Monitoring the temporal and spatial distribution of RNA in living yeast cells.
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.
Long
Roy M
RM
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA.
Urbinati
Carl R
CR
D020413:3' Untranslated Regions; D003001:Cloning, Molecular; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017909:Levivirus / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014170:Transformation, Genetic; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D049452:Green Fluorescent Proteins