hDicer for 1 min at room temperature. (Lanes 5) RNAs treated with RNase V1 in the presence of hDicer for 3 min at room temperature. Sizes of DNA markers are indicated at the left. (B) S1 nuclease cleavage of pre-miR-16 and the indicated mutants in the absence or presence of hDicer. RNA markers are indicated at the right. (C) S1 nuclease cleavage of pre-miR-30, pre-miR-31, and their mutants, as in B. RNA markers are indicated at the right. Shown in A–C are representatives of at least two experiments.
