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PubMed2001

Differences in conformational properties of the second intracellular loop (IL2) in 5HT(2C) receptors modified by RNA editing can account for G protein coupling efficiency.

Adenosine-to-inosine RNA editing events that have been demonstrated for 5HT (2C) receptors resulted in alterations of the amino acid sequence at positions 156, 158 and 160 in the intracellular loop 2 (IL2) region. The edited receptor isoforms were shown to have reduced basal activity, but similar maximum responses to agonist binding. To identify the molecular mechanism of these pharmacological effects of editing we explored the conformational properties of the edited IL2 in comparison with the wild type. The results from conformational studies of the IL2 isoforms, using biased Monte Carlo simulations with an implicit solvent model based on a screened Coulomb potential, show that the compared loops differ in their preferred spatial orientations as a result of differences in the conformational space that is accessible to them by energy criteria. For the IL2 of the unedited (5HT (2C-INI) ) receptor, the preference for structures oriented towards the 7TM bundle is larger than for the 5HT (2C-VGV) edited receptor. This difference in preferred orientation can affect the association of IL2 with other intracellular loop domains involved in G protein coupling and hence the coupling efficiency. The results illustrate the high sensitivity of the system to small changes in the interaction surface presented to other intracellular loops, and/or the G protein.

Amino Acid Sequence↗

PubMed1994

Intron sequence directs RNA editing of the glutamate receptor subunit GluR2 coding sequence.

The Ca2+ permeability and the rectifying properties of the glutamate receptors assembled from the subunits GluR1-GluR4 depend upon a critical Arg in the GluR2 subunit located in a domain that has been proposed to span the membrane. The GluR2 subunit gene encodes a Gln (CAG) at this position, whereas the mRNA is edited so that it encodes an Arg (CGG) at this position [Sommer, B., Kohler, M., Sprengel, R. & Seeburg, P. H. (1991) Cell 67, 11-20]. The editing process is specific since only the GluR2 subunit RNA is edited even though the GluR1, GluR3, and GluR4 RNAs have a similar sequence. We show that this selective RNA editing depends upon a critical intron sequence in the GluR2 gene. This critical intron sequence is sufficient to cause editing of the GluR3 subunit exon in a chimera minigene constructed so that the GluR3 exon is placed upstream to the GluR2 intron sequence. Transfections of a neuronal cell line, N2a, with minigene constructs encoding different fragments of the GluR2 gene demonstrate that the 5' part of the 3' intron is essential for editing. Part of the exon and this critical intron sequence contains an inverted repeat that can fold into a structure consisting of three helical elements. Similar conclusions were reached by Higuchi, M., Single, F. n., Köhler, M., Sommer, B., Sprengel, R. & Seeburg, P. H. [(1993) Cell 75, 1361-1370]. These experiments demonstrate that the low Ca2+ permeability of the ionotropic non-N-methyl-D-aspartate glutamate receptors depends upon RNA editing, which requires a sequence in an intron 3' to the exon.

Algorithms↗

PubMed2004

Adenosine deaminases acting on RNA (ADARs): RNA-editing enzymes.

Adenosine deaminases acting on RNA (ADARs) were discovered as a result of their ability extensively to deaminate adenosines in any long double-stranded RNA, converting them to inosines. Subsequently, ADARs were found to deaminate adenosines site-specifically within the coding sequences of transcripts encoding ion-channel subunits, increasing the diversity of these proteins in the central nervous system. ADARI is now known to be involved in defending the genome against viruses, and it may affect RNA interference. ADARs are found in animals but are not known in other organisms. It appears that ADARs evolved from a member of another family, adenosine deaminases acting on tRNAs (ADATs), by steps including fusion of two or more double-stranded-RNA binding domains to a common type of zinc-containing adenosine-deaminase domain.

Adenosine Deaminase↗

PubMed2003

Differential inhibition of RNA editing in hepatitis delta virus genotype III by the short and long forms of hepatitis delta antigen.

Hepatitis delta virus (HDV) produces two essential forms of the sole viral protein from the same open reading frame by using host RNA editing activity at the amber/W site in the antigenomic RNA. The roles of these two forms, HDAg-S and HDAg-L, are opposed. HDAg-S is required for viral RNA replication, whereas HDAg-L, which is produced as a result of editing, inhibits viral RNA replication and is required for virion packaging. Both the rate and amount of editing are important because excessive editing will inhibit viral RNA replication, whereas insufficient editing will reduce virus secretion. Here we show that for HDV genotype III, which is associated with severe HDV disease, HDAg-L strongly inhibits editing of a nonreplicating genotype III reporter RNA, while HDAg-S inhibits only when expressed at much higher levels. The different inhibitory efficiencies are due to RNA structural elements located ca. 25 bp 3' of the editing site in the double-hairpin RNA structure required for editing at the amber/W site in HDV genotype III RNA. These results are consistent with regulation of amber/W editing in HDV genotype III by a negative-feedback mechanism due to differential interactions between structural elements in the HDV genotype III RNA and the two forms of HDAg.

Cell Line↗

PubMed1994

RNA editing of the glutamate receptor subunits GluR2 and GluR6 in human brain tissue.

Editing of mRNA in the coding region of the second transmembrane domain of glutamate receptor subunits GluR2, GluR5, and GluR6 involves a change of the base A in genomic DNA to the base G in mRNA as described in rat brain. To determine whether this reaction occurs in humans as well as rats, we studied RNA editing of GluR2 and GluR6 in human brain. We compared the extent of editing in controls and cases with Huntington's disease. To assay the extent of editing in brain RNA, first strand cDNA was amplified using the polymerase chain reaction yielding a product across the region of the second transmembrane spanning segment in which editing takes place in rats. The PCR product was incubated with the restriction enzyme BbvI, which recognizes the sequence GCAGC present in the nonedited sequence of the mRNA in subunits GluR2 and GluR6. Thus, BbvI cuts the nonedited version but leaves the edited version intact. As in the rat, the GluR2 subunit mRNA was completely edited in human brain. The GluR6 subunit was nearly completely edited in all gray matter structures investigated including cortex, striatum, thalamus, hippocampus, amygdala, and cerebellum with extent of editing ranging from 89% in the cerebellum to 95% in the cortex and striatum. No significant differences in the extent of RNA editing were apparent in control versus Huntington's disease brains. To compare the extent of editing in neurons and glia in the brain, editing in cerebral cortex (predominantly gray matter and thus neurons) was compared with editing in corpus callosum (white matter and thus nearly completely glial cells).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1993

RNA editing in trypanosome mitochondria: guidelines for models.

Mitochondrial RNAs in trypanosomes are post-transcriptionally altered by uridine insertion and deletion. The information for these RNA editing processes, which are essential for the production of functional messengers, is provided by small guide RNAs. This article discusses how features of partially edited RNAs, gRNAs and chimeric RNAs, in which a gRNA is covalently linked to an editing site of pre-mRNA, have been used for the construction of models.

Animals↗

PubMed2000

Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis.

The members of the ADAR (adenosine deaminase acting on RNA) gene family are involved in site-selective RNA editing that changes adenosine residues of target substrate RNAs to inosine. Analysis of staged chimeric mouse embryos with a high contribution from embryonic stem cells with a functional null allele for ADAR1 revealed a heterozygous embryonic-lethal phenotype. Most ADAR1+/- chimeric embryos died before embryonic day 14 with defects in the hematopoietic system. Our results suggest the importance of regulated levels of ADAR1 expression, which is critical for embryonic erythropoiesis in the liver.

Adenosine Deaminase↗

PubMed1998

Hepatitis delta virus RNA editing is highly specific for the amber/W site and is suppressed by hepatitis delta antigen.

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.

Base Sequence↗

PubMed2001

The human but not the Xenopus RNA-editing enzyme ADAR1 has an atypical nuclear localization signal and displays the characteristics of a shuttling protein.

The RNA-editing enzyme ADAR1 (adenosine deaminase that acts on RNA) is a bona fide nuclear enzyme that has been cloned from several vertebrate species. Putative nuclear localization signals (NLSs) have been identified in the aminoterminal regions of both human and Xenopus ADAR1. Here we show that neither of these predicted NLSs is biologically active. Instead, we could identify a short basic region located upstream of the RNA-binding domains of Xenopus ADAR1 to be necessary and sufficient for nuclear import. In contrast, the homologous region in human ADAR1 does not display NLS activity. Instead, we could map an NLS in human ADAR1 that overlaps with its third double-stranded RNA-binding domain. Interestingly, the NLS activity displayed by this double-stranded RNA-binding domain does not depend on RNA binding, therefore showing a dual function for this domain. Furthermore, nuclear accumulation of human (hs) ADAR1 is transcription dependent and can be stimulated by LMB, an inhibitor of Crm1-dependent nuclear export, indicating that hsADAR1 can move between the nucleus and cytoplasm. Regulated nuclear import and export of hsADAR1 can provide an excellent mechanism to control nuclear concentration of this editing enzyme thereby preventing hyperediting of structured nuclear RNAs.

