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Imaging dynamics of endogenous mitochondrial RNA in single living cells

Nature Methods volume 4, pages 413–419 (2007)Cite this article

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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.

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Figure 1: Detection of a target mRNA based on complementation of split EGFP fragments.
Figure 2: Selective binding of the probes to a target mRNA in mitochondria.
Figure 3: Visualization of MT-ND6 mtRNA, mtDNA and mitochondria in single living cells.
Figure 4: FRAP of MT-ND6 mtRNA and mtDNA probes.
Figure 5: Effects of a reactive oxygen species on the dynamics of MT-ND6 mtRNA in mitochondria.

Change history

  • 04 April 2007

    In the version of this article initially published online, the wording in the third sentence of the introduction was unclear. The error has been corrected for all versions of the article.

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Acknowledgements

This work was supported by Japan Society for the Promotion of Science (JSPS) and Japan Science and Technology Corporation (JST), and by grants from New Energy, Industrial Technology Development Organization (NEDO), Asahi Glass and Uehara Memorial Foundations.

Author information

Authors and Affiliations

  1. Department of Chemistry, School of Science, The University of Tokyo, Hongo Bunkyo-ku, 113-0033, Tokyo, Japan

    Takeaki Ozawa, Yutaka Natori, Moritoshi Sato & Yoshio Umezawa

  2. Japan Science and Technology Corporation, Tokyo, Japan

    Takeaki Ozawa, Yutaka Natori, Moritoshi Sato & Yoshio Umezawa

  3. Department of Molecular Structure, Institute for Molecular Science, 38 Nishigonaka, Myodaiji, 444-8585, Okazaki, Aichi

    Takeaki Ozawa

  4. PREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan

    Takeaki Ozawa

Authors
  1. Takeaki Ozawa
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  2. Yutaka Natori
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  3. Moritoshi Sato
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  4. Yoshio Umezawa
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Contributions

T.O. and Y.N., experimental design, performance of experiments, data analysis and manuscript preparation; M.S., EMSA experiment and data analysis; Y.U., design and principle of the project, and manuscript preparation.

Corresponding author

Correspondence to Yoshio Umezawa.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Structure of PUM-HD protein. (PDF 104 kb)

Supplementary Fig. 2

Schematic structures of major constructs. (PDF 235 kb)

Supplementary Fig. 3

Localization of mPUM1-GN and mPUM2-GC. (PDF 1361 kb)

Supplementary Fig. 4

Chromatography of purified mPUM1 and mPUM2 on a Superdex 200 column. (PDF 278 kb)

Supplementary Video 1

Time-lapse images of HeLa cells expressing mPUM1-GN and mPUM2-GC. (AVI 674 kb)

Supplementary Video 2

Time-lapse images of mtDNA in HeLa cells. (AVI 674 kb)

Supplementary Video 3

Time-lapse images of HeLa cells expressing MTS-EGFP (green). (AVI 674 kb)

Supplementary Video 4

Time-lapse images of HeLa cells expressing mPUM1-GN and mPUM2-GC. (AVI 674 kb)

Supplementary Video 5

Time-lapse images of mtDNA in HeLa cells Upon H2O2 stimulus. (AVI 674 kb)

Supplementary Methods (PDF 77 kb)

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Ozawa, T., Natori, Y., Sato, M. et al. Imaging dynamics of endogenous mitochondrial RNA in single living cells. Nat Methods 4, 413–419 (2007). https://doi.org/10.1038/nmeth1030

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