Abstract
Adenosine deaminases acting on RNA (ADARs) hydrolytically deaminate adenosines (A) in a wide variety of duplex RNAs and misregulation of editing is correlated with human disease. However, our understanding of reaction selectivity is limited. ADARs are modular enzymes with multiple double-stranded RNA binding domains (dsRBDs) and a catalytic domain. While dsRBD binding is understood, little is known about ADAR catalytic domain/RNA interactions. Here we use a recently discovered RNA substrate that is rapidly deaminated by the isolated human ADAR2 deaminase domain (hADAR2-D) to probe these interactions. We introduced the nucleoside analog 8-azanebularine (8-azaN) into this RNA (and derived constructs) to mechanistically trap the protein–RNA complex without catalytic turnover for EMSA and ribonuclease footprinting analyses. EMSA showed that hADAR2-D requires duplex RNA and is sensitive to 2′-deoxy substitution at nucleotides opposite the editing site, the local sequence and 8-azaN nucleotide positioning on the duplex. Ribonuclease V1 footprinting shows that hADAR2-D protects ∼23 nt on the edited strand around the editing site in an asymmetric fashion (∼18 nt on the 5′ side and ∼5 nt on the 3′ side). These studies provide a deeper understanding of the ADAR catalytic domain–RNA interaction and new tools for biophysical analysis of ADAR–RNA complexes.
Highlights
Adenosine deaminases acting on RNA (ADARs) are editing enzymes responsible for hydrolytic deamination of adenosine (A) in the context of RNA polymers, producing inosine (I) at corresponding nucleotide positions [1,2,3]
It occurred to us that these RNAs would be useful in defining catalytic domain–RNA interactions, if they could be shortened to a length accessible by chemical synthesis and retain high hADAR2-D reactivity
No synthetically accessible RNA had been reported that was deaminated by hADAR2-D with a rate high enough to suggest 8-azaN substitution would lead to tight binding
Summary
Adenosine deaminases acting on RNA (ADARs) are editing enzymes responsible for hydrolytic deamination of adenosine (A) in the context of RNA polymers, producing inosine (I) at corresponding nucleotide positions [1,2,3]. This reaction has a variety of functional consequences for the RNA substrates including modulating thermal stability of base-paired structures, changing the meaning of codons in mRNAs (recoding), altering splicing patterns of pre-mRNAs, changing miRNA targeting within 3 untranslated regions (UTRs), etc. While the RNA-binding properties of ADARs’ dsRBDs have been well documented [25,26], few reports have focused on the RNA binding requirements of the ADAR catalytic domains
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