A genomic integration method for the simultaneous visualization of endogenous mRNAs and their translation products in living yeast.
Protein localization within cells can be achieved by the targeting and localized translation of mRNA. Yet, our understanding of the dynamics of mRNA targeting and protein localization, and of how general this phenomenon is, is not clear. Plasmid-based expression systems have been used to visualize exogenously expressed mRNAs and proteins; however, these methods typically produce them at levels greater than endogenous and can result in mislocalization. Hence, a method that allows for the simultaneous visualization of endogenous mRNAs and their translation products in living cells is needed. We previously developed a method (m-TAG) to localize endogenously expressed mRNAs in yeast by chromosomal insertion of the MS2 aptamer sequence between the open-reading frame (ORF) and 3' UTR of any gene. Upon coexpression with the MS2 RNA-binding coat protein (MS2-CP) fused with GFP, the aptamer-tagged mRNAs bearing their 3' UTRs are localized using fluorescence microscopy. Here we describe an advanced method (mp-TAG) that allows for the simultaneous visualization of both endogenously expressed mRNAs and their translation products in living yeast for the first time. Homologous recombination is used to insert the mCherry gene and MS2-CP binding sites downstream from any ORF, in order to localize protein and mRNA, respectively. As proof of the concept, we tagged ATP2 as a representative gene and demonstrated that endogenous ATP2 mRNA and protein localize to mitochondria, as shown previously. In addition, we demonstrate that tagged proteins like Hhf2, Vph1, and Yef3 localize to their expected subcellular location, while the localization of their mRNAs is revealed for the first time.
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Liora
L
Haim-Vilmovsky
Noga
N
Gadir
Rebecca H
RH
Herbst
Jeffrey E
JE
Gerst
D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D016681:Genome, Fungal; D006657:Histones / Q000235:genetics / Q000378:metabolism; D059765:Homologous Recombination; D042541:Intracellular Space / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D034443:RNA Transport; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D006657:Histones; D008565:Membrane Proteins; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D006180:Proton-Translocating ATPases