title: Purification and assembly of human Argonaute, Dicer, and TRBP complexes.
abstract: The RNA-induced silencing complex (RISC) is a programmable gene-silencing machine involved in many aspects of eukaryotic biology. In humans, RISC is programmed or "loaded" with a small-guide RNA by the action of a tri-molecular assembly termed the RISC-loading complex (RLC). The human RLC is composed of the proteins Dicer, TRBP, and Argonaute2 (Ago2). To facilitate structural and biochemical dissection of the RISC-loading process, we have developed a system for the in vitro reconstitution of the human RLC. Here, we describe in detail methods for the expression and purification of recombinant Dicer, TRBP, and Ago2 and protocols for the assembly of RLCs and RLC subcomplexes. We also describe several simple assays to observe the biochemical activities of the assembled protein complexes.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Nabanita
authors.initials: N
authors.lastname: De
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Macrae
mesh_terms: D060565:Argonaute Proteins; D001681:Biological Assay; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D009841:Oligonucleotides / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D009841:Oligonucleotides; D012313:RNA; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D011994:Recombinant Proteins; D043244:Ribonuclease III; D000099253:Trans-Activation Responsive RNA-Binding Protein; C466422:AGO2 protein, human