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Identification of Substrates for Adenosine Deaminases That Act on RNA

  • Daniel P. Morse
Part of the Methods in Molecular Biology book series (MIMB, volume 265)

Abstract

Adenosine deaminases that acts on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine in double-stranded RNA. This chapter provides a detailed protocol for identifying inosine-containing RNAs. Candidate ADAR substrates are identified by cleaving poly (A)+ RNA specifically after inosine and using differential display to detect cleaved molecules. To confirm the presence of inosine, each individual candidate substrate is amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and the PCR product is directly sequenced. Sites that contain inosine at the RNA level appear as a mixture of adenosine and guanosine in the cDNA. The relative peak areas provide an estimate of the extent of editing at each site.

Key Words

Adenosine deaminases that act on RNA RNA editing inosine substrate differential display RNase T1 reverse transcriptase polymerase chain reaction 

References

  1. 1.
    Bass, B. L. (2002) RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846.PubMedCrossRefGoogle Scholar
  2. 2.
    Schaub, M. and Keller, W. (2002) RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie 84, 791–803.PubMedCrossRefGoogle Scholar
  3. 3.
    Gerber, A. P. and Keller, W. (2001) RNA editing by base deamination: more enzymes, more targets, new mysteries. Trends Biochem. Sci. 26, 376–384.PubMedCrossRefGoogle Scholar
  4. 4.
    Seeburg, P. H. (1996) The role of RNA editing in controlling glutamate receptor channel properties. J. Neurochem. 66, 1–5.PubMedCrossRefGoogle Scholar
  5. 5.
    Burns, C. M., Chu, H., Rueter, S. M., Hutchinson, L. K., Canton, H., Sanders-Bush, E., and Emeson, R. B. (1997) Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 387, 303–308.PubMedCrossRefGoogle Scholar
  6. 6.
    Polson, A. G., Bass, B. L., and Casey, J. L. (1996) RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase. Nature 380, 454–456.PubMedCrossRefGoogle Scholar
  7. 7.
    Morse, D. P. and Bass, B. L. (1999) Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA. Proc. Natl. Acad. Sci. USA 96, 6048–6053.PubMedCrossRefGoogle Scholar
  8. 8.
    Morse, D. P., Aruscavage, P. J., and Bass, B. L. (2002) RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA. Proc. Natl. Acad. Sci. USA 99, 7906–7911.PubMedCrossRefGoogle Scholar
  9. 9.
    Brusa, R., Zimmermann, F., Koh, D. S., Feldmeyer, D., Gass, P., Seeburg, P. H., and Sprengel, R. (1995) Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice. Science 270, 1677–1680.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedCrossRefGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    Knight, S. W. and Bass, B. L. (2002) The role of RNA editing by ADARs in RNAi. Mol. Cell 10, 809–817.PubMedCrossRefGoogle Scholar
  15. 15.
    Paul, M. S. and Bass, B. L. (1998) Inosine exists in mRNA at tissue-specific levels and is most abundant in brain mRNA. EMBO J. 17, 1120–1127.PubMedCrossRefGoogle Scholar
  16. 16.
    Liang, P. and Pardee, A. B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.PubMedCrossRefGoogle Scholar
  17. 17.
    Morse, D. P. and Bass, B. L. (1997) Detection of inosine in messenger RNA by inosine-specific cleavage. Biochemistry 36, 8429–8434.PubMedCrossRefGoogle Scholar
  18. 18.
    Moore, M. J. and Sharp, P. A. (1992) Site-specific modification of pre-mRNA: the 2′-hydroxyl groups at the splice sites. Science 256, 992–997.PubMedCrossRefGoogle Scholar
  19. 19.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  20. 20.
    Bantle, J. A., Maxwell, I. H., and Hahn, W. E. (1976) Specificity of oligo (dT)-cellulose chromatography in the isolation of polyadenylated RNA. Anal. Biochem. 72, 413–427.PubMedCrossRefGoogle Scholar
  21. 21.
    Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., and Smith, J. A. (1987) Current Protocols in Molecular Biology, John Wiley & Sons, New York.Google Scholar
  22. 22.
    Broude, N. E. and Budowsky, E. I. (1971) The reaction of glyoxal with nucleic acid components. 3. Kinetics of the reaction with monomers. Biochim. Biophys. Acta 254, 380–388.PubMedGoogle Scholar
  23. 23.
    Whitfeld, P. R. and Witzel, H. (1963) On the mechanism of action of Takadiastase ribonuclease T1. Biochim. Biophys. Acta 72, 338–341.PubMedCrossRefGoogle Scholar
  24. 24.
    Cameron, V. and Uhlenbeck, O. C. (1977) 3′-Phosphatase activity in T4 polynucleotide kinase. Biochemistry 16, 5120–5126.PubMedCrossRefGoogle Scholar
  25. 25.
    Greer, C. L. and Uhlenbeck, O. C., personal communication.Google Scholar
  26. 26.
    Melcher, T., Maas, S., Higuchi, M., Keller, W., and Seeburg, P. H. (1995) Editing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA in vitro reveals site-selective adenosine to inosine conversion. J. Biol. Chem. 270, 8566–8570.PubMedCrossRefGoogle Scholar
  27. 27.
    Polson, A. G. and Bass, B. L. (1994) Preferential selection of adenosines for modification by double-stranded RNA adenosine deaminase. EMBO J. 13, 5701–5711.PubMedGoogle Scholar
  28. 28.
    Herb, A., Higuchi, M., Sprengel, R., and Seeburg, P. H. (1996) Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences. Proc. Natl. Acad. Sci. USA 93, 1875–1880.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Daniel P. Morse
    • 1
  1. 1.Chemistry DepartmentUnited States Naval AcademyAnnapolis

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