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In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. Here, we identify fly Cyclophilin 40 (Cyp4 as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC for