C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation

Abstract

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.

Fig. 1

Purification of C3PO as a RISC activator. (A) The dsRNA- and siRNA-initiated RISC assays were performed in buffer, S2 extract, recombinant Ago2, Dcr-2/R2D2 (D2/R2) complex, and D2/R2 plus wild-type or catalytic-mutant (cm/D965A) Ago2. Herein all siRNA-initiated RISC assays used duplex siRNA if not stated otherwise. (B) The siRNA-initiated RISC assays were performed with untreated or heat-inactivated (HI) S2 extract, or recombinant Dcr-2/R2D2/Ago2 in the absence or presence of S2^HI extract. (C) Purification of C3PO through a seven-step chromatographic procedure. Following the final Mono Q step, individual fractions were assayed with recombinant Dcr-2/R2D2/Ago2 for the RISC-enhancing activity (top) or resolved by SDS-polyacrylamide gel (PAGE) followed by Colloidal-staining (bottom). (D) The siRNA-initiated RISC assays were performed using recombinant Dcr-2/R2D2/Ago2 alone or with increasing amount of recombinant C3PO or Translin.

Fig. 2

C3PO is required for efficient RNAi. (A) Western blots comparing the levels of Dcr-2, R2D2, Ago2, Translin, Trax, and β-tubulin between wild-type (WT) and trsn mutant lysates. (B) The siRNA-initiated RISC assays were performed in buffer, and WT or trsn mutant ovary extracts. The RISC activity was measured by the percentage of cleaved mRNA. (C) The siRNA-initiated RISC assays were performed in buffer, 20μg WT extract, or 20μg trsn mutant extract without or with recombinant C3PO. (D) Images showing segmentation phenotypes of WT and trsn mutant embryos: (a) WT embryo injected with water (8 abdominal cuticle belts); (b) WT embryo injected with ftz-siRNA (severe, 3–4 belts); (c) trsn embryo injected with ftz-siRNA (mild, 5–6 belts); (d) trsn embryo injected with ftz-siRNA (no phenotype, 7–8 belts). (E-F) Graphs showing distribution of WT or trsn mutant embryos with severe, mild, or no phenotype following injection of ftz-siRNA (E, n>100) or ftz-dsRNA (F, n>150).

Fig. 3

C3PO promotes RISC activation by removing siRNA passenger strand cleavage products. (A) Native siRNA gel-shift assays were performed using radiolabeled let-7 siRNA with untreated or heat-inactivated (HI) WT, dcr-2, ago2 mutant ovary extract (lanes 1–4), or 40μg of WT and two preps of trsn mutant ovary extract (lanes 5–7). (B-C) Passenger strand cleavage assays were performed with 40μg of WT or trsn mutant extract. The 9-nt or12-nt cleavage products were detected separately using siRNA whose passenger strand was radiolabeled at the 5′ (B) or 3′ (C) end. (D-E) The data in (B-C) was converted into graphs illustrating different stability of the 9-mer and 12-mer in WT and trsn extracts.

Fig. 4

C3PO is an Mg²⁺-dependent endoribonuclease. (A) 5′-radiolabeled single-stranded (ss)-siRNA was incubated with increasing amounts of recombinant Translin or C3PO complex. (B) The RNase assays were performed with recombinant C3PO in the absence or presence of EDTA or EGTA. (C) The RNase assays were conducted by incubating circular ss-siRNA with increasing amounts of recombinant C3PO. (D) Following sequential chromatography, fractions were assayed for the RISC-enhancing activity (top), RNase activity (middle), or Western blotting to detect Translin and Trax (bottom).

Fig. 5

The RNase activity of C3PO is required for RISC activation. (A) A partial Translin/Trax multi-sequence alignment (top) and a modeled structure of Drosophila Trax based on crystal structure of human Translin from 1j1j (bottom). The comprehensive alignment of Translin/Trax is shown in Figure S9. The putative catalytic D/E residues are colored in black. (B) The RNase activity was compared between WT and three catalytic mutant (m1=E123, m2=E126, m3=D204) C3PO complexes. (C) The RISC-enhancing activity was compared between WT and catalytic mutant C3PO by assaying together with recombinant Dcr-2/R2D2/Ago2.