Volume 39, Issue 2, Pages 282-291 (July 2010) In Vitro Assembly of Plant RNA-Induced Silencing Complexes Facilitated by Molecular Chaperone HSP90  Taichiro Iki, Manabu Yoshikawa, Masaki Nishikiori, Mauren C. Jaudal, Eiko Matsumoto-Yokoyama, Ichiro Mitsuhara, Tetsuo Meshi, Masayuki Ishikawa  Molecular Cell  Volume 39, Issue 2, Pages 282-291 (July 2010) DOI: 10.1016/j.molcel.2010.05.014 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 39, 282-291DOI: (10.1016/j.molcel.2010.05.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 AGO1-mRNA Translation- and siRNA Duplex-Dependent Cleavage of a Target RNA in BYL (A) Structure of gf698 siRNAs and target RNAs (GF-s, GF-s-mut, and GF-as). The siRNAs gf698-A (guide strand) and -B (passenger strand) carried phosphate groups (P) at the 5′ termini and 2′-hydroxymethyl groups (Me) at the 3′-terminal nucleotides, and the gf698 siRNA duplex had 3′ overhangs of 2 nt. Target GF-s RNA and gf698-B siRNA had partial sequences of GFP mRNA, while the sequences of target GF-as and gf698-A siRNA were complementary to GFP mRNA. (B) AGO1-mRNA translation- and gf698 siRNA duplex-dependent cleavage of GF-s RNA in BYL. AGO1 mRNA was translated in BYL, incubated with the gf698 siRNA duplex, and then incubated with 10 nM 32P-labeled GF-s or GF-as target RNA for 5 min. The reaction products were analyzed with denaturing 5% PAGE. The right panel shows an immunoblot of translation products detected with anti-AGO1 antibodies. (C) Formation of AGO1-siRNA complexes in BYL. AGO1 or FLAG-AGO1 mRNA-translated BYL reaction mixtures were incubated with the gf698 siRNA duplexes, in which either gf698-A (guide strand: G) or gf698-B (passenger strand: P) was 32P labeled and analyzed with 4% native PAGE. In the lanes marked “FLAG-IP,” fractions that were immunopurified with anti-FLAG antibody from the samples after incubation with the siRNA duplexes were applied. The asterisk shows the slow band (see text) that corresponds to the AGO1-siRNA complex. (D) Formation of AGO1-siRNA-target RNA complexes. AGO1 mRNA-translated BYL reaction mixture was incubated with the siRNA duplex containing 32P-labeled gf698-A (guide strand), further incubated with 100 nM unlabeled GF-s or GF-s-mut (mut) target RNA for 10 min, and analyzed with 4% native PAGE. The asterisk and arrows show positions of the slow band and smeared bands with increased electrophoretic mobility that correspond to an AGO1-siRNA complex and AGO1-siRNA-target RNA complexes, respectively (see text). Quantification of band intensity suggested that approximately 20 nM of the AGO1-siRNA complex was formed. A similar preparation (e.g., Figure S1C) had the activity to cleave ∼20 fmol target RNA per microliter in 5 min. See Figure S1 for the information about Nicotiana tabacum AGO1 (Figures S1A and S1B), for the contribution of endogenous AGO1 protein in BYL to target RNA cleavage (Figure S1C), and for the specific cleavage of target RNA by the AGO1-siRNA complex (Figure S1D). Molecular Cell 2010 39, 282-291DOI: (10.1016/j.molcel.2010.05.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Effect of a Mutation in AGO1 that Affects Ribonuclease Activity on Passenger-Strand Removal (A) Effect of the D857A mutation in AGO1 on target cleavage. AGO1WT or AGO1D857A mRNA-translated BYL reaction mixtures were incubated with the gf698 siRNA duplex and further incubated with 10 nM 32P-labeled GF-s target for 5 min. RNA was purified from the mixture and analyzed with denaturing 5% PAGE (top panel). Bottom panel shows an immunoblot of the translation products with anti-AGO1 antibodies, confirming that AGO1WT and AGO1D857A were produced at similar levels. (B) Effect of the D857A mutation in AGO1 on binding of the AGO1-siRNA complex to GF-s target RNA. AGO1WT or AGO1D857A mRNA-translated BYL reaction mixtures were incubated with the gf698 siRNA duplex in which the guide strand was 32P labeled and further