title: Monitoring of RNA Dynamics in Living Cells Using PUM-HD and Fluorescent Protein Reconstitution Technique.
abstract: Fluorescence live-cell RNA imaging to monitor the intracellular localization and dynamics of the target RNA is a challenging subject. One of the difficulties to achieve this is to establish a precise method to enable a fluorescent labeling to the target RNA in living cells. Technologies to reduce the background fluorescence and to detect the RNA with high sensitivity are also necessary to visualize and analyze the intracellular localization and dynamic of the target RNA precisely. Especially in monitoring single-molecule motion, a special setup of a microscope system is required. Such technical problems make the live-cell RNA imaging to be a difficult subject. We recently developed a methodology to label and to visualize a target RNA in living cells with low background fluorescence by using a probe that is based on an RNA-binding protein domain PUM-HD (pumilio homology domain) and a fluorescent protein reconstitution method. A noteworthy property of PUM-HD to apply RNA probes is that this protein domain can be modified to recognize a particular 8-base RNA sequence by inducing tailor-made designed mutagenesis. The fluorescent protein reconstitution method allows us to detect the target RNA with high signal-to-noise ratio. Using the probe based on PUM-HD, a fluorescent protein reconstitution method, and a homebuilt fluorescent microscope system, we succeeded in single-molecule observation of a target RNA in living cells. In this chapter, the techniques to establish the probe and to observe the motion of single-molecule RNA are described.
authors.affiliation: School of Science, The University of Tokyo, Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yoshimura
authors.affiliation: School of Science, The University of Tokyo, Tokyo, Japan. Electronic address: ozawa@chem.s.u-tokyo.ac.jp.
authors.forename: T
authors.initials: T
authors.lastname: Ozawa
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods; D061848:Optical Imaging / Q000379:methods*; D000072417:Protein Domains; D012313:RNA / Q000032:analysis* / Q000235:genetics; D015347:RNA Probes / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry*
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D015347:RNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012313:RNA
keywords: Fluorescence imaging; Live-cell imaging; PUM-HD; RNA; Single molecule; TIRF microscopy