In vitro reconstitution of chaperone-mediated human RISC assembly

ATPase activities of Hsp90β and Hsc70 are necessary for RISC assembly initiated with small RNA duplex but not with single-stranded RNA. (A) Coomassie staining of recombinant ATPase mutants of Hsp90β and Hsc70. (B–D) Target RNA cleavage assay by reconstituted human Ago2-RISC using ATPase mutants of Hsp90β and Hsc70 with let-7 siRNA duplex (B), 9GU wobble duplex (C), and single-stranded RNA (D). (E) Quantification of target cleavage in B–D. The mean ± SD from three independent experiments is shown.