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Visualizing adenosine-to-inosine RNA editing in the Drosophila nervous system

Nature Methods volume 9, pages 189–194 (2012)Cite this article

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Abstract

Informational recoding by adenosine-to-inosine RNA editing diversifies neuronal proteomes by chemically modifying structured mRNAs. However, techniques for analyzing editing activity on substrates in defined neurons in vivo are lacking. Guided by comparative genomics, here we reverse-engineered a fluorescent reporter sensitive to Drosophila melanogaster adenosine deaminase that acts on RNA (dADAR) activity and alterations in dADAR autoregulation. Using this artificial dADAR substrate, we visualized variable patterns of RNA-editing activity in the Drosophila nervous system between individuals. Our results demonstrate the feasibility of structurally mimicking ADAR substrates as a method to regulate protein expression and, potentially, therapeutically repair mutant mRNAs. Our data suggest variable RNA editing as a credible molecular mechanism for mediating individual-to-individual variation in neuronal physiology and behavior.

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Figure 1: Molecular design of a fluorescent reporter of RNA editing.
Figure 2: GFPedit–derived fluorescence in Drosophila tissues.
Figure 3: Neuron-specific modulation of GFPedit–derived fluorescence by alterations in dADAR auto-editing.
Figure 4: Variability in dADAR activity between individual male Drosophila.
Figure 5: Nonstereotypical patterns of RNA editing in Drosophila neurons.

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Acknowledgements

We thank C. Lawrence and members of the Reenan laboratory for helpful discussions, C. Staber and G. Williams for expert technical assistance, and K. Koh (Thomas Jefferson University) for reagents. This work was funded by an Ellison Medical Foundation Senior Scholar award.

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Authors and Affiliations

  1. Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, USA

    James E C Jepson, Yiannis A Savva, Kyle A Jay & Robert A Reenan

  2. Department of Neuroscience, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    James E C Jepson

  3. Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA

    Kyle A Jay

Authors
  1. James E C Jepson
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  2. Yiannis A Savva
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  3. Kyle A Jay
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  4. Robert A Reenan
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Contributions

J.E.C.J. performed all imaging experiments and analyzed the data. Y.A.S. and J.E.C.J. performed RNA-editing analysis. R.A.R. designed the GFPedit reporter. K.A.J. and R.A.R. cloned the GFPedit and YFPsplice constructs and performed in vitro validation experiments. Y.A.S. and J.E.C.J. generated the recombinant dAdar alleles. J.E.C.J. and R.A.R. wrote the paper, with contributions from Y.A.S.

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Correspondence to Robert A Reenan.

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The authors declare no competing financial interests.

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Jepson, J., Savva, Y., Jay, K. et al. Visualizing adenosine-to-inosine RNA editing in the Drosophila nervous system. Nat Methods 9, 189–194 (2012). https://doi.org/10.1038/nmeth.1827

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