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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

References

Biology

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Published: December 17th, 2012

DOI:

10.3791/50066

1Department of Biochemistry, University of Toronto

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

In eukaryotes, mRNA encoding secreted and membrane proteins can be targeted to the ER co-translationally by the signal recognition particle1,2 and can be maintained on the ER via the direct interactions between ribosomes and translocons during the translation3,4. However, whether mRNAs can be targeted and maintained on the ER independent of either ribosomes or translation was unclear until very recently. Previous studies attempted to address whether there is translation-independent mRNA association with the ER using cellular fractionation techniques. Because harsh chemical conditions were required to disassociate ribosomes from ER derived vesicle....

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1. Preparation of Materials for Extraction

  1. Preparation of Cells
    1. Seed tissue culture cells on acid-treated coverslips for at least one day prior to the experiment. We use COS-7 or U2OS, as these two cell lines exhibit robust production of secreted protein and have a well defined ER. Note that to achieve high extraction efficiency, cells should not exceed 80% confluency on the coverslip on the day of the experiment.
    2. If a particular exogenous mRNA is being investigated, cells are transfect.......

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To determine the percentage of mRNA that is ER-associated, COS-7 cells that were transfected with plasmids that contained the placental alkaline phosphatase (ALPP) or cytochrome P450-8B1 (CYP8B1) were either extracted with digitonin and then fixed, or directly fixed (see Figure 1, compare "Unextracted Ctrl" to "Extracted Ctrl"). The non-nuclear fluorescence was quantified in both samples and the fraction of ER-associated mRNA was calculated (Figure 2)........

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The localization of mRNAs to various subcellular sites, through the interaction of transcripts with mRNA localization proteins, is a widespread phenomenon important for the proper sorting of proteins to their final destination, and for the fine tuning of gene expression to the local requirements of a subcellular region19,20.

Using the assay described here, we revisited the question of whether mRNAs that encode secretory proteins can be maintained on the ER in the presence or absence.......

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This work was supported by grants from the National Science and Engineering Research Council of Canada to X.A.C. and A.F.P.

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