A genomic integration method to visualize localization of endogenous mRNAs in living yeast.
mRNA localization may be an important determinant for protein localization. We describe a simple PCR-based genomic-tagging strategy (m-TAG) that uses homologous recombination to insert binding sites for the RNA-binding MS2 coat protein (MS2-CP) between the coding region and 3' untranslated region (UTR) of any yeast gene. Upon coexpression of MS2-CP fused with GFP, we demonstrate the localization of endogenous mRNAs (ASH1, SRO7, PEX3 and OXA1) in living yeast (Saccharomyces cerevisiae).
Haim
Liora
L
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Zipor
Gadi
G
Aronov
Stella
S
Gerst
Jeffrey E
JE
D048868:Adaptor Proteins, Signal Transducing; D036022:Capsid Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D017909:Levivirus / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D000074401:Peroxins; D016133:Polymerase Chain Reaction / Q000379:methods*; D012331:RNA, Fungal / Q000032:analysis / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics*
C098733:ASH1 protein, S cerevisiae; D048868:Adaptor Proteins, Signal Transducing; D036022:Capsid Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; C070895:PEX3 protein, S cerevisiae; D000074401:Peroxins; D012331:RNA, Fungal; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C116430:SRO7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins