Structure of C3PO and Mechanism of Human RISC Activation

Purification of C3PO as a key activator of human RISC

To reconstitute human RISC activity, we generated polyhistidine (His)-tagged Dicer-TRBP and hAgo2 recombinant proteins using an insect cell expression system (Supplementary Fig. 1a). Unlike dAgo2, recombinant hAgo2 displayed potent ss-siRNA-initiated RISC activity (Supplementary Fig. 1b). This distinct feature of hAgo2 presented a problem for the reconstitution study due to background activity resulting from ss-siRNA present in synthetic duplex siRNA (Supplementary Fig. 1c). We could eliminate this background activity by using duplex siRNA prepared with excess (1.5-fold) passenger strand relative to the guide strand (Supplementary Fig. 1c).

Recombinant Dicer-TRBP and hAgo2 could not reconstitute duplex siRNA-initiated RISC activity (Supplementary Fig. 1d), suggesting that other factors are required for human RISC activation. To identify activators of human RISC, we took an unbiased biochemical approach by supplementing recombinant hAgo2 with chromatographic fractions of HeLa extract to reconstitute duplex siRNA-initiated RISC activity. A robust RISC-activating activity was observed in the Q30 fraction (0.3 M salt elution of Q-Sepharose column) of HeLa extract (Supplementary Fig. 1e), which was purified to near homogeneity through a seven-step chromatographic procedure. Two proteins, ~25 kD and ~35 kD, which closely correlated with the RISC-activating activity, were identified as Translin and TRAX by mass spectrometric analysis (Fig. 1a). Since this complex was previously purified from S2 extract as a RISC activator¹⁷, we will refer to human Translin/TRAX complex herein as hC3PO similar to the fly system. The unbiased biochemical purification identified hC3PO as the predominant, if not the only, RISC activator from HeLa extract.

Reconstitution of human RISC activity

Recombinant human Ago2 and C3PO proteins could efficiently reconstitute duplex siRNA-initiated RISC activity in the absence of Dicer-TRBP complex (Fig. 1b and Supplementary Fig. 2a). In this system, the RISC activity was abolished when using a catalytic mutant hAgo2, and C3PO associated with Ago2 in the absence or presence of siRNA (Supplementary Fig. 2b and 2c). Moreover, we compared ds-siRNA- and ss-siRNA-initiated RISC assays by incubating recombinant hAgo2 with or without hC3PO. While hC3PO was necessary for ds-siRNA-initiated RISC activity, it did not enhance ss-siRNA-initiated hAgo2-RISC activity (Fig. 1b). These results suggest that hC3PO functions at the step of RISC assembly rather than the RISC-mediated mRNA cleavage.

In Drosophila, Dcr-2-R2D2 complex is absolutely required for recruiting duplex siRNA to dAgo2 for RISC assembly^8–12. However, the role of Dicer-TRBP complex in human RISC activation is controversial. While some studies proposed that Dicer-TRBP promoted human RISC assembly^28–30, others suggested that human RISC assembly was uncoupled from dicing and the efficiency of RNAi was not compromised in dicer knockout cells^23,32,33. Here, our reconstitution studies indicated that Dicer-TRBP complex was not required for, nor did it enhance the RISC activity of recombinant Ago2 and C3PO (Fig. 1b and 1c). Moreover, recombinant hAgo2, but not dAgo2, could directly bind duplex siRNA independent of the Dicer complex (Fig. 1d). These biochemical studies demonstrate that Dicer-TRBP complex is dispensable for recruiting duplex siRNA to hAgo2 for RISC assembly.

