In vitro reconstitution of the human RISC-loading complex.
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.
MacRae
Ian J
IJ
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. macrae@scripps.edu
Ma
Enbo
E
Zhou
Min
M
Robinson
Carol V
CV
Doudna
Jennifer A
JA
D000818:Animals; D060565:Argonaute Proteins; D002268:Carboxypeptidases / Q000737:chemistry*; D002384:Catalysis; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D004330:Drosophila; D004722:Endoribonucleases / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D035683:MicroRNAs / Q000378:metabolism; D008954:Models, Biological; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D034622:RNA Interference; D043244:Ribonuclease III
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D012313:RNA; D004722:Endoribonucleases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D002268:Carboxypeptidases; C436327:SCPEP1 protein, human; D053487:DEAD-box RNA Helicases