Figure 1.

AGO7 specifically associates with miR390. (A) Scheme for the RISC assembly in BY-2 lysate. (B) The structures of miR390/miR390*, miR166/miR166* and miR171/miR171* duplexes. Red and black strands represent the guide and passenger strands, respectively. The 5′ end of the guide strand was radiolabelled with ³²P. (C) RISC assembly in BY-2 lysate. AGO7 specifically associates with miR390/miR390* duplex. In contrast, AGO1 interacts with miR166/miR166* and miR171/miR171* duplex both bearing 5′ U. ‘ds’ and ‘ss’ denote double-stranded miRNAs (pre-RISC) and single-stranded miRNAs (mature RISC), respectively. Western blotting of immunoprecipitated AGO proteins is shown at the bottom. (D) Hsp90 and Hsp70 inhibitors block both AGO1– and AGO7–RISC assembly. RISC was assembled in the presence of an Hsp70 inhibitor (PES), an Hsp90 inhibitor (GA) or DMSO (mock). AGO, ARGONAUTE; DMSO, dimethylsulphoxide; GA, geldanamycin; IP, immunoprecipitation; PES, phenylethynesulphonamide; RISC, RNA-induced silencing complex.

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