Article ; Online: Purification and assembly of human Argonaute, Dicer, and TRBP complexes.
Methods in molecular biology (Clifton, N.J.)
2011 Volume 725, Page(s) 107–119
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- ... ...
| 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. |
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| MeSH term(s) | Argonaute Proteins ; Biological Assay ; Eukaryotic Initiation Factor-2/genetics ; Eukaryotic Initiation Factor-2/isolation & purification ; Eukaryotic Initiation Factor-2/metabolism ; Humans ; Isotope Labeling ; Oligonucleotides/metabolism ; RNA/metabolism ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/isolation & purification ; RNA-Binding Proteins/metabolism ; RNA-Induced Silencing Complex/isolation & purification ; RNA-Induced Silencing Complex/metabolism ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Recombinant Proteins/metabolism ; Ribonuclease III/genetics ; Ribonuclease III/isolation & purification ; Ribonuclease III/metabolism |
| Chemical Substances | AGO2 protein, human ; Argonaute Proteins ; Eukaryotic Initiation Factor-2 ; Oligonucleotides ; RNA-Binding Proteins ; RNA-Induced Silencing Complex ; Recombinant Proteins ; trans-activation responsive RNA-binding protein (136628-24-5) ; RNA (63231-63-0) ; Ribonuclease III (EC 3.1.26.3) |
| Language | English |
| Publishing date | 2011-04-29 |
| Publishing country | United States |
| Document type | Journal Article ; Research Support, N.I.H., Extramural |
| ISSN | 1940-6029 |
| ISSN (online) | 1940-6029 |
| DOI | 10.1007/978-1-61779-046-1_8 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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