Imaging dynamics of endogenous mitochondrial RNA in single living cells.
We developed genetically encoded RNA probes for characterizing localization and dynamics of mitochondrial RNA (mtRNA) in single living cells. The probes consist of two RNA-binding domains of PUMILIO1, each connected with split fragments of a fluorescent protein capable of reconstituting upon binding to a target RNA. We designed the probes to specifically recognize a 16-base sequence of mtRNA encoding NADH dehydrogenase subunit 6 (ND6) and to be targeted into the mitochondrial matrix, which allowed real-time imaging of ND6 mtRNA localization in living cells. We showed that ND6 mtRNA is localized within mitochondria and concentrated particularly on mitochondrial DNA (mtDNA). Movement of the ND6 mtRNA is restricted but oxidative stress induces the mtRNA to disperse in the mitochondria and gradually decompose. These probes provide a means to study spatial and temporal mRNA dynamics in intracellular compartments in living mammalian cells.
Ozawa
Takeaki
T
Department of Chemistry, School of Science, The University of Tokyo, Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
Natori
Yutaka
Y
Sato
Moritoshi
M
Umezawa
Yoshio
Y
D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000494:pharmacology
D005456:Fluorescent Dyes; C471242:PUM1 protein, human; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012313:RNA; C507225:MT-ND6 protein, human; D009245:NADH Dehydrogenase