title: TRICK: A Single-Molecule Method for Imaging the First Round of Translation in Living Cells and Animals.
abstract: The life of an mRNA is dynamic within a cell. The development of quantitative fluorescent microscopy techniques to image single molecules of RNA has allowed many aspects of the mRNA lifecycle to be directly observed in living cells. Recent advances in live-cell multicolor RNA imaging, however, have now made it possible to investigate RNA metabolism in greater detail. In this chapter, we present an overview of the design and implementation of the translating RNA imaging by coat protein knockoff RNA biosensor, which allows untranslated mRNAs to be distinguished from ones that have undergone a round of translation. The methods required for establishing this system in mammalian cell lines and Drosophila melanogaster oocytes are described here, but the principles may be applied to any experimental system.
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
authors.forename: J M
authors.initials: JM
authors.lastname: Halstead
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland; University of Basel, Basel, Switzerland.
authors.forename: J H
authors.initials: JH
authors.lastname: Wilbertz
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Wippich
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA, United States.
authors.forename: T
authors.initials: T
authors.lastname: Lionnet
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Ephrussi
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. Electronic address: jeffrey.chao@fmi.ch.
authors.forename: J A
authors.initials: JA
authors.lastname: Chao
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D036022:Capsid Proteins / Q000235:genetics; D002478:Cells, Cultured; D004331:Drosophila melanogaster / Q000166:cytology* / Q000235:genetics; D017909:Levivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D008164:Luminescent Proteins; D012333:RNA, Messenger
keywords: Fluorescence microscopy; Live-cell imaging; RNA imaging; Single-particle tracking; Translation