Using fluorescent proteins to study mRNA trafficking in living cells.
This chapter presents the MS2-GFP system, a method to study the trafficking of RNA molecules in living cells. This system is based on two components: a fusion of the MS2 coat protein to a fluorescent protein and a reporter mRNA containing multimers of the RNA stem-loop recognized by the MS2 coat protein. The MS2-GFP protein bound to the RNA stem-loops acts as a beacon that allows the detection of this mRNA within a cell by epifluorescence or confocal microscopy. This chapter focuses on the use of this system in mammalian fibroblast cells and in yeast cells, and discusses several technical considerations of the MS2-GFP system. Detailed protocols for validating the MS2-GFP signal in fixed cells by fluorescent in situ hybridization of the target RNA using fluorophore-labeled oligonucleotide probes are also provided.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D017909:Levivirus / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D034443:RNA Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure
D036022:Capsid Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins