Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method

Yuru Wang, Peter A. Beal

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5′ side of the modification site. This 5′ binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure-activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5′ binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure-function relationships in ADAR reactions.

Original languageEnglish (US)
Pages (from-to)9872-9880
Number of pages9
JournalNucleic Acids Research
Volume44
Issue number20
DOIs
StatePublished - 2016

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Mutagenesis
RNA
Deamination
Structure-Activity Relationship
Substrate Specificity
Adenosine
Yeasts
Amino Acids
Messenger RNA
Proteins

ASJC Scopus subject areas

  • Genetics

Cite this

Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method. / Wang, Yuru; Beal, Peter A.

In: Nucleic Acids Research, Vol. 44, No. 20, 2016, p. 9872-9880.

Research output: Contribution to journalArticle

Wang, Yuru ; Beal, Peter A. / Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method. In: Nucleic Acids Research. 2016 ; Vol. 44, No. 20. pp. 9872-9880.
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