Small hairpin (sh)RNA has been widely used as a potent loss-of-function tool for reverse genetics in mammalian systems^34–36. Previous reconstitution studies of human RISC commonly used pre-let-7 to initiate RISC assembly^28–30, which carried let-7 miRNA at the 5′ arm of the stem-loop and could function as a guide similar to ss-let-7 to direct hAgo2-mediated mRNA cleavage. To reconstitute shRNA-initiated RISC activity, we designed a modified let-7 shRNA (sh-let-7) by placing let-7 sequence at the 3′ arm of the stem-loop. Since RISC slices its target at the 10-nt position counting from 5′ of guide RNA, unprocessed sh-let-7 could not direct hAgo2 to cleave the same target mRNA for ss-let-7 (Supplementary Fig. 2d). We observed a robust shRNA-initiated RISC activity only when recombinant human Ago2, C3PO, and Dicer-TRBP complex were all present (Fig. 1e). These reconstitution studies suggest that Dicer-TRBP complex is required to process shRNA to duplex siRNA, which is then assembled by Ago2 and C3PO into active RISC.

C3PO is required for efficient RNAi in mammalian cells

We prepared extracts from immortalized wild-type and translin (trsn)^−/− mouse embryonic fibroblasts (MEF) as well as trsn^−/− MEF expressing a Flag-Translin rescuing transgene. Western blotting showed that Ago2 was equally expressed in different cell lines, whereas the endogenous Translin was missing in trns^−/− MEF cells (Fig. 2a). TRAX was also presumably missing as previously reported³⁷. The lack of C3PO diminished duplex siRNA-initiated RISC activity in trsn^−/− extract (Fig 2b), which could be rescued by addition of recombinant C3PO or by expression of Flag-Translin in trns^−/− MEFs (Fig. 2b and 2c). Thus, C3PO is required for efficient duplex siRNA-initiated RISC activity in mammalian extracts.

To determine if C3PO is required for RNAi in vivo, we co-transfected wild-type, trsn^−/−, and trsn^−/− (Flag-Translin) MEFs with a siRNA and a psiCheck2 reporter construct that contained a firefly Luciferase (Fluc) gene as well as a Renilla luciferase (Rluc) gene carrying two perfect target sites in the 3′ untranslated region. Using the ratio of Rluc/Fluc activity to measure the efficiency of RNAi, we observed a four to six-fold derepression of the reporter gene in trsn^−/− MEFs as compared to wild type MEFs. Moreover, the RNAi defect in trsn^−/− MEFs could be efficiently rescued by transgenic expression of Flag-Translin (Fig. 2d). Similar to MOV10 or RHA knockdown^26,27, depletion of C3PO resulted in a partial defect of siRNA-mediated gene silencing. Taken together, these studies suggest that the helicase and slicer mechanisms may function redundantly to promote RISC activation in mammalian cells.

Both Translin and TRAX are required for C3PO’s nuclease and RISC-activating activities

We compared the binding of recombinant human Translin, TRAX, and C3PO to radiolabeled ssDNA, ss-siRNA, or ds-siRNA by native gel-shift assays (Supplementary Fig. 2a and 3a). Both Translin and hC3PO could efficiently interact with ssDNA and ss-siRNA, but barely with ds-siRNA (Supplementary Fig. 3a). Although TRAX is related to Translin (25% sequence identity and 43% similarity), it exhibited no binding to any substrate (Supplementary Fig. 3a). Like dC3PO¹⁷, hC3PO could efficiently cleave ss-siRNA, but not ds-siRNA (Supplementary Fig. 3b). Additionally, hC3PO exhibited modest nuclease activity toward ssDNA, but not toward 2′-O-methylated oligos (Supplementary Fig. 3b). Moreover, only hC3PO complex, not Translin or TRAX, displayed nuclease activity (Supplementary Fig. 3c). Pre-incubation of Translin and TRAX regenerated ssRNase activity (Supplementary Fig. 3d), suggesting that it was a unique property of C3PO complex, but not of its subunits. Similarly, only C3PO complex, not Translin or TRAX, could function together with hAgo2 to reconstitute duplex siRNA-initiated RISC activity (Supplementary Fig. 3e).

