title: A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.
abstract: Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.
authors.affiliation: Department of Chemistry, University of California, Davis , 1 Shields Ave, Davis, California 95616, United States.
authors.forename: Yuru
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of California, Davis , 1 Shields Ave, Davis, California 95616, United States.
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Havel
authors.affiliation: Department of Chemistry, University of California, Davis , 1 Shields Ave, Davis, California 95616, United States.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Beal
mesh_terms: D000243:Adenosine Deaminase / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D016601:RNA-Binding Proteins; D012313:RNA; C000595356:ADAR protein, human; D000243:Adenosine Deaminase