A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1

Yuru Wang, Jocelyn Havel, Peter A. Beal

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 ADARs editing site selectivity.

Original languageEnglish (US)
Pages (from-to)2512-2519
Number of pages8
JournalACS Chemical Biology
Volume10
Issue number11
DOIs
StatePublished - Nov 20 2015

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RNA Editing
Adenosine Deaminase
Adenosine
RNA
Inosine
Deamination
Saccharomycetales
Codon
Base Pairing
Libraries
Saccharomyces cerevisiae
Plasmids
Bearings (structural)
Enzymes
Yeast
Assays
Screening
Proteins
In Vitro Techniques
human ADA protein

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1. / Wang, Yuru; Havel, Jocelyn; Beal, Peter A.

In: ACS Chemical Biology, Vol. 10, No. 11, 20.11.2015, p. 2512-2519.

Research output: Contribution to journalArticle

Wang, Yuru ; Havel, Jocelyn ; Beal, Peter A. / A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1. In: ACS Chemical Biology. 2015 ; Vol. 10, No. 11. pp. 2512-2519.
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