Crystal structure of human C3PO

Since Translin and TRAX are both required for C3PO’s activities, it is important to study how these two subunits are assembled in the C3PO complex. Previous X-ray structural studies of human and mouse Translin showed that eight Translin subunits form a football-shaped octameric barrel with a large hollow interior^38,39. However, electron microscopic studies suggested a different, open octameric ring structure for Translin⁴⁰. Here we have determined the crystal structure of hC3PO complex at 3.0 A resolution (Table 1), which reveals a hetero-octamer barrel consisting of six Translin and two TRAX subunits with a similar overall architecture as that of Translin homo-octamer (Fig. 3a and 3b)^38,39. The two TRAX subunits are located adjacently on one side of the barrel, resulting in an unusual asymmetric structural arrangement (Fig. 3a and 3b).

Both TRAX and Translin subunits are composed of seven α helices, with six of them forming a two-layered right-handed superhelical bundle (Fig. 3c). TRAX also contains several extra loop sequences, of which the N-terminal 30 amino acids and amino acids 157–176 are disordered in the structure. In hC3PO complex, each of the two TRAX subunits forms a heterodimer with a Translin subunit mainly through hydrophobic interactions between the N-terminal α1 helix of TRAX and theα5, α6 helices of Translin, and vice versa (Fig. 3d). The conformation of Translin-TRAX heterodimer closely resembles that of Translin-Translin homodimer, with root mean square deviation (RMSD) between superimposed C_α atoms to be ~1.1Å. Nearly all of the interacting residues at the dimer interface are conserved between TRAX and Translin, such as Val35, Phe39, Phe42, and Leu46 of TRAX correspond to Val3, Phe7, Leu10, and Leu14 of Tranlsin (Fig. 3d and Supplementary Fig. 4). The highly conserved and hydrophobic inter-subunit interface strongly suggests that the heterodimer of Translin-TRAX and the homodimer of Translin are the secondary building blocks of C3PO complex.

In hC3PO structure, two Translin-TRAX heterodimers and two Translin-Translin homodimers tetramerize side-by-side to form an asymmetric octameric barrel. The two TRAX subunits are placed adjacently and interact with each other through their long α1 helices, whereas the α1 helix of Translin always pairs with the α1 helix of Translin (Fig. 3e). Inspection of the TRAX-TRAX interface reveals a network of salt bridges between the two α1 helices, such as Asp51-Arg69′, Glu54-Arg61′ and Lys58-Asp62′ (Fig. 3f). These charged residues are highly conserved in TRAX, but not between TRAX and Translin, which explains why aα1-α1 interface of TRAX-Translin is not favored in hC3PO complex (Supplementary Fig. 4). In contrast, the distances between the charged or polar residues at the three Translin-Translin α1– α1 interfaces are generally too long to allow for the formation of salt bridges or hydrogen bonds (Fig. 3f).

Asymmetric octameric assembly of C3PO

Intriguingly, the stoichiometry of Translin and TRAX is 6:2 in the asymmetric hC3PO complex. At first glance, the hC3PO structure exhibits a four-fold rotational symmetry that relates the top or bottom four subunits, whereas a two-fold symmetry further relates the top and bottom halves of hC3PO. Then, why does TRAX only occupy two out of the eight possible positions of the hetero-octamer? A close inspection of the interactions among the top four subunits (three Translin and one TRAX) reveals a breakdown of the pseudo-four-fold symmetry resulting from a right-handed superhelical shift between adjacent subunits (Fig. 4a). Among the four subunits, TRAX occupies the lowest position (Position 1), and going counterclockwise, the next subunit shifts slightly higher than the last until Translin (Position 4) comes back to encounter TRAX. Consequently, the position of Translin (4) is two helical turns higher relative to that of TRAX, leading to a breakdown of the pseudo-four-fold symmetry (Fig. 4a).

This breakdown of symmetry can be best illustrated by the disruption of the hydrogen bond between Tyr258 of TRAX (corresponding to Tyr210 of Translin) and Asp211 of adjacent Translin subunit. This hydrogen bond is maintained between TRAX and Translin (2), Translins (2) and (3), and Translins (3) and (4), but completely breaks down between Translin (4) and TRAX (Fig. 4a). Instead, it is replaced by new interactions mediated by two highly conserved residues Arg263 and Glu266 of TRAX. Specifically, Arg263 of TRAX forms salt bridges with Asp211′ and Glu207′ of Translin (4), and Glu266 of TRAX interacts with Arg215′ of Translin (3) (Fig. 4a). We also observed a similar spiral arrangement of Translin subunits in the homo-octameric structure of Translin (Supplementary Fig. 5a and 5b), which had not been described in previous reports^38,39. Because of this asymmetric spiral arrangement, the position of TRAX subunit is thus unique in the octamer, which may explain why the 6:2 ratio of Translin to TRAX is favored in hC3PO complex.

To determine if this asymmetric assembly is critical for C3PO function, we disrupted the interaction between TRAX and Translin (4) by mutating Arg263 to Asp (R263E) on TRAX, and Asp211 and Glu207 to Ala (D211A, E207A, D211A/E207A) on Translin. Recombinant wild type and mutant hC3PO complexes were produced in E. coli by co-expressing His-TRAX and untagged Translin (Supplementary Fig. 5c). Thus, only hC3PO complex could be recovered by nickel purification because Translin was untagged and His-TRAX was insoluble when expressed alone. These mutations significantly affected the oligomeric state of hC3PO, with E207A, R263E, and E207A/D211A mutants showing increasing amount of large protein aggregates (Fig. 4b). Disruption of the asymmetric octameric assembly severely damaged hC3PO’s ssRNA binding and RNase activity (Fig. 4c and 4d). The severity of mutant phenotype was proportional to the percentage of protein aggregates that lacked the RNase activity (Supplementary Fig. 5d).

Catalytic center of C3PO

The RNase activity of hC3PO could be supported by a broad spectrum of divalent ions (e.g. Mg²⁺, Mn²⁺), but not by Ca²⁺ or monovalent ions (e.g. Na⁺ or K⁺) (Supplementary Fig. 6a). Like dC3PO, hC3PO is an endonuclease that could cleave a 21-nt circular ssRNA (Supplementary Fig. 6b). Moreover, the cleavage products migrated slower after phosphatase treatments, suggesting they carried a 5′ phosphate (Supplementary Fig. 6c). Following hC3PO’s cleavage of a linear ss-siRNA, the 5′ cleavage products migrated faster by 2-nt after β-elimination treatment, indicative of a 2′, 3-′ hydroxyl terminus (Supplementary Fig. 6c). Therefore, C3PO is an endonuclease that cleaves a phosphodiester bond into a 5′ phosphate and a 3′ hydroxyl terminus.

To further study the catalytic mechanism of hC3PO, we have determined the structure of hC3PO in complex with Mn²⁺. This new structure is nearly identical to the apo-structure of hC3PO although the two crystals were obtained from different conditions. The anomalous difference electron density map unambiguously reveals the location of a single Mn²⁺ ion (Fig. 5a). Interestingly, there are four extra density peaks in the vicinity of Mn²⁺, which are likely SO₄²⁻ or PO₄³⁻ ions due to the presence of ammonium sulfate and sodium phosphate in the crystallization condition. Previous bioinformatics studies have predicted three Glu or Asp residues, which are invariant in TRAX but absent in Translin, as the putative catalytic sites of C3PO¹⁷. Consistently, mutating the corresponding residue E126, E129 or D193 of human TRAX to Ala abolished the RNase activity but did not affect ssRNA binding of hC3PO (Fig. 5b and 5c). These catalytic residues are clustered around the Mn²⁺, which directly coordinates E129 and E197 of TRAX and one SO₄²⁻, implicating E197 as the fourth catalytic residue. Indeed, the E197A mutant hC3PO exhibited the same phenotype as the other catalytic mutants. Although the specific roles of E126 and D193 are unclear, one residue could potentially coordinate the metal ion through a water molecule, while the other activates a water molecule to attack the scissile phosphate on ssRNA.

