C3PO an endoribonuclease that promotes RNAi by facilitating RISC activation

摘要

The catalytic engine of RNAi is the RNA-induced silencing complex (RISC), wherein the endoribonuclease Argonaute and single-stranded siRNA direct target mRNA cleavage. Here we have reconstituted long dsRNA- and duplex siRNA-initiated RISC activities using recombinant Drosophila Dicer-2, R2D2 and Ago2 proteins. We employ this core reconstitution system to purify an RNAi regulator-component 3 promoter of RISC (C3PO), a complex of Translin and Trax. C3PO is a Mg²⁺-dependent endoribonuclease that promotes RISC activation by removing siRNA passenger strand cleavage products. These studies establish an in vitro RNAi reconstitution system and identify C3PO as a key activator of the core RNAi machinery.