title: Detection of human Dicer and Argonaute 2 catalytic activity.
abstract: The microRNA (miRNA)-guided RNA silencing pathway is a central and well-defined cellular process involved in messenger RNA (mRNA) translational control. This complex regulatory process is achieved by a well orchestrated machinery composed of a relatively few protein components, among which the ribonuclease III (RNase III) Dicer and Argonaute 2 (Ago2) play a central role. These two proteins are essential and it is of particular interest to measure and detect their catalytic activity under various situations and/or conditions. In this chapter, we describe different protocols that aim to study and determine the catalytic activity of Dicer and Ago2 in cell extracts, immune complexes, and size-fractionated cell extracts. Another protocol aimed at assessing miRNA binding to Ago2 is also described. These experimental approaches are likely to be useful to researchers investigating the main steps of miRNA biogenesis and function in human health and diseases.
authors.affiliation: CHUL Research Center/CHUQ and Faculty of Medicine, Université Laval, Quebec, QC, Canada.
authors.forename: Marjorie P
authors.initials: MP
authors.lastname: Perron
authors.forename: Patricia
authors.initials: P
authors.lastname: Landry
authors.forename: Isabelle
authors.initials: I
authors.lastname: Plante
authors.forename: Patrick
authors.initials: P
authors.lastname: Provost
mesh_terms: D060565:Argonaute Proteins; D015152:Blotting, Northern; D002384:Catalysis; D002460:Cell Line; D058645:Denaturing Gradient Gel Electrophoresis; D057075:Enzyme Assays; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007553:Isotope Labeling; D035683:MicroRNAs / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D012313:RNA; D043244:Ribonuclease III