title: Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes.
abstract: Single mRNA imaging in live cells is a useful technique to elucidate its precise localization and dynamics. We developed a method for visualizing endogenous mRNAs in living cells with single molecule sensitivity using genetically encoded probes. An RNA-binding protein of human PUMILIO1 (PUM-HD) was used for recognizing base sequences of a target mRNA, β-actin mRNA. Two PUM-HDs were modified by amino acid mutations to bind specifically to tandem 8-base sequences of the target mRNA. Because each PUM-HD was connected with amino- and carboxyl-terminal fragments of enhanced green fluorescent protein (EGFP), the probes emit fluorescence by reconstitution of EGFP fragments upon binding to β-actin mRNAs. The EGFP reconstituted on the mRNAs was monitored with a total internal reflection fluorescence microscope. Results show that each fluorescent spot in live cells represented a single β-actin mRNA and that distinct spatial and temporal movement of the individual β-actin mRNAs was visualized. We also estimated the average velocity of the movement of the single mRNAs along microtubules in live cells. This method is widely applicable to tracking various mRNAs of interest in the native state of living cells with single-mRNA sensitivity.
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Toshimichi
authors.initials: T
authors.lastname: Yamada
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yoshimura
authors.forename: Asumi
authors.initials: A
authors.lastname: Inaguma
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
mesh_terms: D000199:Actins / Q000235:genetics; D000818:Animals; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D041681:NIH 3T3 Cells; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; C471242:PUM1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins