Abstract
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.
Highlights
When cells are exposed to an assortment of environmental stresses, global translational arrest of housekeeping transcripts is accompanied by the formation of distinct cytoplasmic structures known as stress granules (SGs) and an increase in the number of pbodies (PBs) [1,2]
While SGs and PBs have been extensively studied from the perspective of their protein content and dynamics and progress has been made in understanding their role in translational repression, the study of native mRNA dynamics during translational inhibition has been limited by the difficulty with detecting native mRNA with single RNA sensitivity. mRNA localization within SGs and PBs during stress has been inferred using fluorescence microscopy mainly in three ways i) directly using in situ hybridization (FISH) with immunofluorescence, or ii) using plasmid-derived mRNA systems and iii) indirectly, by monitoring the behavior of known binding proteins such as TIAR/-1 or PABP [7]
The cells were fixed and various proteins were fluorescently-labeled using immunostaining. Both mRNA populations were found to be distributed in diffraction-limited spots or granules within the cytoplasm, clearly visible in comparison to background noise (Figure S1A, B); as previously shown, b-actin mRNA were abundant in the perinuclear region, in protrusions and along the edges of the cells (Figure 1B and S1B) [9], while poly A+ mRNAs appeared to be relatively more abundant in the perinuclear region (Figure 1A)
Summary
When cells are exposed to an assortment of environmental stresses, global translational arrest of housekeeping transcripts is accompanied by the formation of distinct cytoplasmic structures known as stress granules (SGs) and an increase in the number of pbodies (PBs) [1,2]. PBs consist of a core of proteins involved in mRNA repression and degradation, including the mRNA decapping machinery [5], as well as key effectors of microRNA (miRNA)-mediated RNA interference (RNAi), such as Argonaute-2 (Ago2), miRNAs, and their cognate mRNAs [6]. Given their protein content, these cytoplasmic foci are thought to represent key players in the regulation of translation. Other options for the study of native mRNA are required to gain both dynamic potential and single molecule sensitivity
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