Analysis of the RNA-Editing Reaction of ADAR2 with Structural and Fluorescent Analogues of the GluR-B R/G Editing Site

Abstract

ADARs are adenosine deaminases responsible for RNA editing reactions that occur in eukaryotic pre-mRNAs, including the pre-mRNAs of glutamate and serotonin receptors. Here we describe the generation and analysis of synthetic ADAR2 substrates that differ in structure around an RNA editing site. We find that five base pairs of duplex secondary structure 5' to the editing site increase the single turnover rate constant for deamination 17-39-fold when compared to substrates lacking this structure. ADAR2 deaminates an adenosine in the sequence context of a natural editing site >90-fold more rapidly and to a higher yield than an adjacent adenosine in the same RNA structure. This reactivity is minimally dependent on the base pairing partner of the edited nucleotide; adenosine at the editing site in the naturally occurring A.C mismatch is deaminated to approximately the same extent and only 4 times faster than adenosine in an A.U base pair at this site. A steady-state rate analysis at a saturating concentration of the most rapidly processed substrate indicates that product formation is linear with time through at least three turnovers with a slope of 13 +/- 1.5 nM.min(-1) at 30 nM ADAR2 for a k(ss) = 0.43 +/- 0.05 min(-1). In addition, ADAR2 induces a 3.3-fold enhancement in fluorescence intensity and a 14 nm blue shift in the emission maximum of a duplex substrate with 2-aminopurine located at the editing site, consistent with a mechanism whereby ADAR2 flips the reactive nucleotide out of the double helix prior to deamination.

Substrate recognition by ADAR1 and ADAR2

“…In the case of the GluR-B R/G derivative, an A:U base pair at the editing site did not significantly alter ADAR2-catalyzed editing+ This is consistent with a recent report (Stephens et al+, 2000) where only a very modest decrease in the extent of deamination by ADAR2 was observed in vitro when the same mutation in the wild-type R/G site was tested+ Based on this finding, the authors concluded that the identity of the base opposing the edited adenosine exerts a relatively minor effect on editing+ However, if the investigators had tested ADAR1 with the same mutant or ADAR2 with a GluR-B R/G mutant that contained an A:G mismatch at the editing site, they would likely have observed a more severe effect+…”

A Transition State Analogue for an RNA-Editing Reaction

“…We used phosphoramidite 7 and the appropriate deprotection conditions to prepare mimics of the glutamate receptor B-subunit (GluR-B) pre-mRNA near the R/G editing site ( Figure 4A). 17 We chose initially to analyze the effect 8-azanebularine has on the ADAR2 RNA binding affinity using a deletion mutant of the enzyme lacking dsRBM I (ADAR2 216-711 ) ( Figure 4B). We refer to this protein as R-D because it contains one dsRBM and the Deaminase domain.…”

Structure and Sequence Determinants Required for the RNA Editing of ADAR2 Substrates

“…Although subtle determinants of ADAR specificity have been identified in the DRBMs (46), the primary determinants of ADAR site specificity are thought to reside in the characteristics of their dsRNA substrates (38,47). Structural selectivity of ADARs for a particular adenosine in the duplex is thought to be imparted by the pattern of bulges and base-pairing regions in the dsRNA, combined with the overall thermodynamic stability of the RNA (20,21,46,48). According to observations made using artificial substrates, internal loops of greater than six nucleotides define subdomains of ADAR action (22).…”

“…A similar base-flipping strategy has been implicated for ADAR2 function in which the DRBM not only plays a role in the recognition of potential ADAR2 sites, but also an additional role in rendering the nucleotides around the targeted adenosine more conformationally flexible, thus lowering the activation energy for base flipping (19,48). More recent studies have demonstrated that the identity of nucleotides surrounding the targeted adenosine moiety can affect the efficiency of editing, possibly by altering whether the targeted adenosine is stacked in the RNA helix (50), suggesting that local sequences surrounding the edited adenosine moiety can affect its ability to be flipped out of the helix during catalysis.…”