Article

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.

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    ABSTRACT: ADARs (adenosine deaminases acting on RNA) are RNA editing enzymes that bind double helical RNAs and deaminate select adenosines (A). The product inosine (I) is read during translation as guanosine (G) so such changes can alter codon meaning. ADAR-catalyzed A to I changes occur in coding sequences for several proteins of importance to the nervous system. However, these sites constitute only a very small fraction of known A to I sites in the human transcriptome and the significance of editing at the vast majority sites is unknown at this time. Site-selective inhibitors of RNA editing are needed to advance our understanding of the function of editing at specific sites. Here we show that 2'-O-methyl/locked nucleic acid (LNA) mixmer antisense oligonucleotides are potent and selective inhibitors of RNA editing on two different target RNAs. These reagents are capable of binding with high affinity to RNA editing substrates and remodeling the secondary structure by a strand-invasion mechanism. The potency observed here for 2'-O-methyl/LNA mixmers suggests this backbone structure is superior to the morpholino backbone structure for inhibition of RNA editing. Finally, we demonstrate antisense inhibition of editing of the mRNA for the DNA repair glycosylase NEIL1 in cultured human cells providing a new approach to exploring the link between RNA editing and the cellular response to oxidative DNA damage.
    ACS Chemical Biology 02/2013; · 5.44 Impact Factor

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