Abstract
Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.
Highlights
RNA-editing involves altering a transcript’s sequence by insertion, deletion, or modification of nucleotides resulting in a change in information content from that originally encoded in the genome [1]
We compared the binding sites for ADAR2 s deaminase domain (ADAR2d) E488Q and ADAR2 R2D E488Q using footprinting with the nonspecific duplex RNA cleavage reagent methidium-propyl EDTA Fe (II) [50]
It should be noted that we observed a region of weak protection by ADAR2-R2D E488Q approximately 25–32 bp in the 5 direction relative to 8-AN, which is not observed for hADAR2d
Summary
RNA-editing involves altering a transcript’s sequence by insertion, deletion, or modification of nucleotides resulting in a change in information content from that originally encoded in the genome [1]. A-to-I editing can result in the formation of alternative splice variants, alteration of microRNA processing and targeting, a change in codon sequence and suppression of activation of the innate immune system by endogenous double stranded RNAs (dsRNAs) [4,5,6]. Knock down of ADAR1 was found to be lethal to a subset of cancer cells displaying an interferon-stimulated gene signature. These studies have identified ADAR1 as a potential cancer therapeutic target [14,15,16,17,18,19]. ADARs are currently being used in directed RNA editing applications either via recruitment of endogenous ADARs with antisense guide RNAs or in engineered fusion proteins bearing ADAR deaminase domains [20,21,22,23,24,25,26]
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