Hsp90 facilitates accurate pre-piRNA loading in vitro. (A) Experimental procedure for B. (B) Fifty-nucleotide 5′-radiolabeled RNAs with 5′ U, A, G, or C were incubated in lysates prepared from wild-type BmN4 cells (naïve) or Flag-Siwi- or Flag-BmAgo3-expressing cells in the presence or absence of 1 mM 17-AAG. After immunoprecipitation with anti-Flag antibody, Siwi- or BmAgo3-bound RNAs were detected. (C) Quantification for B. The level of Siwi- or BmAgo3-bound 1U-50 RNA in DMSO control was normalized to 1. The average ± standard deviations from three independent trials are shown. 17-AAG abolished the 1U preference of Siwi, while promoting promiscuous incorporation into BmAgo3. (D) Experimental procedure for E. (E) 1U-50 RNA loaded into Flag-Siwi was subjected to the trimming assay in the presence or absence of 1 mM 17-AAG. The 27- to 28-nt trimming products were generated even in the presence of 17-AAG and were resistant to NaIO4-mediated oxidization followed by β-elimination, which is indicative of 2′-O-methylation. As a control, addition of 1 mM S-adenosyl homocysteine (SAH) inhibited 2′-O-methylation of the trimming product, which resulted in its faster gel migration after NaIO4/β-elimination. (F) A function of Hsp90 in the primary piRNA pathway. Hsp90 is required for accurate loading of pre-piRNAs into Siwi, but not for subsequent 3′-end trimming and 2′-O-methylation.
