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In vitro reconstitution of the human RISC-loading complex.

ABSTRACT: 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.

SUBMITTER: MacRae IJ

PROVIDER: S-EPMC2206567 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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In vitro reconstitution of the human RISC-loading complex.

MacRae Ian J IJ Ma Enbo E Zhou Min M Robinson Carol V CV Doudna Jennifer A JA

Proceedings of the National Academy of Sciences of the United States of America 20080104 2

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 ...[more]

PMID: 18178619

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