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Mol Cell. Author manuscript; available in PMC 2016 September 03. Published in final edited form as: Mol Cell. 2015 September 3; 59(5): 807–818. doi:10.1016/j.molcel.2015.07.006.

TAF11 assembles RISC loading complex to enhance RNAi efficiency Chunyang Liang1,5, Yibing Wang1,5, Yukiko Murota2, Xiang Liu1, Dean Smith3, Mikiko C. Siomi2, and Qinghua Liu1,4,*

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1Department

of Biochemistry, UT Southwestern Medical Center, Dallas, TX 75390, USA

2Department

of Biological Sciences, University of Tokyo, Tokyo 113-0032, Japan

3Departments

of Pharmacology and Neuroscience, UT Southwestern Medical Center, Dallas, TX

75390, USA 4International

Institute of Integrated Sleep Medicine (IIIS), University of Tsukuba, Tsukuba, 305-8575, Japan

SUMMARY

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Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains Dicer-2(Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here, we identified the missing factor of RLC as TATA-binding protein associated factor 11 (TAF11) by genetic screen. Although an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11−/− ovary extract, we reconstituted the RLC in vitro using recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of Drosophila RLC and elucidate a novel cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency.

Keywords RISC; RLC; TAF11; Dcr-2-R2D2; Ago2; siRNA

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*

Corresponding author: [email protected]; FAX: (214) 648-8856. 5Co-first author Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AUTHOR CONTRIBUTIONS Q. Liu, D. Smith and C. Liang conceived the project. C. Liang and X. Liu performed genetic screen. C. Liang, Y. Wang and X. Liu performed biochemical experiments. M. C. Siomi and Y. Murota performed immunostaining. Q. Liu wrote the manuscript.

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INTRODUCTION

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RNA interference (RNAi) describes a conserved double-stranded RNA (dsRNA)-induced gene silencing mechanism that protects eukaryotes from invading nucleic acids (Ding and Voinnet, 2007; Fire et al., 1998; Liu and Paroo, 2010). The dsRNA molecules can be synthesized from exogenous viruses and transgenes, or from endogenous transposons and repetitive elements within the host genome (Ding and Voinnet, 2007; Okamura and Lai, 2008). Perceived as aberrant RNA by host cells, they are processed by Dicer, a family of multidomain RNase III enzymes, into small interfering RNAs (siRNA) (Bernstein et al., 2001; Liu et al., 2003). In Drosophila melanogaster, there are two Dicers: Dicer-1 (Dcr-1) generates microRNA from short (~60-nt) hairpin RNA precursor, whereas Dicer-2 (Dcr-2) cleaves long dsRNA into siRNA (Lee et al., 2004; Liu et al., 2003). In addition, the LOQUACIOUS (LOQS) gene encodes four dsRNA-binding protein (dsRBP) isoforms: whereas Loqs-PB functions as a specific cofactor for Dicer-1 in microRNA production (Forstemann et al., 2005; Jiang et al., 2005; Saito et al., 2005), Loqs-PD enhances Dcr-2's siRNA-generating activity (Miyoshi et al., 2010a; Zhou et al., 2009).

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Nascent siRNA duplex is assembled into the effector RNA-induced silencing complex (RISC). In an active RISC, single-stranded siRNA guides Ago2 endoRNase to catalyze target mRNA cleavage (Liu et al., 2004; Martinez et al., 2002; Rivas et al., 2005; Song et al., 2004). Thus, the central step of RISC assembly is to dissociate two strands of siRNA, selectively incorporate one as the “guide strand” into active RISC, and discard the other called the “passenger strand”. Accumulating studies in multiple model systems support a “slicer” model of RISC assembly that consists of two basic steps: RISC loading and RISC activation (Liu and Paroo, 2010). RISC loading describes the process by which both strands of siRNA are loaded onto Ago2 to form an inactive pre-RISC. RISC activation is catalyzed sequentially by two RNases, Ago2 and QIP/C3PO (component 3 promoter of RISC). First, Ago2 nicks the “passenger strand” of duplex siRNA into a 9-nt and a 12-nt fragment (Leuschner et al., 2006; Matranga et al., 2005; Miyoshi et al., 2005; Rand et al., 2005). Second, C3PO or QIP is recruited to selectively degrade the passenger fragments to convert inactive pre-RISC (Ago2/guide strand-passenger strand) into active RISC (Ago2/guide strand) (Liu et al., 2009; Maiti et al., 2007; Ye et al., 2011).

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In Drosophila, formation of the RISC loading complex (RLC) precedes and is required to initiate RISC assembly. Dcr-2 and R2D2 are core components of the RLC and coordinately bind and load duplex siRNA onto Ago2 (Liu et al., 2003; Pham et al., 2004; Tomari et al., 2004a). Assembly of RLC and RISC is abolished or diminished in dcr-2 or r2d2 null extracts, which can be restored by addition of recombinant Dcr-2-R2D2 complex (Liu et al., 2006; Pham et al., 2004; Tomari et al., 2004a). Accordingly, recombinant Dcr-2-R2D2 and Ago2 proteins reconstitute a basal level of dsRNA- or duplex siRNA-initiated RISC activity in vitro, suggesting that they comprise the catalytic core of Drosophila RNAi (Liu et al., 2009). Moreover, heat shock proteins (HSPs), e.g. Hsp90/Hsc70, are required to keep Ago2 conformation flexible to facilitate siRNA loading (Iki et al., 2010; Iwasaki et al., 2010; Iwasaki et al., 2015; Miyoshi et al., 2010b).

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The precise molecular composition and action mechanism of Drosophila RLC remain to be determined. Whereas human RLC was defined as the Dicer-TRBP-Ago2 complex (MacRae et al., 2008), fly RLC was discovered as the siRNA-protein (siRNP) complex containing Dcr-2, R2D2, and possibly other protein(s) (Pham et al., 2004; Pham and Sontheimer, 2005; Tomari et al., 2004a). It is widely believed, however, that Ago2 is not an integral component of fly RLC because RLC forms normally in ago2 null extract (Liu et al., 2009). The other factor(s) of Drosophila RLC has not been identified to date despite of intense genetic and biochemical search in the last decade.

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The TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs or TAFIIs) make up TFIID, a general transcription factor that nucleates the assembly of RNA polymerase II (Pol II) pre-initiation complex over the transcription start site on both TATAcontaining and TATA-less promoters (Albright and Tjian, 2000; Bell and Tora, 1999). Whereas TBP is essential for general transcription, individual TAFs affect transcription of a specific subset of genes in yeast and mammalian cells (Albright and Tjian, 2000; Bell and Tora, 1999; Green, 2000). A subset of TAFs carries histone-fold domain resembling the core histones H2B, H3 or H4 (Burley and Roeder, 1996). It is thought that these histone-like TAFs form a histone octamer-like structure consisting of two dimers of H2B-like TAFs in complex with a tetramer of H3- and H4-like TAFs (Hoffmann et al., 1996; Xie et al., 1996). Here, we identified histone-like TAF11 as the missing factor of Drosophila RLC by forward genetic screening. We showed that TAF11 protein could exist in the nucleus or co-localize with Dcr-2/R2D2 in cytoplasmic foci called D2 bodies. We determined the molecular composition of Drosophila RLC by in vitro reconstitution. Our biochemical studies support a model that TAF11 tetramer assembles the RLC by facilitating Dcr-2-R2D2 tetramerization to enhance RNAi efficiency.

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RESULTS Identification of TAF11 as a new RNAi factor by extensive EMS screening We performed an ethylmethanesulfonate (EMS)-mutagenesis screen to identify RNAideficient mutants using a similar strategy as described (Lee et al., 2004). In brief, we screened EMS-mutagenized GMR-whiteRNAi flies, in which eye-specific expression of a ~400bp white-dsRNA induced silencing of endogenous white+ gene, resulting in light orange eye (Kalidas and Smith, 2002). The RNAi-deficient mutants should revert back to red eyes due to loss of silencing of white+ expression. To enable recovery of lethal or sterile mutations from F1 germline, we employed Flipase/FRT-mediated recombination to generate mosaic flies with homozygous mutant eyes in an otherwise heterozygous background.

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Previously, multiple laboratories had carried out similar EMS screens, but failed to identify new RNAi factors beyond the core RNAi machinery (R. Carthew, E. Lai, and R. Zhou, personal communications). Potential explanations may include: 1) these screens were not saturated; 2) many RNAi mutants did not show a phenotype due to redundancy in the RNAi pathway. Thus, we decided to conduct a near saturated EMS screen of chromosome 2L. After mutagenizing ~50,000 males and screening ~1.5 million F1 mosaic flies (estimated 26x coverage), we isolated 44 dark orange or red eye mutants from this screen. Among those 44 mutants, complementation tests revealed 14 r2d2 alleles, 3 loqs alleles, and two new Mol Cell. Author manuscript; available in PMC 2016 September 03.

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groups of mutants with 7 (G3) and 20 alleles (G4) (Figure 1A). We mapped the causal mutations of G3 mutants to a 38 kb interval of 30C7-C9 by deficiency mapping. We identified genetic lesions in the TAF11 gene in all seven G3 mutants: whereas six taf11 alleles were nonsense mutations, predicted to result in premature termination of TAF11 protein, one was a missense mutation that substituted Alanine 141 with Glutamic acid (A141E) in the histone-fold domain of TAF11 protein (Figure S1A).

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Fly TAF11 is annotated as a homolog of human TAFII28, a subunit of basic transcription factor TFIID (Albright and Tjian, 2000; Bell and Tora, 1999), raising the possibility that the RNAi-deficient phenotype of taf11 mutants was a secondary effect due to reduced transcription of white-dsRNA from the GMR-whiteRNAi transgene. We eliminated this possibility by showing normal level of white-dsRNA expression in the homozygous fly heads of two different taf11 mutant strains by quantitative RT-PCR using both intronic and exonic primers (Figure 1A and 1B). By contrast, Northern blotting showed that endogenous white-siRNA level was diminished in homozygous taf11 mutant heads (Figure 1C), which resembled the phenotype of r2d2 mutants that was previously shown to be specifically defective for RISC loading (Liu et al., 2006; Marques et al., 2007). These results suggest that taf11 mutant flies are indeed defective for dsRNA-induced RNAi silencing.

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Mutants deficient for Dcr-2, R2D2, or Ago2 are homozygous viable, suggesting that the core RNAi machinery is dispensable for fly development and viability (Lee et al., 2004; Liu et al., 2003; Okamura et al., 2004). However, all seven taf11 mutants were homozygous lethal or sub-lethal. This lethality was not due to background mutations because it could not be eliminated by intercrossing different taf11 alleles or with corresponding deficiency strains (data not shown). For hypomorphic taf11 alleles, homozygous taf11−/− flies were weak, sterile, and typically died within five days (Figure S1B). Consistent with the sterility, homozygous taf11−/− females had much smaller ovaries compared to wild type females (Figure S1C). These phenotypes suggest that TAF11 may have dual functions: 1) a nuclear transcription function that is required for viability and development; and 2) a cytoplasmic function in RNAi. TAF11 is required for RNAi-mediated antiviral response in S2 cells

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Flock house virus (FHV) is an RNA virus carrying two plus-strand molecules, RNA1 and RNA2. Whereas RNA2 encodes the single virion structural protein, RNA1 encodes protein A, RNA-dependent RNA polymerase (RdRP), and viral RNAi suppressor B2. In S2 cells, RNA1 can replicate autonomously without RNA2, while a mutant RNA1 (pFR1gfp), in which B2 is replaced by GFP, is strongly suppressed by the RNAi pathway. As previously shown (Wang et al., 2006), following knockdown of Ago2 by RNAi, transfection of pFR1gfp resulted in viral amplification and GFP-positive S2 cells (Figure 1D). However, ~90% knockdown of Dcr-2 or TAF11 by RNAi resulted in no GFP-positive cells in this assay (data not show), although RNAi was clearly defective in dcr-2 or taf11 mutant flies. To overcome this difficulty, we applied the CRISPR/Cas9 technology to knockout the DCR-2 or TAF11 gene in S2 cells (Bassett et al., 2014), and followed by transient pFR1gfp transfection to evaluate RNAi-mediated viral suppression. Despite of low efficiency of CRISPR in S2 cells, cell-autonomous knockout of DCR-2 or TAF11 could result in a Mol Cell. Author manuscript; available in PMC 2016 September 03.

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signficant number of GFP-positive cells (Figure 1D). Since human TAFII28 (TAF11) heterodimerizes with TAFII18 (TAF13) (Birck et al., 1998), we wanted to examine whether fly TAF13 was a co-factor for TAF11 in RNAi. However, CRISPR knockout of TAF13 by multiple constructs failed to yield GFP-positive cells (Figure 1D). Collectively, these genetic studies uncovered an unexpected role of TAF11 in Drosophila RNAi and antiviral defense. TAF11 is a key RLC component

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To map the precise biochemical defect of taf11 mutants, we prepared wild-type and taf11−/− (A141E) mutant ovary extracts to perform in vitro RNAi assays. Western blotting showed that Dcr-2, R2D2 and Ago2 proteins were expressed at wild-type levels, but full-length TAF11 protein was diminished in taf11−/− extract (Figure 2A). Furthermore, taf11−/− extract exhibited normal dsRNA-processing activity, but was defective for duplex siRNA-initiated RISC activity (Figure 2B and 2C). Addition of recombinant TAF11 enhanced the RISC activity in taf11−/− extract (Figure 2D). These studies suggest that TAF11 is required for efficient RISC assembly and or activity. Native gel-shift assays were previously used to study the stepwise RISC assembly by agarose or polyacrylamide gel electrophoresis (PAGE). At least three siRNP complexes, the R2-D2-Initiator complex (RDI)/R1, RLC/complex A/R2, and RISC/R3 were detected in Drosophila embryo or S2 cell extract (Pham et al., 2004; Pham and Sontheimer, 2005; Tomari et al., 2004a). The RDI contains Dcr-2, R2D2 and duplex siRNA (Pham and Sontheimer, 2005). It is believed that the RDI recruits unknown factor(s) to form the RLC, which loads duplex siRNA onto Ago2 to initiate RISC assembly.

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By native agarose gel-shift assay, we showed that assembly of RLC and RISC, but not RDI, was defective in taf11−/− ovary extract, suggesting that TAF11 was required for RLC assembly (Figure 2E and S2A). Importantly, assembly of both RLC and RISC was fully rescued in taf11−/− extract by addition of recombinant TAF11 protein (Figure 2F and 2G), but not by BSA or mutant TAF11A141E protein (Figure S2B and S2C). These biochemical studies strongly suggest that TAF11 is a key RLC component. TAF11 exists in the nucleus/cytoplasm and associates with core RNAi machinery

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The majority of RNAi factors are cytoplasmic and RISC assembly occurs in the cytoplasm. Although annotated as a nuclear transcription factor, the nuclear localization of fly TAF11 has not been validated. Thus, we prepared total, nuclear and cytoplasmic extracts from S2 cells, and examined the distribution of TAF11 protein by Western blotting. The result showed that TAF11 exists in both the nuclear and cytoplasmic fractions (Figure 3A). Consistent with previous report (Cernilogar et al., 2011), Dcr-2, R2D2, and Ago2 proteins were also found in both compartments, although mostly present in the cytoplasm (Figure 3A). To examine whether TAF11 interacted with core RNAi machinery, we co-transfected S2 cells with GFP-TAF11 and Flag-tagged Dcr-2, R2D2, Ago2, or Loqs-PB constructs. Coimmunoprecipitation (IP) showed that TAF11 could specifically associate with Dcr-2, R2D2, and Ago2, but not with Loqs-PB (Figure 3B), a specific co-factor for Dcr-1 in

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microRNA production (Forstemann et al., 2005; Jiang et al., 2005; Saito et al., 2005; Ye et al., 2007). The association of TAF11 and Dcr-2/R2D2 was independent of RNA (Figure S3). Furthermore, we detected self-association of different epitope-tagged TAF11, Dcr-2, or R2D2 proteins in S2 cells (Figure 3C-3E), suggesting that these RNAi factors could potentially form homo-oligomeric complexes in vivo. TAF11 co-localizes with Dcr-2/R2D2 to cytoplasmic D2 bodies

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A portion of endogenous Dcr-2 and R2D2 proteins co-localize to distinct cytoplasmic foci called D2 bodies in S2 cells and ovarian follicle cells (Nishida et al., 2013). Both Dcr-2 and R2D2 are required for the formation of D2 bodies because Dcr-2 stabilizes R2D2, whereas R2D2 localizes Dcr-2 to the D2 bodies (Nishida et al., 2013). We observed frequent colocalization of GFP-TAF11 with RFP-Dcr-2 or RFP-R2D2 in D2 bodies by confocal imaging (Figure 4A). Similarly, we also detected co-localization of GFP-TAF11 with endogenous Dcr-2 or R2D2 in D2 bodies (Figure 4B). In this experiment, three different GFP-TAF11 localization patterns were observed (% of transfected cells): 1) nucleus (~56.7%), D2 body (~36.7%), and both (6.6%) (Figure 4C). It is worth noting that GFPTAF11 tends to concentrate in the nucleus when overexpressed, whereas more of endogenous TAF11 locates in the cytoplasm (Figure 3A). These results suggest a dynamic localization of TAF11 protein between the nucleus and cytoplasmic D2 bodies.

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Furthermore, we examined localization of GFP-TAF11 following knockdown of Dcr-2 by RNAi in S2 cells, which was shown to cause the disappearance of D2 bodies (Nishida et al., 2013). In control siRNA-treated S2 cells, GFP-TAF11 was observed in D2 bodies in ~44% of transfected S2 cells (data not shown). Importantly, no cytoplasmic GFP-TAF11 foci were detected in Dcr-2 siRNA-treated S2 cells (Figure 4D), indicating that GFP-TAF11 could not form cytoplasmic foci by itself. These results suggest that TAF11 localizes to cytoplasmic D2 bodies through its interaction with Dcr-2-R2D2 complex. TAF11 enhances RISC loading activity of Dcr-2-R2D2 complex

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We showed that addition of recombinant TAF11 enhanced the core RISC activity of recombinant Dcr-2-R2D2 and Ago2 proteins by two to threefold (Figure 5A). Moreover, we measured the efficiency of RISC loading by ultra violet (UV) light-induced crosslinking of Ago2 with radiolabeled duplex siRNA (Figure 5B) (Liu et al., 2003; Tomari et al., 2004b). Whereas Ago2 alone showed no siRNA loading, Dcr-2-R2D2 complex bound radiolabeled siRNA and facilitated siRNA loading onto Ago2 (Figure 5B and 5C). Wild-type TAF11, but not mutant TAF11A141E, enhanced crosslinking of both Dcr-2-R2D2 and Ago2 proteins to radiolabeled siRNA by two to threefold (Figure 5B, 5C, and S4B). Notably, TAF11 was not crosslinked with radiolabeled siRNA, suggesting that it did not contact siRNA within the RLC. These in vitro reconstitution studies suggest that TAF11 enhances the RISC loading activity of Dcr-2-R2D2 complex. TAF11 enhances siRNA-binding activity of Dcr-2-R2D2 complex, but switches from a sigmoidal to a hyperbolic siRNA-binding curve We compared siRNA-binding activity of Dcr-2-R2D2 complex in the absence and presence of TAF11 by native PAGE gel-shift assay. It was difficult for native PAGE to resolve the

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RDI and RLC (Figure 5D and S4A), although they showed a dramatic mobility difference on native agarose gel for unknown reasons (Figure 2E and 2F). It is widely believed that Dcr-2-R2D2 complex binds duplex siRNA as a stable heterodimer (Nykanen et al., 2001; Liu et al., 2003; Pham and Sontheimer, 2005). However, here we showed that recombinant Dcr-2-R2D2 complex exhibited a sigmoidal (cooperative) siRNA-binding curve (Figure 5D and 5E). Based on our observation of self-association of Dcr-2 and R2D2 (Figure 3D and 3E), we propose a simple model: at low concentrations, Dcr-2-R2D2 complex exists as a heterodimer with low affinity for duplex siRNA. At high concentrations, Dcr-2-R2D2 heterodimer can spontaneously dimerize to form heterotetramer with higher affinity for duplex siRNA. Thus, the sigmoidal siRNA-binding curve may simply reflect the concentration-dependent formation of Dcr-2-R2D2 heterotetramer.

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Remarkably, the presence of TAF11 not only enhanced the siRNA affinity of Dcr-2-R2D2 complex by tenfold (Kd=2.37μM, -TAF11 → Kd=0.23μM, +TAF11), but also switched Dcr-2-R2D2 complex from a sigmoidal to a hyperbolic siRNA-binding curve (Figure 5D and 5E). It is likely that association with TAF11 facilitates or stabilizes the formation of Dcr-2-R2D2 heterotetramer. This higher order complex can bind duplex siRNA with high affinity as one entity and thus exhibits a non-cooperative siRNA-binding curve. In vitro reconstitution of RLC

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To determine the molecular composition of Drosophila RLC, we tried to reconstitute the RLC in vitro using recombinant Dcr-2-R2D2 complex, TAF11, and radiolabeled duplex siRNA (Figure 6A). We developed a native agarose/PAGE hybrid gel-shift assay and showed that recombinant Dcr-2-R2D2 and TAF11 proteins coordinately bound radiolabeled siRNA duplex to form a large siRNP complex with a mobility similar to that of native RLC formed in S2 extract (Figure 6A). By contrast, mutant TAF11A141E could not cooperate with Dcr-2-R2D2 to form the RLC, and exhibited diminished ability to enhance siRNA-binding activity of Dcr-2-R2D2 complex (Figure 6A and S5A). These in vitro reconstitution studies suggest that Drosophila RLC is comprised of Dcr-2-R2D2, TAF11, and duplex siRNA. TAF11 tetramer assembles the RLC by facilitating Dcr-2-R2D2 tetramerization

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To study the action mechanism of TAF11 in RLC, we compared the elution profile of recombinant Dcr-2-R2D2 complex in the absence or presence of TAF11 by size-exclusion chromatography. The majority of recombinant His6-TAF11 protein migrated as a major peak corresponding to ~600 kDa on Superdex-200 column (Figure S5B). Since both the size and shape of a protein affect its elution profile on Superdex-200 column, we measured molecular weight of recombinant His6-TAF11 by blue native PAGE. Although only a single ~30 kDa band of His6-TAF11 was detected by SDS-PAGE, two His6-TAF11 bands, ~30 kDa and ~120 kDa, were resolved on blue PAGE, which probably corresponded to TAF11 monomer and tetramer, respectively (Figure 6B). Taken together, these results suggest that TAF11 may exist as a tetramer (~120KDa), but have a non-globular (e.g. disc or spindle) shape, such that its molecular weight is overestimated (~600 kDa) by size-exclusion chromatography.

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On the other hand, recombinant Dcr-2-R2D2 complex displayed a strikingly messy elution profile-present in almost every fraction and with a major peak at ~1 kDa (Figure 6C, top, fractions 16-18). A likely explanation for this unusual elution profile was that recombinant Dcr-2-R2D2 complex interacted with the gel-filtration matrix and, thus, was dragged into much later fractions. Remarkably, if recombinant Dcr-2-R2D2 complex was pre-incubated with wild-type TAF11 protein before Superdex-200 chromatography, a single and clean elution peak of Dcr-2-R2D2 complex was detected at the position of TAF11 peak (Figure 6C, middle, fractions 4-6). Importantly, this elution profile of recombinant Dcr-2-R2D2TAF11 complex was consistent with that of endogenous proteins after fractionation of S2/ S100 extract by Superdex-200 chromatography (Figure S5C). By contrast, pre-incubation with TAF11 failed to move the elution peak of BSA (Figure S5D), suggesting that TAF11 was not generally a sticky protein. Moreover, mutant TAF11A141E, which migrated as a major peak of ~30 kDa, was unable to move the elution peak of Dcr-2-R2D2 complex (Figure 6C, bottom). Therefore, this dramatic change in the gel-filtration profile of Dcr-2R2D2 complex is likely caused by direct and specific interactions between TAF11 and Dcr-2/R2D2 proteins.

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We also compared the elution profiles of recombinant RDI (Dcr-2-R2D2-siRNA) and RLC (Dcr-2-R2D2-TAF11-siRNA) on Superdex-200 chromatography (Figure 6D, 6E, and S5E). Recombinant RDI exhibited a major peak between 158 kDa and 474 kDa (Figure 6D, fractions 7-9), which might correspond to a Dcr-2-R2D2 heterodimer in complex with duplex siRNA as previously reported (Nykanen et al., 2001; Pham and Sontheimer, 2005). However, in light of the sigmoidal siRNA-binding curve of Dcr-2-R2D2 complex (Figure 5D and 5E), it is likely that this RDI peak may contain Dcr-2-R2D2 heterotetramer in complex with duplex siRNA. On the other hand, recombinant RLC migrated with a single peak between ~474 kDa and ~670 kDa on Superdex-200 column (Figure 6E, fractions 4-6). This elution profile is consistent with a large siRNP complex that is comprised of a TAF11 tetramer (~120 kDa), a Dcr-2-R2D2 heterotetramer (~480 kDa), and duplex siRNA (~14 kDa). The result further supports our idea that TAF11 exists as a tetramer (~120 kDa) with an irregular shape rather than a globular 20-mer of ~600 kDa (Figure S5B). Otherwise, recombinant RLC [TAF1120-(Dcr-2-R2D2)2-siRNA] should hypothetically migrate close to the void (fractions 1-2) according to its projected molecular weight of ~1.1 MDa. More likely, we suspect that recombinant RLC [TAF114-(Dcr-2-R2D2)2-siRNA] acquires a globular shape and thus exhibits an elution peak consistent with its molecular weight of ~600 kDa.

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Alternatively, an exciting possibility is that TAF11 tetramer could function as a “catalyst” to facilitate Dcr-2-R2D2 tetramerization and then leave the complex. However, this is less likely because substoichiometric amount of TAF11 failed to change the gel-filtration profile of Dcr-2-R2D2 complex (data not shown). We could also detect by Western blotting the presence of both TAF11 and Dcr-2-R2D2 proteins within the recombinant RLC band excised after native gel-shift assay (Figure S5F). Moreover, the dramatic movement of Dcr-2-R2D2 complex on Superdex-200 column suggest that association with TAF11 or siRNA may cause major conformational changes in Dcr-2/R2D2 proteins, such that they can no longer interact with the gel-filtration matrix (Figure 6D and 6E).

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To obtain more direct evidence for our “tetramer” model, we tried to measure the molecular weight of recombinant RDI and RLC by blue native PAGE. However, both RDI and RLC became unstable on blue PAGE probably due to disruption by the Coomassie blue dye. Thus, we attempted UV crosslinking to stabilize recombinant RDI and RLC formed with radiolabeled duplex siRNA before running on blue PAGE. A potential caveat is that UV crosslinking was not 100% efficient and might only crosslink part of the RDI or RLC complex. Nevertheless, recombinant RDI showed a smear between 240 to 480 kDa, possibly corresponding to Dcr-2-R2D2 heterodimer/heterotetramer crosslinked to radiolabeled siRNA (Figure 6F). On the other hand, recombinant RLC showed an enhanced smear between 240 to 480 kDa as well as a new band between 480 kDa and 720 kDa, possibly corresponding to the TAF114-(Dcr-2-R2D2)2-siRNA complex (Figure 6F). Taken together, this collection of biochemical studies strongly suggest that TAF11 tetramer assembles the RLC by facilitating Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities.

