title: Visualizing endogenous mRNAs in living yeast using m-TAG, a PCR-based RNA aptamer integration method, and fluorescence microscopy.
abstract: Localized mRNA translation is involved in cell-fate determination, polarization, and morphogenesis in eukaryotes. While various tools are available to examine mRNA localization, no easy and quick method has allowed for the visualization of endogenously expressed mRNAs in vivo. We describe a simple method (m-TAG) for PCR-based chromosomal gene tagging 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 of any yeast gene. Upon co-expression of MS2-CP fused with GFP, specific endogenously expressed mRNAs can be visualized in vivo for the first time. This method allows for the easy examination of mRNA localization using fluorescence microscopy and leaves the yeast cells amenable for further genetic analysis.
authors: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
authors: Liora
authors: L
authors: Haim-Vilmovsky
authors: Jeffrey E
authors: JE
authors: Gerst
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics*; D018914:Cells, Immobilized / Q000378:metabolism; D011232:Chemical Precipitation; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D019426:Integrases / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012331:RNA, Fungal / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D052157:Aptamers, Nucleotide; D015335:Molecular Probes; D012331:RNA, Fungal; D012333:RNA, Messenger; D004247:DNA; C045073:Cre recombinase; D019426:Integrases