A Screening Protocol for Identification of Functional Mutants of RNA Editing Adenosine Deaminases
Published Online: 1 DEC 2012
Copyright © 2012 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Chemical Biology
How to Cite
Eifler, T., Chan, D. and Beal, P. A. 2012. A Screening Protocol for Identification of Functional Mutants of RNA Editing Adenosine Deaminases. Current Protocols in Chemical Biology. 4:357–369.
- Published Online: 1 DEC 2012
Genetic screens can be used to evaluate a spectrum of mutations and thereby infer the function of particular residues within a protein. The Adenosine Deaminase Acting on RNA (ADAR) family of RNA-editing enzymes selectively deaminate adenosines (A) in double-helical RNA, generating inosine (I). The protocol described here exploits the editing activity of ADAR2 in a yeast-based screen by inserting an editing substrate sequence with a stop codon incorporated at the editing site upstream from the sequence encoding the reporter α-galactosidase. A-to-I editing changes the stop codon to a tryptophan codon, allowing normal expression of the reporter. This technique is particularly well-suited for screening ADAR and ADAR substrate mutant libraries for editing activity. Curr. Protoc. Chem. Biol. 4:357-369 © 2012 by John Wiley & Sons, Inc.
- RNA editing;
- A-to-I editing;
- ADAR substrate