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PubMed2003

Elevated activity of the large form of ADAR1 in vivo: very efficient RNA editing occurs in the cytoplasm.

Mammalian cells express small and large forms of the RNA editing enzyme ADAR1, referred to as ADAR1-S and ADAR1-L, respectively. Here we observed that ADAR1-L was >70-fold more active than was ADAR1-S when assayed with a substrate that could be edited in either the nucleus or cytoplasm, and was also much more active when assayed with a substrate that was generated in the cytoplasm during viral replication. In contrast, when a substrate that could only be edited within the nucleus was assayed, the activity of ADAR1-S was found to be somewhat higher than that of ADAR1-L. We show here not only that editing could occur in the cytoplasm but also that the process was extremely efficient, occurred rapidly, and could occur in the absence of translation. Consistent with the observation that editing in the cytoplasm can be very efficient, deletion of the nuclear localization signal from ADAR2 resulted in a protein with 15-fold higher activity when tested with a substrate that contained an editing site in the mature message. In addition to its potential role in an antiviral response, we propose that ADAR1-L is the form primarily responsible for editing mRNAs in which the editing site is retained after processing.

Adenosine Deaminase↗

PubMed2004

The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1.

Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-A resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site "flap" whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.

APOBEC-1 Deaminase↗

PubMed1999

Mapping contacts between gRNA and mRNA in trypanosome RNA editing.

All guide RNAs (gRNAs) identified to date have defined 5' anchor sequences, guiding sequences and a non-encoded 3' uridylate tail. The 5' anchor is required for in vitro editing and is thought to be responsible for selection and binding to the pre-edited mRNA. Little is known, however, about how the gRNAs are used to direct RNA editing. Utilizing the photo-reactive crosslinking agent, azidophenacyl (APA), attached to the 5'- or 3'-terminus of the gRNA, we have begun to map the structural relationships between the different defined regions of the gRNA with the pre-edited mRNA. Analyses of crosslinked conjugates produced with a 5'-terminal APA group confirm that the anchor of the gRNA is correctly positioning the interacting molecules. 3' Crosslinks (X-linker placed at the 3'-end of a U10tail) have also been mapped for three different gRNA/mRNA pairs. In all cases, analyses indicate that the U-tail can interact with a range of nucleotides located upstream of the first edited site. It appears that the U-tail prefers purine-rich sites, close to the first few editing sites. These results suggest that the U-tail may act in concert with the anchor to melt out secondary structure in the mRNA in the immediate editing domain, possibly increasing the accessibility of the editing complex to the proper editing sites.

Animals↗

PubMed2003

The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana.

The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis: However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis: Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.

Amino Acid Sequence↗

PubMed2005

Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing.

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.

Adenosine Deaminase↗

PubMed2005

A bioinformatic screen for novel A-I RNA editing sites reveals recoding editing in BC10.

MOTIVATION: Recent studies have demonstrated widespread adenosine-inosine RNA editing in non-coding sequence. However, the extent of editing in coding sequences has remained unknown. For many of the known sites, editing can be observed in multiple species and often occurs in well-conserved sequences. In addition, they often occur within imperfect inverted repeats and in clusters. Here we present a bioinformatic approach to identify novel sites based on these shared features. Mismatches between genomic and expressed sequences were filtered to remove the main sources of false positives, and then prioritized based on these features. This protocol is tailored to identifying specific recoding editing sites, rather than sites in non-coding repeat sequences. RESULTS: Our protocol is more sensitive for identifying known coding editing sites than any previously published mammalian screen. A novel multiply edited transcript, BC10, was identified and experimentally verified. BC10 is highly conserved across a range of metazoa and has been implicated in two forms of cancer.

Adenosine↗

PubMed1997

Regulation of serotonin-2C receptor G-protein coupling by RNA editing.

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several receptor subtypes. The 5-HT2 family of receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled receptors and includes three receptor subtypes (5-HT2A, 5-HT(2B) and 5-HT(2C)) which are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol. Here we show that transcripts encoding the 2C subtype of serotonin receptor (5-HT(2C)R) undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT(2C) receptor isoforms encoded by eleven distinct RNA species. Editing of 5-HT(2C)R messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the receptor and can lead to a 10-15-fold reduction in the efficacy of the interaction between receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this post-transcriptional modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled receptor superfamily.

3T3 Cells↗

PubMed1995

Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.

Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.

Adenosine Deaminase↗

PubMed1997

Extensive RNA editing and possible double-stranded structures determining editing sites in the atpB transcripts of hornwort chloroplasts.

Three nonsense codons and an unusual initiation codon were located within the putative coding region of the atpB gene of chloroplast DNA of the hornwort Anthoceros formosae. Nucleotide sequencing of cDNA prepared from transcripts revealed extensive RNA editing. The unusual initiation codon ACG was changed to AUG and three nonsense codons were converted into sense codons. In total 15 C residues of the genomic DNA were replaced by U residues in the mRNA sequences, while 14 U residues were replaced by C residues. This is the highest number of editing events for a chloroplast mRNA reported so far. Partial editing was also shown in a cDNA clone where 23 sites were edited but six sites remained unedited, representing the existence of premature mRNA. The expected two-dimensional structure of the mRNA shows the existence of a sequence complementary to every editing site, which can produce continuous base pairing longer than 5 bp, suggesting that mispairing in the double strand is the site determinant for RNA editing in Anthoceros chloroplasts. Comparison of the cDNA sequence with other chloroplast genes suggests that the mechanism arose in the first land plants and has been reduced during evolution.

Amino Acid Sequence↗

PubMed1992

RNA editing in the mitochondria of a conifer.

A 712-base portion of the mitochondrial gene coxI and the corresponding portion of the coxI transcript were amplified by PCR and by RT-PCR, respectively, from the gymnosperm western red cedar. Sequence comparison of amplified coxI DNA and mRNA revealed 26 C-to-U differences that are best explained by RNA editing of the type known to occur in angiosperms. This finding suggests that mitochondrial RNA-editing of the C-to-U type arose before the divergence of gymnosperms and angiosperms and can be considered a feature common to all higher plants.

Amino Acid Sequence↗

PubMed1993

Determinants of Ca2+ permeability in both TM1 and TM2 of high affinity kainate receptor channels: diversity by RNA editing.

GluR6, a subunit of high affinity kainate receptor channels in the mammalian CNS, carries a glutamine (Q) or arginine (R) residue in a critical position (Q/R site) of the putative channel-forming segment TM2. One form, GluR6(Q), is encoded by the GluR6 gene; the other, GluR6(R), is generated by RNA editing. Further analysis of cloned GluR6 cDNA revealed that two additional positions, located in transmembrane segment TM1, are diversified by RNA editing to generate either isoleucine (I) or valine (V) in one and tyrosine (Y) or cysteine (C) in the other TM1 position. In GluR6 channels, in contrast with AMPA receptor channels, the presence of Q in the TM2 Q/R site determines channels with low Ca2+ permeability, whereas an R determines a higher Ca2+ permeability if TM1 is fully edited. In the TM1 unedited form of GluR6, Ca2+ permeability is less dependent on the presence of either Q or R in TM2. Thus Ca2+ permeability of kainate receptor channels can vary, depending on editing of both TM1 and TM2.

Amino Acid Sequence↗

PubMed2026

A-to-I RNA editing remodels 5'-UTR initiation codons to tune translational output.

A-to-I RNA editing is a prevalent post-transcriptional modification in higher eukaryotes that converts adenosine to inosine within RNA molecules. Because inosine is interpreted as guanosine during translation, editing can alter codon identity and potentially influence translation initiation signals. Here, we examined whether A-to-I editing within the 5' untranslated region (5'-UTR) can remodel upstream initiation codons and thereby tune downstream translation. Using luciferase-based reporter systems, we show that AUA-to-AUI editing generates an initiation-competent inosine-containing codon, whereas AUG-to-IUG editing markedly attenuates initiation and can relieve uORF-mediated repression. Quantitative in vitro and cellular assays establish the initiation hierarchy AUA < AUI < AUG, with IUG exhibiting strongly reduced initiation efficiency. Importantly, AUI-mediated upstream initiation did not behave like a canonical AUG-initiated uORF in the tested contexts; its effect on downstream ORF translation was modest and context-dependent. Transcriptome-wide bioinformatic analysis identified endogenous human transcripts whose 5'-UTRs harbor editing sites compatible with initiation-codon gain or attenuation. Reporter validation using native 5'-UTR sequences supports the possibility that editing-dependent initiation-codon remodeling can tune translational output in living cells, particularly through AUG-to-IUG-mediated derepression. Together, these findings establish a reporter-based framework in which A-to-I editing can remodel 5'-UTR initiation codons, while highlighting the need for endogenous protein-level and native-locus validation to determine physiological relevance.

RNA Editing↗

PubMed2003

Developmental co-variation of RNA editing extent of plastid editing sites exhibiting similar cis-elements.

In tobacco, 30 of 34 sites in chloroplast transcripts that undergo C-to-U RNA editing can be grouped into clusters of 2-5 sites based on sequence similarities immediately 5' to the edited C. According to a previous transgenic analysis, overexpression of transcripts representing one cluster member results in reduction in editing of all cluster members, suggesting that members of an individual cluster share a trans-factor that is present in limiting amounts. To compare leaves and roots, we quantified the editing extent at 34 sites in wild-type tobacco and at three sites in spinach and Arabidopsis. We observed that transcripts of most NADH dehydrogenase subunits are edited inefficiently in roots. With few exceptions, members of the same editing site cluster co-varied in editing extent in chloroplasts versus non-green root plastids, with members of most clusters uniformly exhibiting either a high or low editing extent in roots. The start codon of the ndhD transcript must be created by editing, but the C target is edited inefficiently in roots, and no NDH-D protein could be detected upon immunoblotting. Our data are consistent with the hypothesis that cluster-specific trans-factors exist and that some are less abundant in roots, limiting the editing extent of certain sites in root plastids.

Arabidopsis↗

PubMed1992

RNA Editing of Cytochrome Oxidase Subunit III in Sunflower Mitochondria.

Direct sequencing of cytochrome oxidase subunit III (coxIII) mRNA with a specific primer confirms RNA editing in sunflower (Helianthus annus) mitochondria. Six instances of mRNA editing could be verified, one of these specific to this species. All the editing events involve C to U transitions in the coxIII mRNA causing codon changes that lead to amino acids better conserved in evolution than those encoded in the genomic DNA. This observation confirms RNA editing to be widespread in higher plant mitochondria.

Journal Article↗

PubMed1996

RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase.

Hepatitis delta virus (HDV) is a subviral human pathogen that requires hepatitis B virus (HBV) for packaging. Concurrent infection by HBV and HDV increases the risk of severe liver disease compared to infection with HBV alone. The HDV genome is a closed circular RNA of about 1,700 bases which is replicated through an RNA intermediate, the antigenome. Both RNAs can be folded into highly base-paired, rod-shaped structures, similar to the plant viroid RNAs. Two forms of the sole HDV protein, hepatitis delta antigen, are derived from a single open reading frame by RNA editing; the enzymes responsible for the editing have not been characterized. Here we report that the purified enzyme dsRAD (for double-stranded-RNA-adenosine deaminase) can edit HDV antigenomic RNA in vitro. Most important, we observe that mutations in critical sequences of the antigenome have identical effects on in vitro and in vivo editing, suggesting that dsRAD, or a closely related enzyme, is responsible for editing HDV RNA in vivo.

Adenosine↗

PubMed1998

RNA editing of brain glutamate receptor channels: mechanism and physiology.

Glutamate-gated cation selective channels mediate fast excitatory neurotransmission in the mammalian brain. Functionally critical channel positions contain amino acid residues not predicted from the exonic sequence for the channel subunits. The codons for these residues are created in the respective primary gene transcripts by the site selective deamination of adenosine to inosine. This type of RNA editing requires a short double-stranded RNA structure formed by the exonic sequence around the adenosine targeted for deamination with a complementary sequence in the downstream intron and hence, it precedes splicing. Candidate enzymes for nuclear transcript editing currently comprise three molecularly cloned mammalian RNA-dependent adenosine deaminases. Two of these are expressed in most body tissues, perhaps indicating that adenosine deamination in transcripts is more global than has been recognized. Indeed, numerous mRNAs in different tissues may contain inosine residues and encode proteins with amino acid substitutions and different properties relative to the exonically encoded forms. If so, RNA editing by adenosine deamination may significantly enlarge the functional repertoire of the mammalian genome.

Amino Acid Sequence↗

PubMed1996

RNA editing: how a message is changed.

Considerable progress has been made in unraveling the mechanistic features of RNA editing processes in a number of genetic systems. Recent highlights include the identification of the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme as a zinc-dependent cytidine deaminase that binds to RNA, the demonstration that adenosines in brain glutamate receptor pre-mRNAs are converted into inosines and that double-stranded RNA A deaminase (dsRAD), the candidate enzyme, is another zinc-dependent RNA nucleotide deaminase, and a mounting body of evidence for a cleavage-ligation mechanism for U insertion/deletion editing in kinetoplastid protozoa.

Animals↗

PubMed1996

Occurrence of silent RNA editing in chloroplasts: its species specificity and the influence of environmental and developmental conditions.

We have identified three new C-to-U RNA editing sites, one in atpF and two in atpA transcripts from tobacco chloroplasts. Two of them lead to amino acid substitutions to restore the conserved amino acid found in the corresponding genes of other plants. However, one editing site in the atpA transcript was found to take place partially at the third base of a serine codon (CUC_ to CUU_), thus not leading to an amino acid substitution. This is the first report of silent editing in chloroplasts. The extent of silent editing depends on plastid stage and light conditions, while editing as another site (found 4 nt upstream from the silent editing site) takes place constitutively even in non-photosynthetic cultured cells and bleached white seedlings grown in the presence of spectinomycin and streptomycin. In pea and spinach, despite a conservation in sequence, no editing at the site corresponding to the silent site in tobacco was found. This observation suggests that the silent editing detected in this study is species-specific.

Amino Acid Sequence↗