incubated with 100 nM unlabeled GF-s target RNA for 10 min, then analyzed with 4% native PAGE. The asterisk and arrow are as described in the legend to Figure 1D. (C) Effect of the D857A mutation in AGO1 on AGO1-siRNA complex formation. AGO1WT or AGO1D857A mRNA-translated BYL reaction mixtures were incubated with the gf698 siRNA duplexes in which either guide (G) or passenger (P) strand was 32P labeled and analyzed with 4% native PAGE. Exposure to the imaging plate was shorter for the detection of free siRNA duplexes (lower panels). The asterisk is as described in the legend to Figure 1C. (D) Effect of the D857A mutation in AGO1 on removal of passenger strand from the AGO1-siRNA complex. FLAG-AGO1WT or FLAG-AGO1D857A mRNA-translated BYL reaction mixtures were incubated with the gf698 siRNA duplexes in which either guide (G) or passenger (P) strand was 32P labeled, and RNA was extracted from the mixtures (input lanes). In the lanes marked “FLAG-IP,” samples were immunopurified with anti-FLAG antibody, followed by RNA extraction. RNA samples were analyzed with native 15% PAGE. See Figure S2 for the formation of miRISCs and the effect of the D857A mutation in AGO1 on removal of miRNA∗ strands. Molecular Cell 2010 39, 282-291DOI: (10.1016/j.molcel.2010.05.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Plant siRISC Formation Requires ATP and Endogenous Factors in BYL (A) Effect of ATP depletion before the addition of siRNA duplexes on target RNA cleavage. Small molecules were depleted from AGO1 mRNA-translated BYL reaction mixture by ultrafiltration, and ATP was supplemented at the concentration indicated in the figure. Then, the mixtures were incubated with the gf698 siRNA duplex and further incubated with 10 nM 32P-labeled GF-s target RNA for 5 min. RNA was purified from the mixtures and analyzed with denaturing 5% PAGE. (B) Effect of ATP depletion after incubation with siRNA duplexes on target RNA cleavage. AGO1 mRNA-translated BYL reaction mixture was incubated with the gf698 siRNA duplex, small molecules were depleted from the mixture by ultrafiltration, and ATP was supplemented at the concentration indicated in the figure. The mixtures were then incubated with 50 nM 32P-labeled GF-s target RNA for 15 min, and RNA was purified and analyzed as in (A). (C) Requirement of endogenous factors in BYL for AGO1- and siRNA duplex-dependent target RNA cleavage. The fraction that was immunopurified with anti-FLAG antibodies from the FLAG-AGO1 mRNA-translated BYL reaction mixture was mixed with an equal volume of TR buffer or fresh BYL and incubated with 100 nM ss gf698 siRNA guide strand or the gf698 siRNA duplex (ds) in the presence of 125 μM ATP, 12.5 mM creatine phosphate, 125 ng/μl creatine kinase, and 0.5 unit/μl RNasin Plus RNase Inhibitor (Promega; Madison, WI). The samples were then incubated with 10 nM 32P-labeled GF-s target RNA for 15 min. RNA was purified from the mixtures and analyzed with denaturing 5% PAGE. Molecular Cell 2010 39, 282-291DOI: (10.1016/j.molcel.2010.05.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Effect of an HSP90 Inhibitor, Geldanamycin, on Plant siRISC Formation (A) Effect of geldanamycin (GA) treatment before and after incubation with the siRNA duplex to AGO1 on target RNA cleavage. GA (20 μM) and 2% DMSO or 2% DMSO alone (control) were added to the AGO1 mRNA-translated BYL reaction mixture before or after incubation with the gf698 siRNA duplex. The mixtures were further incubated with 20 nM 32P-labeled GF-s target RNA for the periods indicated in the figure. Then, RNA was purified from the mixtures and analyzed with denaturing 5% PAGE. (B) Effect of GA on the formation of the AGO1-siRNA complex. AGO1WT or AGO1D857A mRNA-translated BYL reaction mixtures were incubated with 20 μM GA and 2% DMSO (+) or 2% DMSO alone (−) and the gf698 siRNA duplex containing 32P-labeled guide strand and analyzed with 4% native