Monitoring the Temporal and Spatial Distribution of RNA in Living Yeast Cells
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RNA localization is a cellular process to spatially restrict translation of specific proteins to defined regions within or
between cells. Most localized mRNAs contain cis-acting localization elements in the 3′-untranslated region (UTR), which are sufficient for localization of an mRNA to a particular
region of the cell. The cis-acting localization elements serve as assembly sites for trans-acting factors which function to sort the mRNA to the correct sub-cellular destination. Although fluorescent in situ hybridization
(FISH) has been widely used to study mRNA localization, FISH has a weakness in that it is a static assay, as FISH requires
that cells be fixed before hybridization. Consequently, FISH is not ideally suited for investigating dynamic mRNA localization
processes. This limitation of FISH has been overcome by the development of techniques that allow the visualization of mRNA
in living cells. Here, we present a protocol that tethers green fluorescent protein (GFP) to an mRNA of interest, allowing
for the visualization of dynamic mRNA localization processes in living cells.
Affiliation(s): (3) Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA
(4) Department of Biology, Loyola Marymount University, Los Angeles, CA, USA
(4) Department of Biology, Loyola Marymount University, Los Angeles, CA, USA
Book Title: Post-Transcriptional Gene Regulation
Series: Methods in Molecular Biology | Volume: 419 | Pub. Date: Dec-21-2007 | Page Range: 187-196 | DOI: 10.1007/978-1-59745-033-1_13
Subject: Genetics/Genomics
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