Methods to analyze microRNA-mediated control of mRNA translation
Date
2007
Authors
Clancy, Jennifer
Nousch, Marco
Humphreys, David T
Westman, Belinda J
Beilharz, TH
Preiss, Thomas
Journal Title
Journal ISSN
Volume Title
Publisher
Academic Press
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.
Description
Keywords
Keywords: messenger RNA; microRNA; messenger RNA; microRNA; confocal microscopy; controlled study; genetic transfection; HeLa cell; human; human cell; internal ribosome entry site; plasmid; polymerase chain reaction; polysome; priority journal; review; ribonuclease
Citation
Collections
Source
Methods in Enzymology
Type
Journal article