Identification of Substrates for Adenosine Deaminases That Act on RNA
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 WordsAdenosine deaminases that act on RNA RNA editing inosine substrate differential display RNase T1 reverse transcriptase polymerase chain reaction
- 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
- 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
- 25.Greer, C. L. and Uhlenbeck, O. C., personal communication.Google Scholar