The hC3PO(Mn²⁺) structure enabled us to model a ssRNA oligo at the catalytic center of hC3PO by superimposing the backbone phosphates onto the positions of the four sulfates/phosphates (Fig. 5d). In this model, the RNA adopts a standard A-conformation with bases point outward and the phosphate backbone directly contacts several positive charged residues, such as K68, R200 of TRAX and R192 of Translin. Mutating each of these residues to Ala attenuated ssRNA binding and abolished the RNase activity of hC3PO (Fig. 5e and 5f). These mutations did not affect protein folding or complex formation as wild-type and mutant hC3PO exhibited similar profiles on size-exclusion chromatography (Supplementary Fig. 6d). Thus, we conclude that these positively charged residues immediately surrounding the active site are critical for properly positioning ssRNA for catalysis.

C3PO cleaves ssRNA at the interior surface

By size-exclusion chromatography, the estimated size of the endogenous fly or human C3PO was consistent with an octameric complex of six Translin and two TRAX subunits (Supplementary Fig. 7). To determine if C3PO functions as an octamer, we compared the gel-filtration profiles of recombinant hC3PO, ss-siRNA, or a mixture of both in the presence of EDTA (to block catalysis, but not RNA binding) (Fig. 6a). On a carefully calibrated Superdex 200 column, the peak of hC3PO corresponded to ~210 kDa, consistent with an octamer (predicted 226 kDa). When analyzing a hC3PO-ss-siRNA mixture, the peak of hC3PO shifted to ~242 kDa and increased in height, indicating the formation of a hC3PO-ssRNA complex. Consistently, the height of free RNA peak was reduced as a significant amount of ss-siRNA now co-migrated with hC3PO (Fig. 6b). Thus, C3PO binds ssRNA as an octamer.

Notably, all of the catalytic residues are located at the concaved surface of TRAX facing the inside of the hC3PO barrel (Fig. 6c and Supplementary Movie 1). Moreover, the electrostatic potential map illustrated many positively charged residues suitable for nucleotide binding at the interior, but not the exterior, surface of hC3PO barrel (Fig. 6d). Specifically, we showed that K68, R200 of TRAX and R192 of Translin are important for ssRNA binding by hC3PO (Fig. 5d-f). These findings collectively suggest that ssRNA binds to the interior surface of hC3PO complex, where catalysis occurs.

C3PO activates human RISC by degrading Ago2-nicked passenger strand

The slicer activity of human Ago2 is required for converting pre-RISC (Ago2/duplex siRNA) to active RISC (Ago2/guide strand) in vitro²³, suggesting that hAgo2-mediated passenger strand cleavage plays a critical role in human RISC activation. However, it remains unclear how Ago2-nicked passenger strand is dissociated from the guide strand to activate RISC. In theory, there are at least three possible mechanisms: 1) passenger fragments spontaneously dissociate; 2) they are unwound by an RNA helicase; 3) they are degraded by an RNase, e.g. C3PO. We have previously shown that mutations of the “EED” catalytic motif of TRAX could abolish dC3PO’s RNase activity and its RISC-enhancing activity¹⁷. Similar results were obtained with the four catalytic mutants (E126A, E129A, D193A or E197A) of hC3PO in our reconstitution studies (Fig. 7a). Additionally, mutations of K68, R200 (TRAX) or R192 (Translin) of hC3PO, which are critical for binding ssRNA for catalysis, also abolished human RISC-activation (Fig. 7b). Collectively, these studies suggest that the RNase activity of C3PO is required for its ability to activate RISC¹⁷.

Because C3PO cleaves ss-siRNA, but not ds-siRNA (Fig. 3b), we hypothesized that that Ago2-mediated passenger cleavage could stimulate the recruitment or activity of C3PO¹⁷. Consistent with this idea, hC3PO preferred to bind and degrade a nicked duplex siRNA that paired a guide strand with radiolabelled 9-nt and/or 12-nt passenger fragments (Fig. 7c and Supplementary Fig. 8a). We showed that this was not due to spontaneous unwinding of the nicked duplex siRNA followed by binding and cleavage of ssRNA by hC3PO (Supplementary Fig. 8b). Importantly, hC3PO was also required for nicked duplex siRNA-initiated RISC activity, but could not activate hAgo2-RISC when using a nicked duplex siRNA carrying 2′-O-methylated passenger fragments that are resistant to C3PO cleavage (Fig. 7d and Supplementary Fig. 3b). Thus, the removal of passenger fragments from Ago2 is not a passive process, but requires active degradation by C3PO. These studies strongly suggest that hC3PO facilitates human RISC activation by specifically degrading the Ago2-nicked passenger strand.

Generation of Recombinant Proteins

Wild type and catalytic mutant His-tagged or His-Flag-tagged hAgo2 were expressed in insect cells using a Baculovirus Expression System (Invitrogen) and purified by Nickel and/or Flag-affinity chromatography. Preparation of recombinant dAgo2, Dcr-2-R2D2, and Dicer-TRBP complexes from insect cells were previously described^{17, 49}. All wild type and mutant His-tagged Translin, Thioredoxin (Trx)-His-tagged TRAX, and C3PO (co-expressing His-tagged TRAX and non-tagged Translin) proteins were expressed in E. coli BL21 (DE3) and purified with nickel affinity, ion exchange, and gel filtration chromatography. All mutants were constructed using “QuickChange” (Stratagene).

Crystallization and structure determination

Recombinant C3PO complex was prepared as described above and used for crystallization trials. The optimal condition for C3PO crystallization contains 12% PEG3350 and 75 mM potassium citrate (pH 5.4). Single crystals were flash frozen in a cryo-solution containing the crystallizing solution plus 30% ethylene glycol. Diffraction data were collected in-house on a Rigaku FR-E SuperBright generator equipped with VariMax mirror and R-Axis IV++ image plate detector. Data were processed with HKL2000⁵⁰. Molecular replacement was performed with program Phaser⁵¹ in the CCP4 package⁵² using human Translin (pdb code 1J1J)³⁸ monomer as the starting model. The TRAX subunit was manually built, guided by the clear electron density after the Translin subunits were located. The model building and adjustment was performed using Coot⁵³. The initial structure refinement was performed using REFMAC5⁵⁴ and the final rounds of refinement were completed using Phenix⁵⁵. The crystal used for soaking Mn²⁺ was grown in solution containing 1.2 M ammonium sulfate, 0.1 M phosphate citrate pH 5.4 and 0.4 M lithium sulfate. Single crystals were chosen and soaked in a cryoprotectant containing 2.3 M lithium sulfate and 50 mM manganese sulfate for 3 min and flash frozen in liquid nitrogen. Anomalous diffraction data were remotely collected on beamline ID-19 at Advanced Photon Source, Argonne National Laboratory, IL. The crystal structure was determined by molecular replacement using apo C3PO as initial model, and the refinements were done using similar procedure as apo C3PO crystal structure. The crystal data and the refinement statistics were listed in Table 1. The structural figures were produced using PYMOL⁵⁶ and the electrostatic potentials were calculated using APBS⁵⁷.

Purification of the RISC-activation activity

Hela cytoplasmic extract (S100) was prepared as previously described⁴⁹. The S100 was loaded onto a HiTrap Q-Sepharose column, and the RISC-activation activity was collected at a step elution Q30 (300 mM NaCl). Ammonium sulfate was added to Q30 to 30% saturation, and after spinning, the supernatant was loaded onto a Phenyl-Sepharose column followed by an elution with 15~0% saturation of ammonium sulfate. After 60% ammonium sulfate precipitation, the pellet containing peak activity was resuspended and loaded onto a Superose 6 gel filtration column. Further purification was performed by sequentially passing the peak activity through a Mono Q column (150~400 mM NaCl), a Heparin column (150~600 mM NaCl), a Hydroxyapatite column (0~200 mM phosphate), and a Smart Q column (150~400 mM NaCl). All purification steps were conducted at 4°C.

