title: Methods to analyze microRNA-mediated control of mRNA translation.
abstract: MicroRNAs (miRs) are an important class of gene regulators that affect a wide range of biological processes. Despite the early recognition of miRs as translational regulators and intense interest in studying this phenomenon, it has so far not been possible to derive a consensus model for the underlying molecular mechanism(s). The potential of miRs to act in a combinatorial manner and to also promote mRNA decay creates conceptual and technical challenges in their study. Here, we discuss critical parameters in design and analysis of experiments used to study miR function including creation of synthetic miR and mRNA partners for assay of translational inhibition using luciferase reporters; measurement of mRNA stability after miR action; defining poly(A) tail length in miR target mRNA; determining the distribution of miRs and their target mRNAs in polysome profiles; and visualization of P-body components. We describe protocols for each of these procedures.
authors.affiliation: Molecular Genetics Program, Victor Chang Cardiac Research Institute (VCCRI), Sydney, Australia.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Clancy
authors.forename: Marco
authors.initials: M
authors.lastname: Nousch
authors.forename: David T
authors.initials: DT
authors.lastname: Humphreys
authors.forename: Belinda J
authors.initials: BJ
authors.lastname: Westman
authors.forename: Traude H
authors.initials: TH
authors.lastname: Beilharz
authors.forename: Thomas
authors.initials: T
authors.lastname: Preiss
mesh_terms: D000818:Animals; D001665:Binding Sites; D019411:Clinical Laboratory Techniques; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000032:analysis / Q000502:physiology*; D010957:Plasmids; D026723:Polyadenylation; D011132:Polyribosomes / Q000737:chemistry; D014176:Protein Biosynthesis; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D014162:Transfection
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger