title: Argonaute pull-down and RISC analysis using 2'-O-methylated oligonucleotides affinity matrices.
abstract: During the last decade, several novel small non-coding RNA pathways have been unveiled, which reach out to many biological processes. Common to all these pathways is the binding of a small RNA molecule to a protein member of the Argonaute family, which forms a minimal core complex called the RNA-induced silencing complex or RISC. The RISC targets mRNAs in a sequence-specific manner, either to induce mRNA cleavage through the intrinsic activity of the Argonaute protein or to abrogate protein synthesis by a mechanism that is still under investigation. We describe here, in details, a method for the affinity chromatography of the let-7 RISC starting from extracts of the nematode Caenorhabditis elegans. Our method exploits the sequence specificity of the RISC and makes use of biotinylated and 2'-O-methylated oligonucleotides to trap and pull-down small RNAs and their associated proteins. Importantly, this technique may easily be adapted to target other small RNAs expressed in different cell types or model organisms. This method provides a useful strategy to identify the proteins associated with the RISC, and hence gain insight in the functions of small RNAs.
authors.affiliation: Laval University Cancer Research Centre, Hôtel-Dieu de Québec (CHUQ), Quebec City, QC, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Jannot
authors.forename: Alejandro
authors.initials: A
authors.lastname: Vasquez-Rifo
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Simard
mesh_terms: D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D008745:Methylation; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*
chemical_list: C558782:ALG-1 protein, C elegans; D029742:Caenorhabditis elegans Proteins; C531976:Let-7 microRNA, Drosophila; D035683:MicroRNAs; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex