C3PO, an Endoribonuclease That Promotes RNAi by Facilitating RISC Activation

RNA Wars During RNA interference (RNAi), the Dicer endonuclease generates small interfering (si)RNAs that, with the help of the protein R2D2, are loaded into the siRNA-induced silencing complex (RISC). Using siRNAs as guides, RISC, and specifically its Argonaute subunit, targets complementary RNAs for destruction. In order to identify other components of the RISC complex, Liu et al. (p. 750) reconstituted the core RISC activity, using purified Drosophila Dicer, R2D2, and Ago-2. The protein C3PO (component 3 promoter of RISC), which consists of heterodimer of Translin and Translin-associated factor X (Trax), was found to enhance RISC activity in this system, and in vivo, with the Trax endonuclease activity activating RISC through the removal of siRNA passenger strand cleavage products. Reconstitution of RNA interference reveals that Slicer activity is enhanced by the protein C3PO. The catalytic engine of RNA interference (RNAi) is the RNA-induced silencing complex (RISC), wherein the endoribonuclease Argonaute and single-stranded small interfering RNA (siRNA) direct target mRNA cleavage. We reconstituted long double-stranded RNA– and duplex siRNA–initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We used this core reconstitution system to purify an RNAi regulator that we term C3PO (component 3 promoter of RISC), 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.