Abstract
<jats:p>In the RNAi pathway, small RNAs processed by Dicer are loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC), which silences target genes. The intermediate complex responsible for this transfer is known as the RISC-loading complex (RLC). However, the molecular mechanism of this delivery process and the identity of all required components have remained elusive. Here we determined the cryo-EM structure of the RISC-loading complex in the Drosophila siRNA pathway. Our high-resolution structural analysis reveals that multiple domains, including the RIIIai of Dicer-2 and the dsRBDs of R2D2, play critical roles in loading the siRNA duplex into Ago2. The Hsp90 machinery reorganizes the spatial arrangement of Ago2 domains, maintaining Ago2 in an open conformation to receive the nascent siRNA duplex. Furthermore, this structure uncovers the mechanism of siRNA strand selection. During loading, the strand whose 5′ end is anchored in the Dicer-2 Platform-binding pocket is everted – a process facilitated by R2D2 – allowing its 5′ end to engage the binding pocket in the MID domain of Ago2. This strand becomes the guide strand in the mature RISC. </jats:p>