DISCUSSION A working model for Drosophila RISC loading complex (RLC)

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Our genetic, cell biological, and biochemical studies suggest a working model for Drosophila RLC assembly and function (Figure 7). The Dcr-2-R2D2 complex exists as an equilibrium of heterodimer and heterotetramer, with the latter showing higher siRNA binding activity. It has been suggested that R2D2 preferentially binds the more stable end, whereas Dcr-2 prefers the less stable end of an asymmetric siRNA (Tomari et al., 2004b). Thus, we propose that a Dcr-2-R2D2 heterotetramer should contain two heterodimers in a parallel orientation. Association with TAF11 tetramer facilitates or stabilizes formation of Dcr-2-R2D2 tetramer, resulting in a higher order [(TAF11)4-(Dcr-2-R2D2)2] complex that binds duplex siRNA with tenfold higher affinity. Therefore, in contrast to the common belief that the RDI recruits another factor (TAF11) to form the RLC, we hypothesize that TAF11 may bypass the RDI and directly assemble the RLC by facilitating Dcr-2-R2D2 tetramerization, which is a far more efficient process. Additionally, association with TAF11 may convert the RDI into RLC, whereas the RLC may be reverted back to RDI under certain conditions. Furthermore, Dcr-2-R2D2 heterotetramer may have a different configuration within the RLC due to association with TAF11, such that the RLC is qualitatively distinct from the RDI and loads duplex siRNA onto Ago2 much more efficiently. While this is the simplest model that fits all available experimental data, we cannot exclude the possibility that there may be alternative explanations. It is paramount to determine the structure of the RLC by electron microscopy (EM) and X-ray crystallography for in-depth understanding of RLC assembly and function. It will also be interesting to examine whether Dicer complexes from other organisms bind duplex siRNA or precursor microRNA in a similar tetramer mode. Dynamic localization of TAF11 to D2 bodies, an in vivo hotspot for RLC assembly? Co-IP and co-immunostaining studies indicate that cytoplasmic GFP-TAF11 localizes to D2 bodies through its association with Dcr-2-R2D2 complex. The three different localization patterns (nucleus, D2 body, or both) of GFP-TAF11 suggest dynamic localization of TAF11

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protein between the nucleus and cytoplasmic D2 bodies. Furthermore, we only detected GFP-TAF11 in D2 bodies in ~44% of transfected S2 cells, of which GFPTAF11 was always seen in a subset (one or two, rarely three) of D2 bodies (data not shown). Therefore, we speculate that unlike Dcr-2/R2D2, TAF11 is a transient visitor rather than permanent resident of D2 bodies, and can be viewed as a licensing factor for specific D2 body to engage in RISC assembly. Thus, the TAF11-positive D2 body may transiently recruit Ago2/ HSPs to execute RISC assembly in vivo. Once RISC assembly is completed, the RLC may be disassembled and TAF11 will leave the D2 body. We propose that the TAF11-positive D2 body functions as the in vivo hotspot for dynamic RLC and RISC assembly. How does the RLC transfer duplex siRNA from Dcr-2-R2D2 to Ago2?

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It remains unclear exactly how the RLC transfers duplex siRNA from Dcr-2-R2D2 to Ago2 during RISC loading. At some point in this process, the Dcr-2-R2D2 complex has to let go of siRNA and pass it on to Ago2. Because it is energetically unfavorable for Ago2 to directly bind duplex siRNA, a number of heat shock proteins (HSPs) are required to keep Ago2 conformation flexible for it to receive duplex siRNA (Iki et al., 2010; Iwasaki et al., 2010; Iwasaki et al., 2015; Miyoshi et al., 2010b). To make this an efficient process, it is likely that these series of events should occur in a highly coordinated fashion.

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Our studies suggest that there are at least two functional states of Dcr-2-R2D2 complex: 1) Dcr-2-R2D2 heterodimer, which has low affinity for duplex siRNA; 2) Dcr-2-R2D2 heterotetramer, which binds duplex siRNA with high affinity. Thus, the conversion between these two states may drive the binding and release of siRNA from Dcr-2-R2D2 complex. On one hand, TAF11 facilitates Dcr-2-R2D2 tetramerization to make it bind duplex siRNA with tenfold higher affinity. On the other hand, other factor(s) may trigger the release of siRNA from Dcr-2-R2D2 heterotetramer by converting it back to heterodimer. Alternatively, a major conformational switch of Dcr-2-R2D2 complex may also result in the binding and release of duplex siRNA. These two mechanisms are not mutually exclusive and may occur simultaneously during RISC loading.

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One simple possibility is that Ago2/HSPs could serve as the siRNA-releasing factors. TAF11 could bridge the RLC with Ago2/HSPs to facilitate siRNA transfer from Dcr-2R2D2 to Ago2. It is also plausible that additional factor(s) may be involved in this process for the following reasons. First, TAFs typically do not act alone. It is believed that histonelike TAFs form a core histone-like octameric complex within TFIID to promote the transcription of target genes. Whereas human TAF11 heterodimerizes with TAF13 in the nucleus (Birck et al., 1998), our CRISPR studies suggest that fly TAF11 does not function with TAF13 in RNAi-mediated antiviral response in S2 cells. Accordingly, even though TAF13 is also located on chromosome 2L, no taf13 RNAi-deficient mutant was identified in our saturated genetic screening. Thus, it is possible that fly TAF11 has a novel cofactor in cytoplasmic RNAi. Secondly, we showed that TAF11 increased the siRNA-binding activity of Dcr-2-R2D2 complex by tenfold, but only enhanced the RISC loading activity by two to threefold. One interpretation is that TAF11 may need a cofactor to maximize the efficiency of RISC loading in this reconstitution system. Thirdly, we observed two RISC-enhancing activities by supplementing recombinant Dcr-2-R2D2 and Ago2 proteins with fractions of

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S2 extract. We identified C3PO as one RISC activator from S2 extract by sequential chromatography (Liu et al., 2009). However, it was difficult to purify another RISC activator because its activity peak was quickly lost after two to three chromatographic steps. This purification difficulty often suggests the existence of an unstable protein complex that can be disrupted by sequential chromatography. Therefore, future studies are required to identify the potential missing factor(s) and elucidate the detailed mechanism by which duplex siRNA is transferred from Dcr-2-R2D2 to Ago2 during RISC loading.

EXPERIMENTAL PROCEDURES Genetic screening

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The GMR-whiteRNAi transgene was previously described (Kalidas and Smith, 2002). w+; FRT40A; GMR-Gal4, UAS-whiteRNAi male flies were treated by ethylmethanesulfonate (25 mM) and crossed with w+; GMR-hid FRT40A; eye-FLP females. The pro-apoptotic GMRhid transgene ensured that only homozygous mutant eye cells survived to adulthood. F1 mosaic flies were screened under the microscope for dark orange or red eye flies, which were deemed as potential RNAi-deficient mutants. Complementation test was performed to establish the r2d2 and loqs groups of mutants by crossing candidate mutant strains with known r2d2 and loqs alleles, and to classify novel mutant strains into two new groups (G3 and G4) by crossing with one another. Native siRNA gel-shift assay

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Synthetic siRNA duplex were 5’ radiolabeled with [γ-32P] ATP by T4 polynucleotide kinase and PAGE purified as previously described (Liu et al., 2009). Recombinant proteins or ovary extracts were incubated with radiolabeled siRNA (2×104 cpm) at 30 °C for 30 min in a 10- l reaction in buffer 12 (100 mm KOAc, 10 mm HEPES, pH 7.4, 2 mM Mg(OAc)2, 5mM DTT, 1mM ATP). After adding 1.5 μl of glycerol, reaction mixtures were resolved by 1.5% native agarose gel made in 0.5 x TBE containing 1.5 mM Mg(OAc)2. For native hybrid gel (Figure 6A), 1% agarose and 4% PAGE (37.5:1 acrylamide:bisacrylamide) were mixed in 0.5 × TBE buffer/1.5 mM Mg(OAc)2, and 10% APS and TEMED were added and the mixture was quickly poured into glass plate pre-warmed at 50 °C. Both gels were run at 4°C with chilled 0.5 × TBE buffer/1.5 mM Mg(OAc)2, and dried onto a Zeta-probe membrane (Bio-Rad) and exposed to X-ray film. Immunofluorescence

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GFP-TAF11 (PAGW) and RFP-Dcr-2/R2D2 (PARW) were placed under the control of Actin promoter using the Gateway system (Invitrogen). Four days after co-transfection of GFP-TAF11 and/or RFP-Dcr-2/R2D2 constructs, S2 cells were fixed with 2% formaldehyde in PBS at room temperature for 15 min and then permeabilized with 0.1% Triton X-100 in PBS. The samples were stained with anti-Dcr-2 (Miyoshi et al., 2009) or anti-R2D2 (Nishida et al., 2013) antibodies. Anti-Dcr-2 and anti-R2D2 antibodies were used at 1:500 dilution. Alexa Fluor546-conjugated anti-mouse antibodies (Molecular Probes) were used at 1:1,000 dilution as secondary antibody. The samples were mounted in VectaShield containing DAPI (Vector Laboratories). All images were collected using a Zeiss LSM510 laser-scanning microscope. Mol Cell. Author manuscript; available in PMC 2016 September 03.

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Size-exclusion chromatography and Blue Native PAGE

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All size-exclusion chromatography were conducted on Superdex-200 10/300 GL column by AKTA HPLC (GE Healthcare) at 4°C. We loaded 0.5 ml of 0.4 μM recombinant Dcr-2R2D2 complex or 1 μM TAF11 proteins onto Superdex-200 column in buffer A (10 mM KOAc, 10 mM HEPES, pH7.4, 2 mM Mg(OAc)2, 5 mM DTT). Alternatively, 0.4 μM recombinant Dcr-2-R2D2 complex and 1 μM wild-type or mutant (A141E) TAF11 were incubated in buffer A at 30°C for 30 minutes before loading onto Superdex-200 column. For recombinant RDI and RLC, 0.5 ml of 0.4 μM recombinant Dcr-2-R2D2 complex was incubated with 1 μM of let-7 duplex siRNA in the absence (RDI) or presence (RLC) of 1μM of TAF11 in buffer 12. Western blotting was performed to detect Dcr-2, R2D2 and TAF11 proteins, whereas Northern blotting was performed to detect let-7 siRNA among chromatographic fractions.

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NativeMark™ Unstained Protein Standard and highly purified His6-TAF11 recombinant protein were resolved by 4-16% Bis-Tris native PAGE (Life Technology) and visualized by Coomassie Blue staining. Radiolabeled let-7 siRNA duplex was incubated with different combination of recombinant Dcr-2-R2D2 and TAF11 in buffer 12 at 30 °C for 30 minutes. The reaction mixtures were exposed to 302nm UV light for 20 minutes on ice before mixing with 0.125% Coomassie Blue G250, resolved by 4-16% Bis-Tris native PAGE (Anode Buffer: 50mM Tris, PH 7.0; Cathode Buffer: 50 mM Tris, 0.02% G250, pH 7.0), and exposed to X-ray film.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

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ACKNOWLEDGEMENTS We are thankful for Drs. S-W. Ding and R. Carthew for generously sharing reagents, Drs. J. Albanesi, Y. Liu, H. Siomi, and J. Ma for helpful comments on the manuscript; and H. Liu, T. Liang, J. Lin, Y. Li, J. Chang, J. Hancock, L. Chen and M. Buszczak for technical assistance. Q. Liu is a W.A. “Tex” Moncrief Jr. Scholar in Medical Research. This work is supported by a Grant-in-Aid for Scientific Research awarded to M.C.S. and the World Premium Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and grants to Q.L. from the Welch foundation (I-1608), America Heart Association (13GRNT16270022), and National Institute of Health (GM111367).

