title: Fluorescent probes for imaging endogenous β-actin mRNA in living cells using fluorescent protein-tagged pumilio.
abstract: Subcellular localization and dynamics of mRNAs control various physiological functions in living cells. A novel technique for visualizing endogenous mRNAs in living cells is necessary for investigation of the spatiotemporal movement of mRNAs. A pumilio homology domain of human pumilio 1 (PUM-HD) is a useful RNA binding protein as a tool for mRNA recognition because the domain can be modified to bind a specific 8-base sequence of target mRNA. In this study, we designed PUM-HD to match the sequence of β-actin mRNA and developed an mRNA probe consisting of two PUM-HD mutants flanking full-length enhanced green fluorescent protein (EGFP). Fluorescence microscopy with the probe in living cells revealed that the probe was labeled precisely with the β-actin mRNA in cytosol. Fluorescent spots from the probe were colocalized with microtubules and moved directionally in living cells. The PUM-HD mutants conjugated with full-length EGFP can enable visualization of β-actin mRNA localization and dynamics in living cells.
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yoshimura
authors.forename: Asumi
authors.initials: A
authors.lastname: Inaguma
authors.forename: Toshimichi
authors.initials: T
authors.lastname: Yamada
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
mesh_terms: D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002470:Cell Survival; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D015335:Molecular Probes / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D015335:Molecular Probes; C471242:PUM1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins