High-Throughput Screening for Functional Adenosine to Inosine RNA Editing Systems

摘要

Deamination of adenosines within messenger RNAs catalyzed by adenosine deaminases that act on RNA (ADAR) enzymes generates inosines at the corresponding nucleotide positions. Because inosine is decoded as guanosine, this reaction can lead to codon changes and the introduction of amino acids into a gene product not encoded in the gene. Translation of the different coding strands created by this process leads to protein structural diversity in the parent organism and is necessary for nervous system function in metazoa. The basis for selective editing of adenosines within certain codons is not well understood at the structural/biochemical level. Here we describe a high-throughput screen for ADAR/substrate combinations capable of RNA editing that can be carried out in the yeast Saccharomyces cerevisiae growing on agar plates. Results from the screening of libraries of human ADAR2 mutants and libraries of RNA substrates shed light on structure–activity relationships in the ADAR-catalyzed adenosine to inosine RNA editing reaction.