PAGE. Exposure to the imaging plate was shorter for the detection of free siRNA duplexes (lower panels). The asterisk is as described in the legend to Figure 1C. (C) Effect of GA on the stability of the AGO1 protein in BYL. After the translation of AGO1 mRNA in BYL, puromycin was added at a concentration of 2 μM to the reaction mixture to stop translation. The mixture was then incubated with 20 μM GA and 2% DMSO (+) or 2% DMSO alone (−) at 25°C for 1, 2, or 4 hr, and the AGO1 protein was detected by immunoblotting. See Figure S3 for the effect of GA on AGO1-miRNA complex formation (Figure S3A) and for the involvement of HSP70 in AGO1-siRNA complex formation (Figure S3B). Molecular Cell 2010 39, 282-291DOI: (10.1016/j.molcel.2010.05.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Analyses of the Roles of HSP90 in Plant siRISC Formation (A) Copurification of HSP90 with AGO1. FLAG-AGO1 mRNA-translated BYL reaction mixture was further incubated at 25°C for 30 min in the presence of additional 2 mM ATP and 3 mM MgCl2, 2 mM ATPγS and 3 mM MgCl2, 2% DMSO, or 20 μM GA and 2% DMSO, then immunopurified with anti-FLAG antibody. The purified samples were analyzed by immunoblotting with anti-HSP90 and anti-AGO1 antibodies. Samples before immunopurification were also analyzed in parallel (“input” panels). In the right panel, the incubation was performed in the presence of both ATPγS and GA (GA + γS) or in the presence of ATPγS (30 min) followed by GA addition (γS → GA). (B) Copurification of siRNA with AGO1. FLAG-AGO1 mRNA-translated BYL reaction mixture was further incubated with the gf698 siRNA duplex containing 32P-labeled guide strand at 25°C for 30 min or 120 min in the presence of additional 2 mM ATP and 3 mM MgCl2, 2 mM ATPγS and 3 mM MgCl2, 2% DMSO, or 20 μM GA and 2% DMSO, then immunopurified with anti-FLAG antibody. In the right panel, the incubation with the siRNA duplex was performed in the presence of both ATPγS and GA (GA + γS) or in the presence of ATPγS (30 min) followed by GA addition (γS → GA). Copurified RNA was analyzed with 15% native PAGE. (C) Copurification of the siRNA duplex with the AGO1-HSP90 complex. FLAG-AGO1 mRNA-translated BYL reaction mixture was further incubated with the gf698 siRNA duplex containing 32P-labeled guide strand at 25°C for 30 min or 120 min in the presence of additional 2 mM ATPγS and 3 mM MgCl2, then immunopurified with anti-FLAG antibody (1st-IP). The purified fraction was incubated with the antisera indicated above the lanes for 20 min and further incubated with protein G-conjugated magnetic beads (Invitrogen; Carlsbad, CA) for 10 min (2nd-IP), and the mixture was then separated to the supernatant fraction and bead-bound fraction. “IgG”: anti-tomato mosaic virus 130 kDa protein antibody (Hagiwara et al., 2003) used as a negative control. Copurified RNA was extracted and analyzed with 15% native PAGE. See Figure S4 for the effect of exogenous siRNA on the association of AGO1 with HSP90 (Figure S4A) and for the effect of ATP and ATPγS on AGO1 RNase activity (Figure S4B). Molecular Cell 2010 39, 282-291DOI: (10.1016/j.molcel.2010.05.014) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 A Possible Mechanism of Plant siRISC Formation An HSP90 dimer binds to AGO1, and ATP binding to the HSP90 dimer causes conformational change in HSP90 and AGO1 (step 1). This conformational change makes AGO1 competent for siRNA binding, and a small RNA duplex binds to AGO1 complexed with ATP-bound HSP90 dimer (step 2). The nucleotide hydrolysis by HSP90 causes dissociation of AGO1 from HSP90 and further conformational change in AGO1 (step 3). Such change results in proper positioning of small RNA duplexes to the endonucleolytic reaction center of the AGO1 protein, which facilitates passenger strand cleavage (step 4). Molecular Cell 2010 39, 282-291DOI: (10.1016/j.molcel.2010.05.014) Copyright © 2010 Elsevier Inc. Terms and Conditions