Assembly and function of RNA silencing complexes

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Abstract
RNA interference (RNAi) is a eukaryotic gene-regulatory pathway that silences the expression of specific genes in response to homologous double-stranded (ds)RNA. The RNA-induced silencing complex (RISC) assembles on short interfering (si)RNA fragments and cleaves target mRNAs that hybridize with the siRNA. In Drosophila melanogaster, the RNase-III enzyme Dicer2 (Dcr2) — which cleaves dsRNAs into siRNAs — is important for RISC assembly, even when pre-cleaved siRNAs are used to trigger silencing. Dcr2 and the associated dsRNA-binding protein R2D2 are components of a RISC-loading complex (RLC) that binds siRNAs and escorts them into RISC. RISC forms through an ATP-dependent assembly pathway that includes an siRNA-unwinding step. The relative thermodynamic stabilities of the ends of the siRNA duplex help to specify the strand that will ultimately assemble into RISC. RISC is a multiple-turnover, divalent-metal-ion-dependent enzyme that hydrolyses the target phosphodiester linkage, leaving 3′-hydroxyl and 5′-phosphate termini. All RISCs that have been characterized so far contain a member of the Argonaute (Ago) family of proteins, as defined by the presence of PAZ and PIWI domains. The PIWI domain has recently been shown to have structural homology to ribonuclease-H enzymes, which implicates it as the endonuclease that cleaves the target mRNA.