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Published on: June 4, 2020
Imaging dynamics of endogenous mitochondrial RNA in single living cells
Takeaki Ozawa1, Yutaka Natori, Moritoshi Sato
1Department of Chemistry, School of Science, The University of Tokyo, Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
Abstract:
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.
Insights
Researchers created RNA probes to track mitochondrial RNA (mtRNA) in living cells. These probes reveal ND6 mtRNA localizes to mitochondria and disperses under stress, offering new insights into cellular RNA dynamics.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Mitochondrial RNA (mtRNA) plays crucial roles in cellular energy production.
- Understanding the spatial and temporal dynamics of mtRNA within living cells is essential but challenging.
- Existing methods lack the resolution to observe mtRNA localization and movement in real-time.
Purpose of the Study:
- To develop and validate novel genetically encoded RNA probes for visualizing mitochondrial RNA in living cells.
- To investigate the localization and dynamics of NADH dehydrogenase subunit 6 (ND6) mtRNA within the mitochondrial matrix.
- To observe the effects of oxidative stress on ND6 mtRNA distribution and stability.
Main Methods:
- Genetically encoded RNA probes were constructed using RNA-binding domains of PUMILIO1 and split fluorescent proteins.
- Probes were designed to target a specific 16-base sequence of ND6 mtRNA within the mitochondrial matrix.
- Real-time imaging techniques were employed to monitor ND6 mtRNA localization and movement in living mammalian cells under normal and stressed conditions.
Main Results:
- The developed probes successfully visualized ND6 mtRNA localization within mitochondria, with concentration observed near mitochondrial DNA (mtDNA).
- ND6 mtRNA exhibited restricted movement under normal conditions.
- Oxidative stress induced dispersion of ND6 mtRNA within mitochondria and subsequent decomposition.
Conclusions:
- Genetically encoded RNA probes offer a powerful tool for studying mtRNA localization and dynamics in real-time within living cells.
- ND6 mtRNA exhibits dynamic behavior influenced by cellular conditions like oxidative stress.
- This technology opens new avenues for investigating intracellular RNA biology in various cellular compartments.
