Analysis of Xenopus dsRNA adenosine deaminase cDNAs reveals similarities to DNA methyltransferases.

We isolated two similar, but distinct, cDNA classes that encode Xenopus double-stranded RNA (dsRNA) adenosine deaminase. The longest, full-length open reading frame (ORF) predicts a 1,270-amino acid protein of 138,754 Da that is similar in size and about 50% identical to proteins encoded by mammalian cDNAs, yet larger than the 120-kDa protein purified from Xenopus eggs. Alignments of the Xenopus and mammalian ORFs show N-terminal heterogeneity, three conserved dsRNA binding motifs (dsRBMs), and strongly conserved carboxyl termini. Consistent with the observation of two cDNA classes, northern analyses of Xenopus oocyte poly A+ RNA show at least three mRNA species. Multiple nuclear polyadenylation hexamers and putative cytoplasmic polyadenylation elements were found in the 3'' UTRs of cDNAs corresponding to the largest mRNA. In vitro translation experiments show that the cDNAs encode active deaminases and that the entire N-terminus and first dsRBM are dispensable for deaminase activity. Importantly, an analysis of the C-termini of five known dsRNA adenosine deaminases, and two putative deaminases, reveals motifs that are strikingly similar to the conserved motifs of the DNA-(adenine-N6alpha)-aminomethyltransferases and the DNA-(cytosine-5)-methyltransferases.     

Double-stranded RNA adenosine deaminase as a potential mammalian RNA editing factor

Double-stranded RNA (dsRNA) adenosine deaminase, or DRADA, is a cellular enzyme that modifies adenosine residues to inosines in dsRNA by hydrolytic deamination, replacing A-U with mismatched I-U base pairs. Since it alters the base composition in its substrate RNA, one possible role played by DRADA is to participate in RNA editing. In this article, a brief review is given of characteristics of DRADA. Its possible involvement in RNA editing is also discussed in detail, including specific cases in which DRADA has been implicated as an RNA editing factor.     

Regulation of glutamate receptor B pre-mRNA splicing by RNA editing

RNA-editing enzymes of the ADAR family convert adenosines to inosines in double-stranded RNA substrates. Frequently, editing sites are defined by base-pairing of the editing site with a complementary intronic region. The glutamate receptor subunit B (GluR-B) pre-mRNA harbors two such exonic editing sites termed Q/R and R/G. Data from ADAR knockout mice and in vitro editing assays suggest an intimate connection between editing and splicing of GluR-B pre-mRNA.

By comparing the events at the Q/R and R/G sites, we can show that editing can both stimulate and repress splicing efficiency. The edited nucleotide, but not ADAR binding itself, is sufficient to exert this effect. The presence of an edited nucleotide at the R/G site reduces splicing efficiency of the adjacent intron facilitating alternative splicing events occurring downstream of the R/G site.

Lack of editing inhibits splicing at the Q/R site. Editing of both the Q/R nucleotide and an intronic editing hotspot are required to allow efficient splicing. Inefficient intron removal may ensure that only properly edited mRNAs become spliced and exported to the cytoplasm.

     

Studies of nucleosides and nucleotides.: LVIII. Deamination of adenosine analogs with calf intestine adenosine deaminase

Several synthetic adeonosine analogs: 8-fluoro-, 8-azido-, 8-iodo-, 8-methylthioadenosine; 8-bromo-2′-deoxyadenosine, 8-bromoxylofuranosyladenine, 5′-benzoly-8-bromoadenosine; 8,2′-S-, 8,2′-O-, 8,2′-NH-, 8,2′-N-CH₃-, 8,3′,-S-, 8,3′-O-, 8,5′-S- and 8,5′O-cycloadenosine; 1-deaza- and 3-deazaadenosine, as well as tubercidine (7-deazaadenosine), were tested as substrates of calf intestine adenosine deaminase.It was found that the adenine base of adenosine should be in the range φ_rmCN = 0–120° (anti to syn-anti) and 8-fluoroadenosine was hydroylzed very slowly. The purine base should have N¹, N³ or N⁷ atoms for the hydrolysis and only 1-deazaadenosine revealed an inhibitory effect toward the hydrolysis of adenosine.5′-OH group should be in the position of S-configuration and must not be substituted.     

Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain.     

Preferential selection of adenosines for modification by double-stranded RNA adenosine deaminase.

Double-stranded RNA adenosine deaminase (dsRAD), previously called the double-stranded RNA (dsRNA) unwinding/modifying activity, modifies adenosines to inosines within dsRNA. We used ribonuclease U2 and a mutant of ribonuclease T1 to map the sites of modification in several RNA duplexes. We found that dsRAD had a 5' neighbor preference (A = U > C > G) but no apparent 3' neighbor preference. Further, the proximity of the strand termini affected whether an adenosine was modified. Most importantly, dsRAD exhibited selectivity, modifying a minimal number of adenosines in short dsRNAs. Our results suggest that the specific editing of glutamate receptor subunit B mRNA could be performed in vivo by dsRAD without the aid of specificity factors, and support the hypothesis that dsRAD is responsible for hypermutations in certain RNA viruses.     

Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA.

RNA editing plays a central role in the life cycle of hepatitis D virus (HDV), a subviral human pathogen. Previous studies (J.L. Casey, K.F. Bergmann, T.L. Brown, and J.L. Gerin, Proc. Natl. Acad. Sci USA 89:7149-7153, 1992; H. Zheng, T.-B. Fu, D. Lazinski, and J. Taylor, J. Virol. 66:4693-4697, 1992) had concluded that the genomic RNA of HDV was the target for RNA editing and that the editing reaction was a conversion of U to C. However, we show here that the antigenomic RNA of HDV is in fact the target for HDV RNA editing, which is therefore a conversion of A to G. This result is verified by using an assay specific for editing on the antigenomic RNA and by analyzing the editing of site-directed mutant RNAs in transfected cells and in cell extracts. Because editing occurs in the absence of viral antigens and the specificity for the HDV editing target site is present even in extracts from Drosophila cells, it is likely that HDV RNA is edited by one or more cellular factors that are conserved among higher eukaryotes. These results raise the likelihood that double-stranded RNA adenosine deaminase specifically edits HDV antigenomic RNA.     

Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2

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.     

Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2.

We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine. The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested. Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37. Transformation of the mutant cells with the TAD1 gene restored enzyme activity. Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA. These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification.     

Theoretical study of antagonists and inhibitors of mammalian adenosine deaminase. II. Isomeric aza-deazaanalogues of adenosine

Isomeric aza-deazaanalogues of adenosine and their N1-protonated forms (except for that of 8-aza-1-deazaadenosine) were studied by computer modeling to find a relationship between their molecular structures and the properties as substrates for the mammalian adenosine deaminase. The atomic charge distribution and maps of the electrostatic potential around their van der Waals molecular surface were calculated using the ab initio STO-3G method. The conformational studies were carried out by the MM+ method of molecular mechanics. The previously proposed mechanism of the substrate acceptance in the active site of mammalian adenosine deaminase was refined, and the potential substrate properties were predicted for two previously unstudied adenosine analogues, 5-aza-9-deazaadenosine and 8-aza-3-deazaadenosine.     

RNA editing catalyzed by ADAR1 and its function in mammalian cells

In mammalian cells two active enzymes, ADAR1 and ADAR2, carry out A-to-I RNA editing. These two editases share many common features in their protein structures, catalytic activities, and substrate requirements. However, the phenotypes of the knockout animals are remarkably different, which indicate the distinct functions they play. The most striking effect of ADAR1 knockout is cell death and interruption of embryonic development that are not observed in ADAR2 knockout. Evidences have shown that ADAR1 plays critical roles in the differentiating cells in embryo and adult tissues to support the cell’s survival and permit their further differentiation and maturation. However, our knowledge in understanding of the mechanism by which ADAR1 exerts its unique effects is very limited. Many efforts had been made trying to understand why ADAR1 is so important that it is indispensible for animal survival, including studies that identify the RNA editing substrates and studies on non-editing mechanisms. The interest of this review is focused on the question why ADAR1 and not ADAR2 is required for cell survival. Therefore, only the data, published and unpublished, potentially connecting ADAR1 to its cell death effect is selectively cited and discussed here. The features of cell death caused by ADAR1 deletion are summarized. Potential involvement of interferon and protein kinase RNA-activated (PKR) pathways is proposed, but obviously more experimental evaluations are needed.     

Extent of RNA editing of glutamate receptor subunit GluR5 in different brain regions of the rat

Summary 1. The structure and function of glutamate receptor subunits GluR2, GluR5, and GluR6 are changed by RNA editing. This reaction produces a base transition in the second transmembrane spanning region. The triplet CAG (coding for glutamine) is changed to CGG (coding for arginine). This transition has a pronounced effect on calcium fluxes through the respective ion channels, because calcium currents decrease with the rate of editing.2. In the present study the extent of RNA editing of the glutamate receptor subunit GluR5 was studied in different brain regions of control rats using a newly developed analysis system. This system is based on restriction analysis of the polymerase chain reaction (PCR) product, derived from reverse-transcribed mRNA as template, with the enzymeBbv1.Bbv1 recognizes the sequence of the nonedited receptor subunit around the edited base (sequence GCAGC) but not that of the edited subunit (sequence GCGGC; A edited to G).3. Total RNA was isolated from the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, pons/medulla oblongata, and white matter and reverse transcribed into cDNA. The region across the edited sequence was amplified by PCR using GluR5-specific primers and the cDNA as template. PCR products were cleaned by ethanol precipitation, incubated withBbv1, and electrophoresed on an agarose gel together with standards. Gels were photographed and the extent of GluR5 mRNA editing was quantified using an image analysis system. A calibration curve was obtained using PCR products amplified from plasmids with edited and nonedited GluR5 as inserts.4. In the brain of control rats the extent of RNA editing of the GluR5 subunit amounted to 62±6.0% of total (cortex), 43±5.3% (striatum), 52±5.3% (hippocampus), 91±6.3% (thalamus), 85±10.2% (hypothalamus), 82±6.5% (cerebellum), 88±6.8% (pons/medulla oblongata), and 41±2.7% (white matter).5. The extent of RNA editing varied, thus, considerably in different brain regions, being lowest in the white matter and striatum and highest in the thalamus and pons/medulla oblongate. RNA editing of glutamate receptor subunits may play an important role in the control of calcium fluxes through non-N-methyl-D-aspartate receptor channels in different physiological and/or pathological states of the brain.     

Estimating RNA editing efficiency of five editing sites in the serotonin 2C receptor by pyrosequencing

Accumulating evidence suggests that altered RNA editing of the serotonin 2C receptor (HTR2C) is involved in the pathophysiology of mental disorders and the action of antidepressants. Estimating RNA editing of HTR2C in various samples is a first step to understanding its pathophysiological roles. Here, we developed a high-throughput quantification method of RNA editing efficiency by pyrosequencing. By optimizing the dispensation order, the RNA editing efficiency of all five RNA editing sites including consecutively ordered sites in HTR2C was obtained. More importantly, our method made it possible to determine the content of partial HTR2C isoforms, which enabled us to monitor possible functional changes of HTR2C. This method was validated in both oligonucleotide and RT-PCR product templates, and showed good correlation with conventional cloning-sequencing analysis. Our method could be a valuable tool in the rapid assessment of RNA editing status, including assessment of natural variations, alterations in disease tissues, and responses to drugs.