Abstract
Evidence for the chemical conversion of adenosine-to-inosine (A-to-I) in messenger RNA (mRNA) has been detected in numerous metazoans, especially those “most successful” phyla: Arthropoda, Mollusca, and Chordata. The requisite enzymes for A-to-I editing, ADARs (adenosine deaminases acting on RNA) are highly conserved and are present in every higher metazoan genome sequenced to date. The fruit fly, Drosophila melanogaster, represents an ideal model organism for studying A-to-I editing, both in terms of fundamental biochemistry and in relation to determining adaptive downstream effects on physiology and behavior. The Drosophila genome contains a single structural gene for ADAR (dAdar), yet the fruit fly transcriptome has the widest range of conserved and validated ADAR targets in coding mRNAs of any known organism. In addition, many of the genes targeted by dADAR have been genetically identified as playing a role in nervous system function, providing a rich source of material to investigate the biological relevance of this intriguing process. Here, we discuss how recent advances in the use of ends-out homologous recombination (HR) in Drosophila make possible both the precise control of the editing status for defined adenosine residues and the engineering of flies with globally altered RNA editing of the fly transcriptome. These new approaches promise to significantly improve our understanding of how mRNA modification contributes to insect physiology and ethology.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Higuchi, M., Maas, S., Single, F. N., Hartner, J., Rozov, A., Burnashev, N., Feldmeyer, D., Sprengel, R., and Seeburg, P. H. (2000) Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406, 78–81
Wang, Q., Khillan, J., Gadue, P., and Nishikura, K. (2000) Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis. Science 290, 1765–1768
Palladino, M. J., Keegan, L. P., O’Connell, M. A., and Reenan, R. A. (2000) A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity. Cell 102, 437–449
Tonkin, L. A., Saccomanno, L., Morse, D. P., Brodigan, T., Krause, M., and Bass, B. L. (2002) RNA editing by ADARs is important for normal behavior in Caenorhabditis elegans. EMBO J 21, 6025–6035
Bass, B. L. (2002) RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846
Gallo, A., Keegan, L. P., Ring, G. M., and O’Connell, M. A. (2003) An ADAR that edits transcripts encoding ion channel subunits functions as a dimer. EMBO J 22, 3421–3430
Basillo C, Wahba A. J, Lengyel P, Speyer J. F., and Ochoa S. (1962) Synthetic polynucleotides and the amino acid code. V. PNAS 48, 613–616
Hanrahan, C. J., Palladino, M. J., Ganetzky, B., and Reenan, R. A. (2000) RNA editing of the Drosophila para Na+ channel transcript. Evolutionary conservation and developmental regulation. Genetics 155, 1149–1160
Reenan, R. A. (2005) Molecular determinants and guided evolution of species-specific RNA editing. Nature 434, 409–413
Hoopengardner, B., Bhalla, T., Staber, C., and Reenan, R. (2003) Nervous system targets of RNA editing identified by comparative genomics. Science 301, 832–836
Grauso, M., Reenan, R. A., Culetto, E., and Sattelle, D. B. (2002) Novel putative nicotinic acetylcholine receptor subunit genes, Dalpha5, Dalpha6 and Dalpha7, in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing. Genetics 160, 1519–1533
Smith, L. A., Peixoto, A. A., and Hall, J. C. (1998) RNA editing in the Drosophila DMCA1A calcium-channel alpha 1 subunit transcript. J Neurogenet. 12, 227–240
Semenov, E. P., and Pak, W. L. (1999) Diversification of Drosophila chloride channel gene by multiple post-transcriptional mRNA modifications. J Neurochem 72, 66–72
Hanrahan, C. J., Palladino, M. J., Bonneau, L. J., and Reenan, R. A. (1999) RNA editing of a Drosophila sodium channel gene. Ann N Y Acad Sci 868, 51–66
Bhalla, T., Rosenthal, J. J., Holmgren, M., and Reenan, R. (2004) Control of human potassium channel inactivation by editing of a small mRNA hairpin. Nat Struct Mol Biol 11, 950–956
Ingleby, L., Maloney, R., Jepson, J., Horn, R., and Reenan, R. (2009) Regulated RNA editing and functional epistasis in Shaker potassium channels. J Gen Phys 133, 17–27
Rong, Y. S., and Golic, K. G. (2000) Gene targeting by homologous recombination in Drosophila. Science 288, 2013–2018
Rong, Y. S., Titen, S. W., Xie, H. B., Golic, M. M., Bastiani, M., Bandyopadhyay, P., Olivera, B. M., Brodsky, M., Rubin, G. M., and Golic, K. G. (2002) Targeted mutagenesis by homologous recombination in D. melanogaster. Genes Dev 16, 1568–1581
Wong, S. K., Sato, S., and Lazinski, D. W. (2001) Substrate recognition by ADAR1 and ADAR2. RNA 7, 846–858
Keegan, L. P., Brindle, J., Gallo, A., Leroy, A., Reenan, R. A., and O’Connell, M. A. (2005) Tuning of RNA editing by ADAR is required in Drosophila. EMBO J 24, 2183–2193
Palladino, M. J., Keegan, L. P., O’Connell, M. A., and Reenan, R. A. (2000) dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing. RNA 6, 1004–1018
Jones, A. K., Buckingham, S. D., Papadaki, M., Yokota, M., Sattelle, B. M., Matsuda, K., and Sattelle, D. B. (2009) Splice-variant- and stage-specific RNA editing of the Drosophila GABA receptor modulates agonist potency. J Neurosci 29, 4287–4292
Ashburner, M. (1989) Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory, New York
Staber C. and Mann R. Quickly and Easily Isolate Genomic DNA from Drosophila With No Preprocessing Using the Maxwell(r) 16 Instrument. Promega Corporation Web site. http://www.promega.com/pubs/tpub_017.htm Updated February 2010. New York
Greenspan, R. J. (1997) Fly Pushing: the theory and practice of Drosophila genetics. Cold Spring Harbor Laboratory, New York
Maggert, K. A., Gong, W. J., and Golic, K. G. (2008) Methods for homologous recombination in Drosophila. Methods Mol Biol 420, 155–174
Siegal, M. L. and Hartl, D. L. (1996) Transgene coplacement and high efficiency site-specific recombination with the Cre/loxP system in Drosophila. Genetics 144, 715–726
O’Keefe, L. V., Smibert, P., Colella, A., Chataway, T. K., Saint, R., and Richards, R. I (2007) Know thy fly. TIGS 23, 238–242
Acknowledgments
The authors wish to thank members of the Reenan lab for helpful discussions and suggestions, especially Sarah Goldgar, and Leila Rieder.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Staber, C.J., Gell, S., Jepson, J.E.C., Reenan, R.A. (2011). Perturbing A-to-I RNA Editing Using Genetics and Homologous Recombination. In: Aphasizhev, R. (eds) RNA and DNA Editing. Methods in Molecular Biology, vol 718. Humana Press. https://doi.org/10.1007/978-1-61779-018-8_3
Download citation
DOI: https://doi.org/10.1007/978-1-61779-018-8_3
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-017-1
Online ISBN: 978-1-61779-018-8
eBook Packages: Springer Protocols