Characterizing mRNA interactions with RNA granules during translation initiation inhibition

• Published in: PloS one 2011-05, Vol.6 (5), p.e19727
• Main Authors: Zurla, Chiara, Lifland, Aaron W, Santangelo, Philip J
• Format: Article
• Language: English
• Subjects: Actin; Actins - genetics; Actins - metabolism; Arsenic compounds; Arsenite; Arsenites - pharmacology; Biology; Biomedical engineering; Cell Line, Tumor; Cell lines; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Cell Survival - drug effects; Cycloheximide; Cycloheximide - pharmacology; Cytoplasm; Cytoplasmic Granules - drug effects; Cytoplasmic Granules - metabolism; DNA probes; Environmental stress; Enzymes; Epithelial cells; Epoxy Compounds - pharmacology; Exosomes - drug effects; Exosomes - metabolism; Fibroblasts; Gene expression; Granular materials; Humans; Hybridization; Immunofluorescence; Inhibition; Macrolides - pharmacology; Messenger RNA; Microscopy; Microtubule-Organizing Center - drug effects; Microtubule-Organizing Center - metabolism; Molecular Probes - metabolism; Muscle proteins; Nocodazole - pharmacology; Oxidative stress; Peptide Chain Initiation, Translational - drug effects; Physics; Poly A - metabolism; Proteins; Puromycin; Puromycin - pharmacology; Ribonucleic acid; RNA; RNA probes; RNA Transport - drug effects; RNA, Messenger - genetics; RNA, Messenger - metabolism; Saccharomyces cerevisiae; Sodium; Sodium arsenite; Sodium Compounds - pharmacology; Stress response; Stress, Physiological - drug effects; Thiazoles - pharmacology; Translation; Translation (Genetics); Translation initiation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0019727

When cells experience environmental stresses, global translational arrest is often accompanied by the formation of stress granules (SG) and an increase in the number of p-bodies (PBs), which are thought to play a crucial role in the regulation of eukaryotic gene expression through the control of mRNA translation and degradation. SGs and PBs have been extensively studied from the perspective of their protein content and dynamics but, to date, there have not been systematic studies on how they interact with native mRNA granules. Here, we demonstrate the use of live-cell hybridization assays with multiply-labeled tetravalent RNA imaging probes (MTRIPs) combined with immunofluorescence, as a tool to characterize the polyA+ and β-actin mRNA distributions within the cytoplasm of epithelial cell lines, and the changes in their colocalization with native RNA granules including SGs, PBs and the RNA exosome during the inhibition of translational initiation. Translation initiation inhibition was achieved via the induction of oxidative stress using sodium arsenite, as well as through the use of Pateamine A, puromycin and cycloheximide. This methodology represents a valuable tool for future studies of mRNA trafficking and regulation within living cells.