m-TAG: a PCR-based genomic integration method to visualize the localization of specific endogenous mRNAs in vivo in yeast.
This protocol describes m-TAG, a novel method for the visualization of endogenously expressed mRNAs in live yeast (Saccharomyces cerevisiae). First, a gene of interest is tagged with multiple binding sites for the RNA-binding MS2 coat protein (MS2-CP), using a PCR-based genomic-tagging strategy and homologous recombination. Next, MS2-CP fused to GFP(x3) is expressed in cells; binding of this fusion protein to the tagged mRNA yields an RNA granule that can be visualized by fluorescence microscopy. While existing methods necessitate cell fixation (for in situ hybridization) or the detection of exogenously expressed mRNAs (from plasmids), or give transient signals (i.e., with fluorescent hybridization probes), m-TAG allows for the robust and stable visualization of endogenously expressed mRNAs in vivo and facilitates the study of mRNA dynamics under different growth conditions. The m-TAG procedure is simple, easy to perform and takes <3 weeks to yield cultured yeast strains for mRNA visualization.
Haim-Vilmovsky
Liora
L
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Gerst
Jeffrey E
JE
D016681:Genome, Fungal; D016133:Polymerase Chain Reaction / Q000379:methods*; D012331:RNA, Fungal / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics*
D012331:RNA, Fungal; D012333:RNA, Messenger
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