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The posttranscriptional recoding of nuclear RNA transcripts has emerged as an important regulatory mechanism during eukaryotic gene expression. In particular the deamination of adenosine to inosine (interpreted by the translational machinery as a guanosine) is a frequent event that can recode the meaning of amino acid codons in translated exons, lead to structural changes in the RNA fold, or may affect splice consensus or regulatory sequence sites in noncoding exons or introns and modulate the biogenesis of small RNAs. The molecular mechanism of how the RNA editing machinery and its substrates recognize and interact with each other is not understood well enough to allow for the ab initio delineation of bona fide RNA editing sites. However, progress in the identification of various physiological modification sites and their characterization has given important insights regarding molecular features and events critical for productive RNA editing reactions. In addition, structural studies using components of the RNA editing machinery and together with editing competent substrate molecules have provided information on the chemical mechanism of adenosine deamination within the context of RNA molecules. Here, I give an overview of the process of adenosine deamination RNA editing and describe its relationship to other RNA processing events and its currently established roles in gene regulation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

RNA editing by adenosine deamination, catalyzed by adenosine deaminases acting on RNA (ADAR), is a post-transcriptional modification that contributes to transcriptome and proteome diversity and is widespread in mammals. Here we administer a bioinformatics search strategy to the human and mouse genomes to explore the landscape of A-to-I RNA editing. In both organisms we find evidence for high excess of A/G-type discrepancies (inosine appears as a guanosine in cloned cDNA) at non-polymorphic, non-synonymous codon sites over other types of discrepancies, suggesting the existence of several thousand recoding editing sites in the human and mouse genomes. We experimentally validate recoding-type A-to-I RNA editing in a number of human genes with high scoring positions including the coatomer protein complex subunit alpha (COPA) as well as cyclin dependent kinase CDK13., (Copyright © 2011 Elsevier Inc. All rights reserved.)

RNA editing by adenosine deamination is a posttranscriptional mechanism for the regulation of gene expression and is particularly widespread in mammals. A-to-I RNA editing generates transcriptome and proteome diversity allowing organisms to produce many more gene products and functions than predicted based on the number of genes within their genome. Also, it regulates important functional properties of neurotransmitter receptor genes in the central nervous system by changing single codons in pre-mRNA. The deficiency or misregulation of editing has been implicated in the etiology of neurological diseases, such as epilepsy, amyotrophic lateral sclerosis (ALS), depression and tumor progression. Widespread A-to-I modification of repeat sequences in the human transcriptome suggests additional roles for RNA editing and links it to other processes of gene regulation, such as RNA splicing as well as siRNA mediated gene silencing and miRNA function. Here, I am reviewing main features of this epigenetic phenomenon, its relevance for health and disease, as well as potential prospects for using RNA editing as a therapeutic tool.

RNA editing by adenosine deamination fuels the generation of RNA and protein diversity in eukaryotes, particularly in higher organisms. This includes the recoding of translated exons, widespread editing of retrotransposon-derived repeat elements and sequence modification of microRNA (miRNA) transcripts. Such changes can bring about specific amino acid substitutions, alternative splicing and changes in gene expression levels. Although the overall prevalence of adenosine-to-inosine (A-to-I) editing and its specific functional impact on many of the affected genes is not yet known, the importance of balancing RNA modification levels across time and space is becoming increasingly evident. In particular, transcriptome instabilities in the form of too much or too little RNA editing activity, or misguided editing, manifest in several human disease phenotypes and can disrupt that balance., (Copyright 2010 Elsevier Ltd. All rights reserved.)

An important molecular mechanism to create protein diversity from a limited set of genes is A-to-I RNA editing. RNA editing converts single adenosines into inosines in pre-mRNA. These single base conversions can have a wide variety of consequences. Editing can lead to codon changes and, consequently, altered protein function. Moreover, editing can alter splice sites and influences miRNA biogenesis and target recognition. The two enzymes responsible for editing in mammals are adenosine deaminase acting on RNA (ADAR) 1 and 2. However, it is currently largely unknown how the activity of these enzymes is regulated in vivo. Editing activity does not always correlate with ADAR expression levels, suggesting posttranscriptional or posttranslational mechanisms for controlling activity. To investigate how editing is regulated in mammalian cells, we have developed a straightforward quantitative reporter system to detect editing levels. By employing luciferase activity as a readout, we could easily detect different levels of editing in a cellular context. In addition, increased levels of ADAR2 correlated with increased levels of luciferase activity. This reporter system therefore sets the stage for the effective screening of cDNA libraries or small molecules for strong modulators of intracellular editing to ultimately elucidate how A-to-I editing is regulated in vivo., (Copyright 2010 Elsevier Inc. All rights reserved.)

RNA editing by A-to-I modification is a widespread mechanism in complex organisms that leads to the posttranscriptional alteration of protein coding as well as noncoding sequences. MiRNA transcripts have been recognized as a major target for RNA editing enzymes, and single-nucleotide changes through editing can impact the biogenesis of mature miRNAs, as well as the target specificity of the regulatory RNA. Bona fide A-to-I RNA editing events are validated experimentally through parallel analysis of genomic DNA and transcribed sequences of miRNA genes isolated from the same specimen through gene-specific amplification and sequencing of endogenous transcripts.

Genetic variability is considered a key to the evolvability of species. The conversion of an adenosine (A) to inosine (I) in primary RNA transcripts can result in an amino acid change in the encoded protein, a change in secondary structure of the RNA, creation or destruction of a splice consensus site, or otherwise alter RNA fate. Substantial transcriptome and proteome variability is generated by A-to-I RNA editing through site-selective post-transcriptional recoding of single nucleotides. We posit that this epigenetic source of phenotypic variation is an unrecognized mechanism of adaptive evolution. The genetic variation introduced through editing occurs at low evolutionary cost since predominant production of the wild-type protein is retained. This property even allows exploration of sequence space that is inaccessible through mutation, leading to increased phenotypic plasticity and provides an evolutionary advantage for acclimatization as well as long-term adaptation. Furthermore, continuous probing for novel RNA editing sites throughout the transcriptome is an intrinsic property of the editing machinery and represents the molecular basis for increased adaptability. We propose that higher organisms have therefore evolved to systems with increasing RNA editing activity and, as a result, to more complex systems.

A-to-I RNA editing can lead to recoding of pre-mRNAs with profound functional consequences for the ensuing proteins. Here we show that complement component 1, q subcomponent-like 1 (C1QL1) undergoes RNA editing in vivo causing non-synonymous amino acid substitutions in human, mouse as well as zebrafish. The major editing site had previously been annotated as a single-nucleotide polymorphism in human, but our analysis reveals that post-transcriptional modification is the cause for the sequence variation. Remarkably, although editing of C1QL1 is conserved across vertebrate species, the predicted RNA secondary structure mediating editing involves different regions in zebrafish versus mammals.

Conversion of adenosine into inosine in RNA molecules constitutes an important post-transcriptional mechanism for generating transcript diversity and is catalyzed by adenosine deaminases acting on RNA (ADARs). Intriguingly, we observed that the editing enzyme ADAR1 enhances reporter gene expression in a cellular, plasmid-based system. The induction of gene expression is independent of the used reporter transgene or the promoter type, but relies on the RNA editing activity and specificity of ADAR1. More detailed analysis indicates that the effect is due to enhanced reporter gene transcription. Induction of gene expression by ADAR1 is lost when the reporter expression cassette is placed in a chromosomal environment. Our results suggest that a cellular, ADAR1-specific RNA editing substrate causes upregulation of plasmid-based gene expression.

Single nucleotide polymorphisms (SNPs) are DNA sequence variations that can affect the expression or function of genes. As a result, they may lead to phenotypic differences between individuals, such as susceptibility to disease, response to medications, and disease progression. Millions of SNPs have been mapped within the human genome providing a rich resource for genetic variation studies. Adenosine-to-inosine RNA editing also leads to the production of RNA and protein sequence variants, but it acts on the level of primary gene transcripts. Sequence variations due to RNA editing may be misannotated as SNPs when relying solely on expressed sequence data instead of genomic material. In this study, we screened the human SNP database for potential cases of A-to-I RNA editing that cause amino acid changes in the encoded protein. Our search strategy applies five molecular features to score candidate sites. It identifies all previously known cases of editing present in the SNP database and successfully uncovers novel, bona fide targets of adenosine deamination editing. Our approach sets the stage for effective and comprehensive genome-wide screens for A-to-I editing targets.

Adenosine Deaminases that act on RNA (ADARs) edit gene transcripts through site-specific conversion of adenosine to inosine by hydrolytic deamination at C6 of the adenosine. ADAR2 gene transcripts are substrates for the ADAR1 and ADAR2 enzymes and their expression is regulated by editing at the - 1 and - 2 sites. Our previous experiments demonstrated up-regulation of type I interferon (IFN) inducible 150 kDa ADAR1 in systemic lupus erythematosus (SLE) T cells. In this study we investigate the role of ADAR1 and ADAR2 in editing of ADAR2 gene transcripts of healthy controls and SLE patients. The ADAR2 gene transcripts were cloned into pCR2.1-TOPO vectors. A total of 150 clones from SLE and 150 clones from controls were sequenced. Sequence analysis demonstrated A to I editing at - 1, + 10, + 23 and + 24 in normal T cells. In SLE clones site-selective editing of the - 2 site was observed as a result of type I IFN-inducible 150 kDa ADAR1 expression. These results are confirmed by analysing ADAR2 transcripts of normal T cells activated with type I IFN-alpha. Editing of the + 23 and + 24 sites was decreased in SLE T cells compared to normal controls. In addition to A to G changes, U to C discrepancies were observed in normal and SLE T cells. In SLE cells, positions - 6 and + 30 were frequently edited from U to C compared to normal controls. Taken together, these results demonstrate altered and site-selective editing in ADAR2 transcripts of SLE patients. Based on these results, it is proposed that altered transcript editing contributes to the modulation of gene expression and immune functions in SLE patients.

The post-transcriptional modification of mammalian transcripts by A-to-I RNA editing has been recognized as an important mechanism for the generation of molecular diversity and also regulates protein function through recoding of genomic information. As the molecular players of editing are characterized and an increasing number of genes become identified that are subject to A-to-I modification, the potential impact of editing on the etiology or progression of human diseases is realized. Here we review the recent knowledge on where disturbances in A-to-I RNA editing have been correlated with human disease phenotypes.

RNA editing by A-to-I modification has been recognized as an important molecular mechanism for generating RNA and protein diversity. In mammals, it is mediated by a family of adenosine deaminases that act on RNAs (ADARs). The large version of the editing enzyme ADAR1 (ADAR1-L), expressed from an interferon-responsible promoter, has a Z-DNA/Z-RNA binding domain at its N-terminus. We have tested the in vitro ability of the enzyme to act on a 50 bp segment of dsRNA with or without a Z-RNA forming nucleotide sequence. A-to-I editing efficiency is markedly enhanced in presence of the sequence favoring Z-RNA. In addition, an alteration in the pattern of modification along the RNA duplex becomes evident as reaction times decrease. These results suggest that the local conformation of dsRNA molecules might be an important feature for target selectivity by ADAR1 and other proteins with Z-RNA binding domains.

RNA editing by adenosine deamination generates RNA and protein diversity through the posttranscriptional modification of single nucleotides in RNA sequences. Few mammalian A-to-I edited genes have been identified despite evidence that many more should exist. Here we identify intramolecular pairs of Alu elements as a major target for editing in the human transcriptome. An experimental demonstration in 43 genes was extended by a broader computational analysis of more than 100,000 human mRNAs. We find that 1,445 human mRNAs (1.4%) are subject to RNA editing at more than 14,500 sites, and our data further suggest that the vast majority of pre-mRNAs (greater than 85%) are targeted in introns by the editing machinery. The editing levels of Alu-containing mRNAs correlate with distance and homology between inverted repeats and vary in different tissues. Alu-mediated RNA duplexes targeted by RNA editing are formed intramolecularly, whereas editing due to intermolecular base-pairing appears to be negligible. We present evidence that these editing events can lead to the posttranscriptional creation or elimination of splice signals affecting alternatively spliced Alu-derived exons. The analysis suggests that modification of repetitive elements is a predominant activity for RNA editing with significant implications for cellular gene expression., Competing Interests: The authors have declared that no conflicts of interest exist.

In mammals, RNA editing by site-selective adenosine deamination regulates key functional properties of neurotransmitter receptors in the central nervous system. Glutamate receptor subunit B is nearly 100% edited at one position (the Q/R-site), which is essential for normal receptor function. Its significance is apparent from mouse models in which a slightly reduced rate of Q/R-site editing is associated with early onset epilepsy and premature death. Here we report that in tissues from malignant human brain tumors, this editing position of glutamate receptor subunit B is substantially underedited compared with control tissues. We also observe alterations in editing and alternative splicing of serotonin receptor 5-HT(2C) transcripts. These changes correlate with a decrease in enzymatic activity of the editing enzyme adenosine deaminase acting on RNA (ADAR) 2, as deduced from analysis of ADAR2 self-editing. Our results suggest a role for RNA editing in tumor progression and may provide a molecular model explaining the occurrence of epileptic seizures in association with malignant gliomas.

RNA editing, the post-transcriptional alteration of a gene-encoded sequence, is a widespread phenomenon in eukaryotes. As a consequence of RNA editing, functionally distinct proteins can be produced from a single gene. The molecular mechanisms involved include single or multiple base insertions or deletions as well as base substitutions. In mammals, one type of substitutional RNA editing, characterized by site-specific base-modification, was shown to modulate important physiological processes. The underlying reaction mechanism of substitutional RNA editing involves hydrolytic deamination of cytosine or adenosine bases to uracil or inosine, respectively. Protein factors have been characterized that are able to induce RNA editing in vitro. A supergene family of RNA-dependent deaminases has emerged with the recent addition of adenosine deaminases specific for tRNA. Here we review the developments that have substantially increased our understanding of base-modification RNA editing over the past few years, with an emphasis on mechanistic differences, evolutionary aspects and the first insights into the regulation of editing activity.

The Zab domain of the editing enzyme ADAR1 binds tightly and specifically to Z-DNA stabilized by bromination or supercoiling. A stoichiometric amount of protein has been shown to convert a substrate of suitable sequence to the Z form, as demonstrated by a characteristic change in the CD spectrum of the DNA. Now we show that Zab can bind not only to isolated Z-forming d(CG)(n) sequences but also to d(CG)(n) embedded in B-DNA. The binding of Zab to such sequences results in a complex including Z-DNA, B-DNA, and two B-Z junctions. In this complex, the d(CG)(n) sequence, but not the flanking region, is in the Z conformation. The presence of Z-DNA was detected by cleavage with a Z-DNA specific nuclease, by undermethylation using Z-DNA sensitive SssI methylase, and by circular dichroism. It is possible that Zab binds to B-DNA with low affinity and flips any favorable sequence into Z-DNA, resulting in a high affinity complex. Alternatively, Zab may capture Z-DNA that exists transiently in solution. The binding of Zab to potential as well as established Z-DNA segments suggests that the range of biological substrates might be wider than previously thought.

RNA editing by site-selective deamination of adenosine to inosine alters codons and splicing in nuclear transcripts, and therefore protein function. ADAR2 (refs 7, 8) is a candidate mammalian editing enzyme that is widely expressed in brain and other tissues, but its RNA substrates are unknown. Here we have studied ADAR2-mediated RNA editing by generating mice that are homozygous for a targeted functional null allele. Editing in ADAR2-/- mice was substantially reduced at most of 25 positions in diverse transcripts; the mutant mice became prone to seizures and died young. The impaired phenotype appeared to result entirely from a single underedited position, as it reverted to normal when both alleles for the underedited transcript were substituted with alleles encoding the edited version exonically. The critical position specifies an ion channel determinant, the Q/R site, in AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor GluR-B pre-messenger RNA. We conclude that this transcript is the physiologically most important substrate of ADAR2.

The mammalian adenosine deaminases acting on RNA (ADARs) constitute a family of sequence-related proteins involved in pre-mRNA editing of nuclear transcripts through site-specific adenosine modification. We report here the identification and characterization of a human ADAR protein, hADAT1, that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala). It represents the functional homologue of the recently identified yeast protein Tad1p [Gerber, A., Grosjean, H., Melcher, T. & Keller, W. (1998) EMBO J. 17, 4780-4789]. The hADAT1 cDNA predicts a protein of 502 aa whose sequence displays strongest overall homology to a Drosophila melanogaster ORF (50% similarity, 32% identity), and the catalytic domain is closely related to the other ADAR proteins. In vitro, the recombinantly expressed and purified hADAT1 protein efficiently and specifically deaminates A(37) in the anticodon loop of tRNA(Ala) from higher eukaryotes and with lower efficiency from lower eukaryotes. It does not modify adenosines residing in double-stranded RNA or in pre-mRNAs that serve as substrates for ADAR1 or ADAR2. The anticodon stem-loop of tRNA(Ala) alone is not a functional substrate for hADAT1. The enzyme is expressed ubiquitously in human tissues and is represented by a single gene. The identification and cloning of hADAT1 should help to elucidate the physiological significance of this unique modification in tRNA(Ala), which is conserved from yeast to man.

The past year has witnessed major progress in the field of mammalian nuclear RNA editing. Two new sequence-related RNA-dependent adenosine deaminases, distantly related to the previously characterized double-stranded RNA adenosine deaminase DRADA/dsRAD, have been molecularly characterized. One of these deaminases edits in vitro with precision for the molecular determinant that controls the Ca2+ permeability of fast synaptic glutamate-gated cation channels. This deaminase, like DRADA, is expressed in many tissues and the search is now on for more substrates of these RNA-editing enzymes. Moreover, the physiological role of the apolipoprotein B RNA editing enzyme APOBEC-1 has been investigated in genetically manipulated mice.

Pre-mRNAs for brain-expressed ionotropic glutamate receptor subunits undergo RNA editing by site-specific adenosine deamination, which alters codons for molecular determinants of channel function. This nuclear process requires double-stranded RNA structures formed by exonic and intronic sequences in the pre-mRNA and is likely to be catalyzed by an adenosine deaminase that recognizes these structures as a substrate. DRADA, a double-stranded RNA adenosine deaminase, is a candidate enzyme for L-glutamate-activated receptor channel (GluR) pre-mRNA editing. We show here that DRADA indeed edits GluR pre-mRNAs, but that it displays selectivity for certain editing sites. Recombinantly expressed DRADA, both in its full-length form and in an N-terminally truncated version, edited the Q/R site in GluR6 pre-mRNA and the R/G site but not the Q/R site of GluR-B pre-mRNA. This substrate selectivity correlated with the base pairing status and sequence environment of the editing-targeted adenosines. The Q/R site of GluR-B pre-mRNA was edited by an activity partially purified from HeLa cells and thus differently structured editing sites in GluR pre-mRNAs appear to be substrates for different enzymatic activities.

Editing of RNA by site-selective adenosine deamination alters codons in brain-expressed pre-messenger RNAs for glutamate receptor (GluR) subunits including a codon for a channel determinant (Q/R site) in GluR-B, which controls the Ca2+ permeability of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Editing of GluR pre-mRNAs requires a double-stranded RNA (dsRNA) structure formed by exonic and intronic sequences and is catalysed by an unknown dsRNA adenosine deaminase. Here we report the cloning of complementary DNA for RED1, a dsRNA adenosine deaminase expressed in brain and peripheral tissues that efficiently edits the Q/R site in GluR-B pre-mRNA in vitro. This site is poorly edited by DRADA, which is distantly sequence-related to RED1. Both deaminases edit the R/G site in GluR-B pre-mRNA, indicating that members of an emerging gene family catalyse adenosine deamination in nuclear transcripts with distinct but overlapping substrate specificities.

In neurons of the mammalian brain primary transcripts of genes encoding subunits of glutamate receptor channels can undergo RNA editing, leading to altered properties of the transmitter-activated channel. Editing of these transcripts is a nuclear process that targets specific adenosines and requires a double-stranded RNA structure configured from complementary exonic and intronic sequences. We show here that the two independent editing sites in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA are edited with positional accuracy by nuclear extract from HeLa cells. Nucleotide analysis by thin layer chromatography of the edited RNA sequences revealed selective adenosine to inosine conversion, most likely reflecting the participation of double-stranded RNA adenosine deaminase. Our results predict the presence of inosine-containing codons in other mammalian mRNAs.