Open AccessA Screening Protocol for Identification of Functional Mutants of RNA Editing Adenosine Deaminases
Author(s) -
Eifler Tristan,
Chan Dalen,
Beal Peter A.
Publication year - 2012
Publication title -
current protocols in chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.503
H-Index - 14
ISSN - 2160-4762
DOI - 10.1002/9780470559277.ch120139
Subject(s) - adar , rna editing , rna , biology , stop codon , inosine , genetics , computational biology , biochemistry , adenosine , gene
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.