TAF11 Assembles the RISC Loading Complex to Enhance RNAi Efficiency Chunyang Liang, Yibing Wang, Yukiko Murota, Xiang Liu, Dean Smith, Mikiko C. Siomi, Qinghua Liu  Molecular Cell  Volume 59, Issue 5, Pages 807-818 (September 2015) DOI: 10.1016/j.molcel.2015.07.006 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 59, 807-818DOI: (10.1016/j.molcel.2015.07.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Identification of TAF11 as a New RNAi Factor by EMS Screen (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 parentheses. A schematic of the GMR-whiteRNAi transgene is shown above. Arrows refer to the positions of three PCR primers for checking white-dsRNA expression in (B). UAS, upstream activating sequence. (B) Quantitative analysis of white-dsRNA expression between heterozygous and homozygous taf111 or taf115 mutant fly heads by real-time RT-PCR. Error bars ± SEM. (C) Comparison 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 hr 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 of 6) and TAF11 (5 of 6), but none for TAF13 (0 of 4), showed GFP-positive cells in this assay. Molecular Cell 2015 59, 807-818DOI: (10.1016/j.molcel.2015.07.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 TAF11 Is a Key RLC Component (A) Western blots comparing the levels of Dcr-2, R2D2, Ago2, TAF11, and Actin between taf11+/− and taf11−/− ovary extracts. All experiments shown here used the 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 the fraction of cleaved mRNA) between taf11+/− and taf11−/− extracts (triplicate experiments, ∗∗∗p < 0.001. The p value was calculated by t test using GraphPad Prism). Error bars ± SEM. (D) The duplex siRNA-initiated RISC assay was performed using 20 μg taf11−/− ovary extract in the absence and presence of increasing concentrations of recombinant TAF11. (E) A native agarose gel shift assay was performed by incubating radiolabeled let-7 duplex siRNA in buffer alone (lane 1) or 40 μg of WT (lane 2), or taf11−/− (lanes 3 and 4) ovary extract. The asterisk refers to a non-specific shift that was not defined. (F) A 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, or 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 < 0.01, ∗∗∗p < 0.001). Error bars ± SEM. Molecular Cell 2015 59, 807-818DOI: (10.1016/j.molcel.2015.07.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 TAF11 Exists in the Nucleus/Cytoplasm and Associates with Core RNAi Machinery (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 (WB) with anti-Flag and anti-GFP antibodies. Stars in (B) and (C) refer to nonspecific bands in the blots. (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 co-IP 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 co-IP using anti-Flag antibody and western blotting with anti-Flag and anti-Myc antibodies. Molecular Cell 2015 59, 807-818DOI: (10.1016/j.molcel.2015.07.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 GFP-TAF11 Co-localizes with Dcr-2/R2D2 in Cytoplasmic D2 Bodies (A) Representative images showing the co-localization of GFP-TAF11 and RFP-Dcr-2 or RFP-R2D2 in cytoplasmic D2 bodies in S2 cells 96 hr 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, 2 μm. (C) A pie chart showing the percentage of transfected S2 cells (n = 30) and showing the localization of GFP-TAF11 in the nucleus (56.7%), D2 body (36.7%), or both (6.6%). (D) Representative images showing the localization of GFP-TAF11 and endogenous Dcr-2 in control siRNA-treated (siluc) or Dcr-2 siRNA-treated (siDcr-2) S2 cells. Molecular Cell 2015 59, 807-818DOI: (10.1016/j.molcel.2015.07.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 TAF11 Enhances Dcr-2-R2D2’s siRNA-Binding and RISC Loading Activities (A) The duplex siRNA-initiated RISC assay was performed with recombinant Dcr-2-R2D2 and Ago2 in the absence or presence of increasing concentrations of recombinant TAF11. The percentage of cleaved mRNA and fold enhancement are 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 the 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 < 0.01, ∗∗∗p < 0.001). Error bars ± SEM. (D) Native PAGE gel shift assays comparing the siRNA-binding activity of increasing concentrations of Dcr-2-R2D2 complex in the absence or presence of 1.5 μM TAF11. (E) Quantitative analysis of data in (D) showing the sigmoidal and hyperbolic siRNA binding curves of the Dcr-2-R2D2 complex in the absence and presence of TAF11, respectively. The intensities of free and bound siRNA were quantified by ImageJ software. The fraction of bound siRNA was expressed (triplicate experiments). The estimated KD values are listed above the graph. Error bars ± SEM. Molecular Cell 2015 59, 807-818DOI: (10.1016/j.molcel.2015.07.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 TAF11 Assembles the RLC by Facilitating Dcr-2-R2D2 Tetramerization (A) Native agarose/PAGE hybrid gel shift assays were performed by incubating radiolabeled let-7 duplex siRNA in buffer alone (lane 1), recombinant Dcr-2-R2D2 complex (lane 2), recombinant Dcr-2-R2D2 complex plus WT (lane 3) or A141E mutant (lane 4) TAF11 protein, or S2 extract (lane 5). Arrows mark the positions of free siRNA, the RDI complex, and the RLC. (B) Highly purified recombinant His6-TAF11 protein was resolved by SDS-PAGE or by blue native PAGE, followed by Coomassie blue staining. (C) Western blotting was performed to detect Dcr-2, R2D2, and TAF11 proteins among individual fractions with corresponding antibodies, following Superdex-200 chromatography of the recombinant Dcr-2-R2D2 complex (top), or a pre-incubated mixture of the recombinant Dcr-2-R2D2 complex with WT (center) or A141E mutant (bottom) TAF11. (D and E) Western blotting was performed to detect Dcr-2, R2D2, and TAF11 proteins, whereas northern blotting was used to detect let-7 siRNA among individual fractions following Superdex-200 chromatography of the recombinant RDI complex (D) and the RLC (E). (F) The recombinant RDI complex or the RLC were assembled by incubating radiolabeled duplex siRNA with the recombinant Dcr-2-R2D2 complex in the absence or presence of TAF11 and exposed to UV crosslinking before resolution by blue native PAGE. The gel was directly exposed to X-ray film. Molecular Cell 2015 59, 807-818DOI: (10.1016/j.molcel.2015.07.006) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 A Working Model for the Drosophila RISC Loading Complex It should be noted that, although our data (Figure 6C) suggest that TAF11 facilitates Dcr-2-R2D2 tetramerization in the absence of siRNA, the existence of this TAF114-(Dcr-2-R2D2)2 complex without siRNA needs more vigorous proof, such as structure determination by EM or X-ray crystallography. Molecular Cell 2015 59, 807-818DOI: (10.1016/j.molcel.2015.07.006) Copyright © 2015 Elsevier Inc. Terms and Conditions