In vitro RISC assays, nuclease assays, and native gel shift assays

In vitro RISC assays were carried out essentially as described¹⁷. Reactions containing recombinant hAgo2, Hela S100 fractions, or MEF cell extracts were incubated at 37°C for 30 minutes except for the time course experiments. Reactions containing dAgo2 were incubated at 30°C. A G-capped labeled 300 nt mRNA containing a perfect let-7 target site was used as target mRNA (~5 nM, 4×10⁴ cpm). 1.5× duplex let-7 siRNA (25 nM), which was prepared with 1.5-fold of passenger strand relative to the guide strand, was used as trigger in duplex siRNA-initiated RISC assays.

All nuclease assays and native gel shift assays were preformed as previously described¹⁷ except for the temperature is 37°C. In native gel shift assays, 10 mM EDTA (final concentration) was added to block the DNA/RNA cleavage. DNA or RNA oligos were radiolabelled at the 5′ end with [γ-³²P] ATP by T4 polynucleotide kinase (NEB), or at the 3′ end with [α-³²P] pCp by T4 RNA ligase (NEB). A 21-nt circular ss-siRNA was generated by self-ligation of a 5′ radiolabelled ss-siRNA. Radiolabelled let-7 duplex siRNA was prepared by annealing a 5′ radiolabelled strand and 1.5× of unlabelled complementary strand. To prepare the nicked duplex siRNA, the guide strand was annealed with a 9-nt and a 12-nt passenger strand fragments. In different experiments, either the guide strand, or the 9-nt or 12-nt fragment was radiolabelled before annealing.

In vivo RNAi assay

A psiCheck2 reporter system (Promega) was used for in vivo RNAi assay. Two perfect target sites for a siRNA (5′-CGAAGGAAGTATCGAATTTAC-3′) were inserted at the 3′ untranslated region (UTR) of the Renilla luciferase (Rluc) gene, with which we could use this siRNA to specifically knockdown Rluc without affecting the Firefly luciferase (Fluc) expression. Wild type and trsn^−/− primary MEFs were transformed by infecting retrovirus expressing a Ras and a dominant negative p53⁵⁸. In addition, a 3× Flag-tagged human Translin (Flag-Translin) rescuing transgene was introduced into trsn^−/− MEFs by retrovirus intergration. For in vivo RNAi assay, wild type, trsn^−/−, and trsn^−/− (Flag-Translin) MEFs were co-transfected with the siRNA and the modified psiCheck2 plasmid. A non-specific siRNA (5′-GGAAAGGGCAGAUUGUGUGGA-3′) was used as control. After 24 hours post-transfection, cells were lysed to conduct dual luciferase assays (Promega). The relative luciferase activities were calculated from the ratio of Rluc/Fluc of the specific siRNA-transfected samples compare to that of the control samples.

Western blotting and immunoprecipitation (IP)

Translin antibody was generated against recombinant Translin. The Ago2 antibody was obtained from Millipore. The Actin antibody and Anti-Flag Agarose resin were purchased from Sigma. Western blot and Flag-IP were performed as previously described⁵⁹. For experiments in Fig. 1d, the RISC reactions were prepared by incubating recombinant His-Flag-tagged hAgo2 or dAgo2 with radiolabelled duplex siRNA in absence or presence of, respectively, Dicer-TRBP or Dcr-2-R2D2 complex. After incubated with anti-Flag agarose for 2 hours at 4°C and washed with buffer A containing 0.5 M NaCl, the associated siRNA were extracted by Trizol (Invitrogen), ethanol precipitated, and resolved on a 16% native polyacrylamide gel.