Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America.
PLoS One. 2011 May 5;6(5):e19727. 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.
当细胞经历环境压力时，全局翻译暂停通常伴随着应激颗粒 (SG) 的形成和 p 体 (PB) 的数量增加，这些颗粒被认为通过控制 mRNA 翻译和降解在真核基因表达的调控中发挥关键作用。从蛋白质含量和动力学的角度来看，SG 和 PB 已经得到了广泛的研究，但迄今为止，还没有关于它们如何与天然 mRNA 颗粒相互作用的系统研究。在这里，我们展示了使用多标记四价 RNA 成像探针 (MTRIPs) 结合免疫荧光的活细胞杂交测定作为一种工具，用于表征上皮细胞系细胞质中多 A+和β-肌动蛋白 mRNA 的分布，以及它们与天然 RNA 颗粒（包括 SG、PB 和 RNA 外切酶）的共定位变化在翻译起始抑制期间。通过使用亚砷酸钠诱导氧化应激以及使用 Pateamine A、嘌呤霉素和环己酰亚胺来实现翻译起始的抑制。该方法代表了未来在活细胞中研究 mRNA 运输和调控的有价值的工具。
