title: m-TAG: a PCR-based genomic integration method to visualize the localization of specific endogenous mRNAs in vivo in yeast.
abstract: 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.
authors.affiliation: Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Liora
authors.initials: L
authors.lastname: Haim-Vilmovsky
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Gerst
mesh_terms: 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*
chemical_list: D012331:RNA, Fungal; D012333:RNA, Messenger
references.citation: Nat Struct Mol Biol. 2008 Dec;15(12):1263-71
references.pmid: 19011635
references.citation: Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):263-8
references.pmid: 16384914
references.citation: Nat Methods. 2007 May;4(5):413-9
references.pmid: 17401370
references.citation: Nature. 2003 Oct 16;425(6959):737-41
references.pmid: 14562106
references.citation: Biol Cell. 2005 Jan;97(1):75-86
references.pmid: 15601259
references.citation: Nat Methods. 2007 May;4(5):409-12
references.pmid: 17417645
references.citation: Annu Rev Biochem. 1998;67:335-94
references.pmid: 9759492
references.citation: PLoS Biol. 2007 Apr;5(4):e81
references.pmid: 17373856
references.citation: Nature. 2002 Jul 25;418(6896):387-91
references.pmid: 12140549
references.citation: Mol Cell. 1998 Oct;2(4):437-45
references.pmid: 9809065
references.citation: Curr Biol. 1999 Jun 3;9(11):569-78
references.pmid: 10359695
references.citation: Nat Methods. 2007 May;4(5):421-7
references.pmid: 17401371
references.citation: Nat Rev Mol Cell Biol. 2001 Apr;2(4):247-56
references.pmid: 11283722
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13308-13
references.pmid: 14583593
references.citation: Nature. 2003 Oct 16;425(6959):686-91
references.pmid: 14562095
references.citation: Cell. 2002 Feb 22;108(4):533-44
references.pmid: 11909524
references.citation: Trends Genet. 2002 Dec;18(12):636-42
references.pmid: 12446149
references.citation: Curr Biol. 1996 Dec 1;6(12):1599-608
references.pmid: 8994824