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Author Manuscript Author Manuscript Figure 1. Identification of TAF11 as a new RNAi factor by EMS screen

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(A) Representative photos showing the eye color of wild-type (WT), loqs, r2d2, or taf11 mutant flies. The number of alleles for each complementation group is listed in parenthesis. A schematic diagram of the GMR-whiteRNAi transgene is shown above. Arrows refer to the positions of three PCR primers for checking white-dsRNA expression in (B). (B) Quantitative analysis of white-dsRNA expression between heterozygous and homozygous taf111 or taf115 mutant fly heads by real time RT-PCR. (C) Quantitative analysis of white-siRNA expression between heterozygous and homozygous r2d21 and taf111 mutant fly heads by Northern blotting. (D) Representative images showing the number of GFP-positive S2 cells 48 hours after pFR1gfp transfection following knockdown of Ago2 by RNAi or knockout of DCR-2, TAF11, or TAF13 by CRISPR/Cas9. Multiple guide RNAs for DCR-2 (4/6) and TAF11 (5/6), whereas none for TAF13 (0/4), showed GFP-positive cells in this assay.

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Author Manuscript Author Manuscript Figure 2. TAF11 is a key RLC component

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(A) Western blots comparing the levels of Dcr-2, R2D2, Ago2, TAF11, and Actin between taf11+/− and taf11−/− ovary extracts. All experiments here used taf115 (A141E) mutant strain. (B) The dsRNA-processing assay was performed using 4 μg of taf11+/− and taf11−/− ovary extracts. (C) (Left) The duplex siRNA-initiated RISC assay was performed using 20 μg taf11+/− and taf11−/− ovary extracts. (Right) Quantitative analysis of the RISC activity (measured by fraction of cleaved mRNA) between taf11+/− and taf11−/− extracts (triplicate experiments, ***, P value < 0.001. P value was calculated by t test using GraphPad Prism). (D) The duplex siRNA-initiated RISC assay was performed using 20 μg taf11−/− ovary extract in the absence and presence of increasing concentration of recombinant TAF11. (E) Native agarose gel-shift assay was performed by incubating radiolabeled let-7 duplex siRNA in buffer alone (lane 1), 40 μg of WT (lane 2) or taf11−/− (lanes 3 and 4) ovary extract. Star refers to a non-specific shift that was not defined. (F) Native agarose gel-shift assay was performed by incubating radiolabeled duplex siRNA in buffer alone (lane 1), 40 μg of WT (lane 2) or taf11−/− (lane 3) extract, 40 μg of taf11−/− extract plus 50 ng recombinant TAF11 (lane 4). (G) Quantitative analysis of RLC formation between WT or taf11−/− ovary extract, or taf11−/− extract plus recombinant TAF11 (triplicate experiments, **, P value < 0.05; ***, P value < 0.001).

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Figure 3. TAF11 exists in the nucleus/cytoplasm and associates with core RNAi machinery

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(A) Western blots showing the distribution of TAF11, Dcr-2, R2D2, Ago2, Actin, and Histone H1 in total, nuclear, and cytoplasmic extracts. (B) S2 cells were co-transfected with constructs expressing GFP-TAF11 and Flag-tagged Dcr-2, R2D2, Ago2, or Loqs-PB followed by co-IP using anti-GFP antibody and Western blotting with anti-Flag and anti-GFP antibodies. (C) S2 cells were co-transfected with GFP-TAF11 and Flag-TAF11 constructs followed by co-IP using anti-GFP antibody and Western blotting with anti-Flag and anti-GFP antibodies. (D) S2 cells were co-transfected with Flag-R2D2 and Myc-R2D2 constructs followed by coIP using anti-Flag antibody and Western blotting with anti-Flag and anti-Myc antibodies. (E) S2 cells were co-transfected with Flag-Dcr-2 and Myc-Dcr-2 constructs followed by coIP using anti-Flag antibody and Western blotting with anti-Flag and anti-Myc antibodies.

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Figure 4. GFP-TAF11 co-localizes with Dcr-2/R2D2 in cytoplasmic D2 bodies

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(A) Representative images showing co-localization of GFP-TAF11 and RFP-Dcr-2 or RFPR2D2 in cytoplasmic D2 bodies in S2 cells 96 hours after co-transfection with corresponding expression constructs. (B) Representative images showing co-localization of GFP-TAF11 and endogenous Dcr-2 or R2D2 in cytoplasmic D2 bodies in S2 cells. Scale bar is 2μm. (C) A pie chart showing the percentage of transfected S2 cells (n=30) showing localization of GFP-TAF11 in the nucleus (56.7%), D2 body (36.7%), or both (6.6%). (D) Representative images showing localization of GFP-TAF11 and endogenous Dcr-2 in the control siRNA (siluc) or Dcr-2 siRNA (siDcr-2)-treated S2 cells.

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Figure 5. TAF11 enhances Dcr-2-R2D2's siRNA binding and RISC loading activities

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(A) The duplex siRNA-initiated RISC assay was performed with recombinant Dcr-2-R2D2 and Ago2 in the absence or presence of increasing concentration of recombinant TAF11. The percentage of cleaved mRNA and fold of enhancement were shown below. (B) Autoradiograph showing recombinant Dcr-2, R2D2, and Ago2 proteins that were UV crosslinked to radiolabeled siRNA after duplex siRNA-initiated RISC assembly. (C) Quantitative analysis of data in (B) comparing the amount of siRNA-crosslinked Dcr-2, R2D2, and Ago2 proteins in the absence or presence of TAF11. The intensities of radiolabeled proteins were quantified by ImageJ software (**, P value

TAF11 Assembles the RISC Loading Complex to Enhance RNAi Efficiency.

Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 com...
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