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Targeted gene silencing by RNAi requires the RNA-induced silencing complex (RISC), whose core component is the protein Argonaute (Ago) bound to a microRNA (miRNA) or an siRNA. In humans, Ago2 is loaded with miRNAs by the action of a specialized assembly called the RISC-loading complex (RLC), comprising the proteins Ago2, Dicer, and TRBP. Here we show that the human RLC assembles spontaneously in vitro from purified components. No cofactors or chaperones are required for the complex to form. The reconstituted RLC, containing one copy of each protein, has the dicing, slicing, guide-strand selection, and Ago2-loading activities observed for the endogenous RLC. Furthermore, once Ago2 is loaded with an miRNA, it tends to dissociate from the rest of the complex. These results lay the groundwork for future structural and functional dissection of RISC loading in humans.

Original publication

DOI

10.1073/pnas.0710869105

Type

Journal article

Journal

Proc Natl Acad Sci U S A

Publication Date

15/01/2008

Volume

105

Pages

512 - 517

Keywords

Animals, Argonaute Proteins, Carboxypeptidases, Catalysis, Cell Line, DEAD-box RNA Helicases, Drosophila, Endoribonucleases, Eukaryotic Initiation Factor-2, Gene Silencing, Humans, Mass Spectrometry, MicroRNAs, Models, Biological, Protein Conformation, RNA, RNA Interference, Ribonuclease III