TRBP contributes to isomiR generation and guide strand selection. (A) Dicer–TRBP generates 1-nt-longer isomiRs for both strands when processing pre-miR-200a ([Dicer], [Dicer–TRBP] = 5 nM, [RNA] = 50 nM). (B) Different dsRNA are produced from Dicer and Dicer–TRBP processing of pre-miR-200a. The exact cleavage sites were identified by sequencing the product small RNA from Dicer and Dicer–TRBP processing reaction. The product dsRNA from Dicer is indicated as dsRNA-D, and the unique product from Dicer–TRBP processing is indicated as dsRNA-T. (C) The effect of differential Dicer cleavage on guide strand selection. dsRNA-D and dsRNA-T show different guide strand selection bias in HEK293 cells. A firefly luciferase construct containing either four tandem 5′-strand miR-200a binding sites in the 3′ UTR (pHL93) or four tandem 3′-strand miR-200a binding sites in the 3′ UTR (HL94) was cotransfected with a control construct that express Renilla luciferase (pRL-TK) (50:1) and with a product dsRNA, either dsRNA-M or dsRNA-I, to HEK293 cells. Dual luciferase assays were performed 36–40 h post-transfection. (Graphs represent data from three experimental replicates.)
