Nucleoside analog studies indicate mechanistic differences between RNA-editing adenosine deaminases.

Department of Chemistry, University of California, Davis, CA 95616, USA.
Nucleic Acids Research (Impact Factor: 8.81). 08/2012; 40(19):9825-35. DOI: 10.1093/nar/gks752
Source: PubMed

ABSTRACT Adenosine deaminases acting on RNA (ADAR1 and ADAR2) are human RNA-editing adenosine deaminases responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. Since inosine is recognized during translation as guanosine, this often results in the expression of protein sequences different from those encoded in the genome. While our knowledge of the ADAR2 structure and catalytic mechanism has grown over the years, our knowledge of ADAR1 has lagged. This is due, at least in part, to the lack of well defined, small RNA substrates useful for mechanistic studies of ADAR1. Here, we describe an ADAR1 substrate RNA that can be prepared by a combination of chemical synthesis and enzymatic ligation. Incorporation of adenosine analogs into this RNA and analysis of the rate of ADAR1 catalyzed deamination revealed similarities and differences in the way the ADARs recognize the edited nucleotide. Importantly, ADAR1 is more dependent than ADAR2 on the presence of N7 in the edited base. This difference between ADAR1 and ADAR2 appears to be dependent on the identity of a single amino acid residue near the active site. Thus, this work provides an important starting point in defining mechanistic differences between two functionally distinct human RNA editing ADARs.

  • Source
    [Show abstract] [Hide abstract]
    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.
    Nucleic Acids Research 01/2015; 43(2). DOI:10.1093/nar/gku1345 · 8.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ribonucleoside analogs bearing terminal alkynes, including 7-ethynyl-8-aza-7-deazaadenosine (7-EAA), are useful for RNA modification applications. However, while alkyne- and triazole-bearing ribonucleosides are in wide spread use, very little information is available on the impact of these modifications on RNA structure. By solving crystal structures for RNA duplexes containing these analogs, we show that, like adenosine, 7-EAA and a triazole derived from 7-EAA base pair with uridine and are well accommodated within an A-form helix. We show copper-catalyzed azide/alkyne cycloaddition (CuAAC) reactions with 7-EAA are sensitive to the RNA secondary structure context with single stranded sites reacting faster than duplex sites. 7-EAA and its triazole products are recognized in RNA template strands as adenosine by avian myoblastosis virus reverse transcriptase (AMV-RT). In addition, 7-EAA in RNA is a substrate for an active site mutant of the RNA editing adenosine deaminase ADAR2. These studies extend our understanding of the impact of these novel nucleobase analogs and set the stage for their use in probing RNA structure and metabolism.
    ACS Chemical Biology 06/2014; 9(8). DOI:10.1021/cb500270x · 5.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The 8-azapurines, and their 7-deaza and 9-deaza congeners, represent a unique class of isosteric (isomorphic) analogues of the natural purines, frequently capable of substituting for the latter in many biochemical processes. Particularly interesting is their propensity to exhibit pH-dependent room-temperature fluorescence in aqueous medium, and in non-polar media. We herein review the physico-chemical properties of this class of compounds, with particular emphasis on the fluorescence emission properties of their neutral and/or ionic species, which has led to their widespread use as fluorescent probes in enzymology, including enzymes involved in purine metabolism, agonists/antagonists of adenosine receptors, mechanisms of catalytic RNAs, RNA editing, etc. They are also exceptionally useful fluorescent probes for analytical and clinical applications in crude cell homogenates.
    Molecular BioSystems 08/2014; 10(11). DOI:10.1039/c4mb00233d · 3.18 Impact Factor

Full-text (3 Sources)

Available from
May 26, 2014