Structural basis for chaperone-guided assembly of RNA-induced silencing complex

a, AGO2 pulldown experiments. Strep pulldown was performed for the indicated time periods and analysed by SDS-PAGE and Coomassie staining. HSP90 and p23 were identified by Western blot and HSP70 was identified by mass spectrometry. b, AGO2 pulldown experiment using HEK293T cells, analysed by SDS-PAGE and silver staining. Strep-Flag-AGO2 was pulled down using the Strep-tag and detected with anti-Flag antibody. HSP90 and p23 were identified by Western blot. c, AMC purification scheme. d, SEC analysis of purified AMCs containing different AGO paralogs. e, AMCs analysed by SDS-PAGE and Coomassie staining. f, RNA analysis of AMCs by urea-PAGE and SYBR Gold staining. Synthetic let-7a-1 duplex was loaded at an equimolar amount to AMCs. g, RNA analysis of high- and low-MW fractions collected in Fig. 1e by urea-PAGE and SYBR Gold staining. h, Three complementary assays for AGO folding. i, Limited proteolysis assay. AMC was incubated with varying amounts of thermolysin in the presence or absence of let-7a-1 duplex and analysed by SDS-PAGE and Coomassie staining. The assay was performed directly on the reaction mixture. j, Sedimentation assay. AMC was incubated with or without let-7a-1 duplex for the indicated times, then centrifuged to separate supernatant and pellet fractions, which were analysed by SDS-PAGE and Coomassie staining. k, SEC-based folding analysis with let-7a-1. Left, AMC was incubated with or without let-7a-1 duplex for the indicated times and analysed by SEC. Shaded areas indicate regions used for AUC calculation. Top right, AGO folding yield is calculated by dividing AUC (280 nm) of low-MW fraction by that of high-MW fraction. Bottom right, analysis of high- and low-MW fractions collected from SEC by SDS-PAGE and silver staining. Bars indicate mean ± s.e.m. (n = 3, independent experiments). P values were determined by two-sided Student’s t-test. All gel images are representative of two independent experiments.