In the current study, we have purified C3PO as an activator of hAgo2-RISC activity from HeLa extract by chromatographic fractionation. Although many nucleases exist in the crude extract, only C3PO possesses this robust RISC-activation activity. Thus, the unbiased biochemical purification underscores the specific role of C3PO as a key activator of human RISC. Furthermore, we have reconstituted for the first time duplex siRNA-initiated human RISC activity using recombinant Ago2 and C3PO proteins in the absence of Dicer-TRBP complex. It should be noted that more robust RISC activity was obtained with nickel purified hAgo2 than with highly purified hAgo2 recombinant protein following chromatographic purification. Recent studies have implicated heat shock proteins as the molecular chaperones for Ago folding and siRNA loading41–43
. Thus, the nickel-purified hAgo2 preparation might contain insect cell-derived heat shock proteins that could promote hAgo2-RISC activity, but were removed by chromatography. Nevertheless, our previous and current studies establish that C3PO plays an evolutionarily conserved role in Drosophila
and human RISC activation17
Our studies highlight important differences between the fly and human RISC systems. A notable distinction is that hAgo2, but not dAgo2, can be efficiently programmed by ssRNA, such as 21-nt ss-siRNA or 60-nt pre-miRNA, into active minimal RISC. Moreover, Dcr-2-R2D2 complex is absolutely required for recruiting duplex siRNA to dAgo2 for Drosophila
. Recombinant Dcr-2-R2D2 and dAgo2 are sufficient to reconstitute the core RISC activity that is greatly enhanced by dC3PO17
. However, in the absence of Dicer-TRBP complex, recombinant hAgo2 can directly interact with duplex siRNA and function together with hC3PO to reconstitute human RISC activity in the absence of Dicer-TRBP complex. These key differences can be attributed to the unique characteristic of Drosophila
Ago2 as all four human Ago proteins are more homologous to fly Ago1 than Ago223
It has been established that Ago2-mediated passenger cleavage plays a critical role in RISC activation in flies, fungi, and humans14–16,18,23
. The RISC-enhancing activity of C3PO depends on the slicer activity of dAgo2 when using a duplex siRNA-unwinding assay to monitor Drosophila
. In both fly and human systems, the intrinsic RNase activity of C3PO is required for its ability to activate RISC17
. Moreover, we observed inefficient RISC assembly accompanied with increased stabilities of passenger strand fragments in C3PO-deficient fly ovary extract, 17
. Here we showed that human C3PO was required for the nicked duplex siRNA-initiated hAgo2-RISC activity. Thus, the removal of Ago2-nicked passenger strand is not a spontaneous process, but requires active assistance from C3PO. Together, our studies support a Dicer-independent mechanism for human RISC activation: 1) hAgo2 directly binds to duplex siRNA, and nicks the passenger strand; 2) hC3PO activates RISC by degrading hAgo2-nicked passenger strand. This Dicer-independent mechanism may represent a more general model for RISC activation in diverse eukaryotes.
Our structural based mutagenesis studies of C3PO identify four catalytic residues (E126, E129, D193 and E197 of human TRAX) and several key residues (K68 and R200 of TRAX and R192 of Translin) that are critical for binding ssRNA at the catalytic center. The nucleotide binding groove of hC3PO is located at an interface between TRAX and Translin, with residues from both subunits participate in RNA binding and/or catalysis. This also explains why the asymmetric spatial arrangement of TRAX and Translin subunits is critical for C3PO’s function.
Both RNA-binding and catalytic residues of C3PO are located inside the football-shaped barrel, suggesting that C3PO may cleave ssRNA at its hollow interior (maximal height 70 Å and diameter 40 Å). However, it is difficult to envision how C3PO recruits ssRNA to the interior of a largely enclosed barrel. An alternative hypothesis is that active C3PO is a tetramer (3 Translin + 1 TRAX). This possibility is less likely because C3PO exists and binds ssRNA as an octamer. Previous studies have shown that Translin also binds ssDNA as an octamer. Notably, a point mutation of Drosophila
Translin results in a tetrameric form lacking ssDNA-binding activity44
. It is possible that the oligomeric state of C3PO is more dynamic in solution than in the crystal. C3PO may transiently adopt a tetrameric conformation to contact ssRNA initially, and the nucleotide binding may stabilize the octamer conformation that is required for RNA cleavage.
Previous studies suggest that the interaction between Ago and RNA is a highly dynamic process associated with large conformational changes in both components45–48
: 1) duplex siRNA is loaded onto Ago2; 2) Ago2-nicked passenger strand has to be removed to activate RISC; 3) target mRNA then comes in to form a duplex with the guide strand; 4) the sliced mRNA products need to dissociate in order for RISC to cleave another target. Therefore, the nicked duplex siRNA is not all deeply buried within Ago2 at all time, and parts of it are accessible to other regulatory factors. We think it is unlikely that the entire nicked duplex siRNA is transferred from Ago2 to the interior of C3PO, where the passenger fragments are degraded and the guide strand is transported back to Ago2. We prefer a model that the guide strand remains bound to Ago2, while the passenger fragments are transferred to the active site of C3PO to be degraded. It is possible that the dissociation and degradation of passenger fragments are closely coupled and jointly promoted by C3PO and Ago2. It will be exciting and challenging for future studies, including structural determination of C3PO-RNA complexes, to unravel the precise mechanism of this dynamic process.