Volume 39, Issue 2, Pages 292-299 (July 2010) Hsc70/Hsp90 Chaperone Machinery Mediates ATP-Dependent RISC Loading of Small RNA Duplexes  Shintaro Iwasaki, Maki Kobayashi, Mayuko Yoda, Yuriko Sakaguchi, Susumu Katsuma, Tsutomu Suzuki, Yukihide Tomari  Molecular Cell  Volume 39, Issue 2, Pages 292-299 (July 2010) DOI: 10.1016/j.molcel.2010.05.015 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 39, 292-299DOI: (10.1016/j.molcel.2010.05.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 An Unknown ATP-Dependent Factor(s) Is Required for Fly Ago1- and Ago2-RISC Assembly (A) Experimental strategy for (B). (B) Immunopurified fly Ago1 or Ago2 can utilize small RNA duplex triggers for target cleavage only when RISC assembly was performed in the presence of S2 cell lysate. (C) Experimental strategy for (D). (D) Preincubation in the immunopurified Ago proteins with lysate in the ATP+ condition before RISC assembly in the ATP– condition did not rescue target cleavage. (E) Target cleavage activity was reconstituted only when immunopurified Ago2, recombinant Dcr-2/R2D2, and dcr-2;ago2 double mutant lysate were all present. (F and G) Immunoprecipitation of FLAG-tagged fly Ago1 (F) or Ago2 (G) with increasing concentrations of NaCl in the washing buffer. Both Ago proteins associated with a set of Hsc70/Hsp90 chaperone machinery. Also see Figure S1 for initial attempts to purify the RISC-loading factor(s). Molecular Cell 2010 39, 292-299DOI: (10.1016/j.molcel.2010.05.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Hsp70 and Hsp90 Inhibitors Block RISC Assembly, but Not Target Cleavage or RISC Turnover (A) Experimental strategy for (B–D). (B–D) Target cleavage by endogenous fly Ago1 (B), fly Ago2 (C), or human Ago2 (D). RISC was assembled in the presence of increasing concentrations of a Hsp70 inhibitor (PES), a negative control analog (MS), and a Hsp90 inhibitor (17-AAG). Figure S2A shows that PES blocks the interaction between fly Hsc70-4 and Ago1 or Ago2. The raw data for (B)–(D) are shown in Figures S2B–S2D. Figure S2E indicates that the inhibitors do not affect the protein levels of Ago1, Ago2, and their related proteins during the course of the experiments. (E) FLAG-tagged fly Ago1 or Ago2 was immunopurified and RISC was assembled in S2 cell lysate as in Figures 1A and 1B in the presence of 1 mM inhibitors. Fifty nanomolar of small RNA duplexes or 500 nM of single-stranded small RNAs were used as triggers. (F) Experimental strategy for (G)–(J). (G–I) Target cleavage by endogenous fly Ago1 (G), fly Ago2 (H), or human Ago2 (I). RISC was assembled in the absence of the inhibitors, and the target cleavage was monitored in the presence of increasing concentrations of the inhibitors. (J) Target cleavage assay by fly Ago2 was performed in a multiple turnover condition in the presence of 1 mM of the inhibitors. Molecular Cell 2010 39, 292-299DOI: (10.1016/j.molcel.2010.05.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Hsp70/90 Chaperone Machinery Is Required Specifically for RISC Loading of Small RNA Duplexes (A) Experimental strategy for (B), (C), and (G). (B and C) Native gel analyses for RISC loading of fly Ago1 (B) and human Ago2 (C). Pre-RISC was formed in the presence of 1 mM of the inhibitors at 15°C where unwinding is virtually blocked. Stars denote complexes unrelated to fly Ago1- or human Ago2-RISC assembly (Kawamata et al., 2009; Yoda et al., 2010). (D) Experimental strategy for (E) and (F). (E and F) Native gel analyses for unwinding within fly Ago1 (E) and human Ago2 (F). Pre-RISC was formed in the absence of the inhibitors and “chased” into mature RISC in the presence of 1 mM of the inhibitors. Stars denote complexes unrelated to RISC assembly of fly Ago1- or human Ago2-RISC (Kawamata et al., 2009; Yoda et al., 2010). (G) Native gel analysis for the formation of RLC for fly Ago2 and mature Ago2-RISC in the presence of 1 mM inhibitors. The identity of complex B, a putative precursor of RLC, is not well characterized. See also Figure S3, which shows that the inhibitors do not affect dicing in flies and humans. Molecular Cell 2010 39, 292-299DOI: (10.1016/j.molcel.2010.05.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Hsc70-4 Is an Important Factor for Fly Ago1- and Ago2-RISC Loading (A) FLAG-tagged fly Ago1 was immunopurified, RISC was assembled in S2 cell lysate supplemented with increasing concentrations of recombinant WT Hsc70-4 or its dominant-negative ATPase mutant (D206S) (see also Figure S4), and target cleavage was monitored. (B) Quantification of (A). (C) The same experiment as in (A) was performed for fly Ago2. (D) Quantification of (C). Molecular Cell 2010 39, 292-299DOI: (10.1016/j.molcel.2010.05.015) Copyright © 2010 Elsevier Inc. Terms and Conditions