title: An efficient system for let-7 microRNA and GW182 protein-mediated deadenylation in vitro.
abstract: Experiments with cell cultures have been useful in analyzing microRNA action. However, miRNA-mediated effects are often assayed many hours or days after miRNA target recognition. Consequently, this has made it difficult to analyze early events of miRNA-mediated repression. The development of cell-free systems that recapitulate miRNA action in vitro has been instrumental in dissecting the molecular mechanisms of miRNA action. Here we describe such a system, derived from mouse Krebs II ascites carcinoma cells, termed Krebs cell-free system. As an example, the protocol for assaying let-7 and GW182 (TNRC6) protein-mediated deadenylation of mRNA in vitro is described.
authors.affiliation: Department of Biochemistry, Goodman Cancer Research Center, McGill University, Montreal, QC, Canada. marc.fabian@mail.mcgill.ca
authors.forename: Marc R
authors.initials: MR
authors.lastname: Fabian
authors.forename: Yuri V
authors.initials: YV
authors.lastname: Svitkin
authors.forename: Nahum
authors.initials: N
authors.lastname: Sonenberg
mesh_terms: D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D023061:Gene Order; D007553:Isotope Labeling; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger; D016601:RNA-Binding Proteins