3T3 Cells↗

PubMed2006

Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of a gRNA bound to its cognate mRNA.

Expression of mitochondrial genes in Trypanosoma brucei requires RNA editing of its mRNA transcripts. During editing, uridylates are precisely inserted and deleted as directed by the gRNA template to create the protein open reading frame. This process involves the bimolecular interaction of the gRNA with its cognate pre-edited mRNA and the assembly of a protein complex with the enzymatic machinery required. While a considerable amount of work has been done identifying the protein components of the editing complex, very little is known about how a functional editosome is assembled. In addition, the importance of RNA structure in establishing a functional editing complex is poorly understood. Work in our lab suggests that different mRNA/gRNA pairs can form similar secondary structures suggesting that a common core architecture may be important for editosome recognition and function. Using solution structure probing, we have investigated the structure of the initiating gRNA, gCYb-558, in the mRNA/gRNA complex with pre-edited apocytochrome b mRNA. Our data indicate that the stem-loop formed by the guiding region of the gRNA alone is maintained in its interaction with the pre-edited message. In addition, our data suggest that a gRNA stem-loop structure is maintained through the first few editing events by the use of alternative base-pairing with the U-tail.

Animals↗

PubMed1998

T. brucei RNA editing: adenosine nucleotides inversely affect U-deletion and U-insertion reactions at mRNA cleavage.

In the currently envisioned mechanism of trypanosome mitochondrial RNA editing, U-insertion and U-deletion cycles begin with a common kind of gRNA-directed cleavage. However, natural, altered, and mutationally interconverted editing sites reveal that U-deletional cleavage is inefficient without and activated by ATP and ADP, while U-insertional cleavage shows completely reverse nucleotide effects. The adenosine nucleotides' effects appear to be allosteric and determined solely by sequences immediately adjacent to the anchor duplex. Both U-deletional and U-insertional cleavages are reasonably active at physiological mitochondrial ATP concentration. Notably, ATP and ADP markedly stimulate complete U-deletion and inhibit U-insertion reactions, reflecting their effects on cleavage. These plus previous results suggest that U deletion and U insertion are remarkably distinct.

Adenosine↗

PubMed1999

Serotonin 5-HT2C receptor RNA editing alters receptor basal activity: implications for serotonergic signal transduction.

Rat and human serotonin 5-HT2C receptor isoforms were evaluated for agonist-independent activation of inositol phosphate production in COS-7 cells. The nonedited isoform (5-HT(2C-INI)) displayed the greatest basal activity, stimulating inositol phosphate production fourfold over the fully edited isoform (5-HT(2C--VGV)). All of the other isoforms tested displayed intermediate levels of basal activity. Decreasing receptor expression levels by 50% produced a parallel decrease in basal activity. 5-HT stimulated inositol phosphate production twofold over basal levels through the 5-HT(2C-INI) receptor and eightfold over basal levels through the 5-HT(2C-VGV) receptor but produced similar maximal levels of inositol phosphate. 5-HT competition for [3H]mesulergine binding to 5-HT(2C-INI) best fit a two-site analysis with K(H) = 7.6 nM and K(L) = 160 nM, whereas 5-HT(2C-VGV) best fit a one-site model with Ki = 163 nM. [3H]5-HT labeled 36% of the total population of 5-HT(2C-INI) receptors labeled by [3H]mesulergine but only 12% of 5-HT(2C-VGV) receptors. [H]5-HT K(D) values increased from 5.1 nM for 5-HT(2C-INI) to 20 nM for 5-HT(2C-VGV). [3H]Mesulergine K(D) values were the same for both isoforms. 5-HT EC50 values for inositol phosphate production increased from 6.1 nM for 5-HT(2C-INI) to 30 nM for 5-HT(2C-VGV). These results demonstrate that RNA editing decreases 5-HT2C receptor basal activity, agonist affinity, and potency, indicating that RNA editing may play a role in regulating serotonergic signal transduction and response to drug therapy.

Animals↗

PubMed2004

Persistent tetrodotoxin-sensitive sodium current resulting from U-to-C RNA editing of an insect sodium channel.

The persistent tetrodotoxin (TTX)-sensitive sodium current, detected in neurons of many regions of mammalian brains, is associated with many essential neuronal activities, including boosting of excitatory synaptic inputs, acceleration of firing rates, and promotion of oscillatory neuronal activities. However, the origin and molecular basis of the persistent current have remained controversial for decades. Here, we provide direct evidence that U-to-C RNA editing of an insect sodium channel transcript generates a sodium channel with a persistent current. We detected a persistent TTX-sensitive current in a splice variant of the cockroach sodium channel gene BgNa(v) (formerly para(CSMA)). Site-directed mutagenesis experiments revealed that an F-to-S change at the C-terminal domain of this variant was responsible for the persistent current. We demonstrated that this F-to-S change was the result of a U-to-C RNA editing event, which also occurred in the Drosophila para sodium channel transcript. Our work provides direct support for the hypothesis that posttranscriptional modification of a conventional transient sodium channel produces a persistent TTX-sensitive sodium channel.

Animals↗

PubMed1994

Developmental changes in the extent of RNA editing of glutamate receptor subunit GluR5 in rat brain.

The extent of RNA editing of the glutamate receptor subunit GluR5 mRNA was evaluated in the cortex, hippocampus and cerebellum of embryonic rat brains, aged 19 days, and adult brains, aged 2-3 months. RNA was isolated and transcribed into cDNA, which was used as template for amplifying a PCR product across the edited region of the GluR5 subunit. Quantification of editing was performed by restriction digest of PCR products with BbvI and image analysis of bands obtained after electrophoresis. The extent of editing was significantly lower in the embryonic state as compared to the adult state. These differences were most pronounced in the cerebellum where the extent of editing amounted to 27 +/- 8% and 78 +/- 2% in the embryonic and adult state, respectively (P < 0.001). Since expression of GluR5 is relatively high in brain areas of neuronal differentiation and synapse formation it is concluded that un-edited GluR5 may play a role in the development of the nervous system.

Animals↗

PubMed1993

RNA Editing in Plant Mitochondria: [alpha]-Phosphate Is Retained during C-to-U Conversion in mRNAs.

RNA editing in higher plant mitochondria frequently results in the post-transcriptional conversion of specific cytidine residues to uridine residues and infrequently results in the reverse conversion. The mechanisms by which this transition could occur are deamination or transamination of the amide at C-4 of cytosine, transglycosylation of the ribosyl residue, or deletion of a CMP residue and insertion of a UMP residue. Intact maize or petunia mitochondria were supplied with [alpha]-32P-CTP to radiolabel CMP residues in the nascent transcripts, and the fate of the [alpha]-phosphate was examined by digestion of the RNA to nucleotide monophosphates and analysis by two-dimensional chromatography. A small fraction of radioactivity comigrated with UMP on two different two-dimensional thin-layer chromatography systems, and the amount of radiolabeled UMP increased between l0-min and 2-hr incubations. The conversion of cytidine-to-uridine residues was detected in the highly edited mRNA fraction but was not detected in the rRNA fraction. Recovery of radiolabeled UMP residues suggests that the [alpha]-phosphate is retained during the editing reaction. These results are consistent with either deamination or transamination, or transglycosylation mechanisms for RNA editing.

Journal Article↗

PubMed1994

RNA editing in the Wilms' tumor susceptibility gene, WT1.

Rat kidney WT1 cDNAs contain either a thymidine or a cytosine residue at position 839. Genomic WT1 DNA contains only T839. To explain these results, we propose the WT1 transcript undergoes RNA editing in which U839 is converted to C, resulting in the replacement of leucine 280 in WT1 by proline. RNA editing at the same nucleotide was observed in WT1 cDNAs from human testis. In functional assays, the WT1-leucine280 polypeptide repressed the EGR-1 promoter in in vitro assays approximately 30% more efficiently than WT1-proline. Edited WT1-C839 mRNA was barely detectable in neonatal kidney, whereas adult rat kidneys contained both U839 and C839-WT1 mRNA, suggesting a role for the two protein isoforms in growth and differentiation.

Animals↗

PubMed1997

Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.

Non-NMDA glutamate receptor subunits of the AMPA-preferring subfamily combine to form ion channels with heterogeneous functional properties. We have investigated the effects of RNA editing at the Q/R site, splice variation of the "flip/flop" cassette, and multimeric subunit assembly on the single-channel conductance and kinetic properties of the recombinant AMPA receptors formed from GluR2 and GluR4 expressed in HEK 293 cells. We found that AMPA receptor single-channel conductance was dependent on the Q/R site editing state of the subunits comprising the channel. Calcium-permeable (unedited) channels had resolvable single-channel events with main conductance states of 7-8 pS, whereas fully edited GluR2 channels had very low conductances of approximately 300 fS (estimated from noise analysis). Additionally, the flip splice variant of GluR4 conferred agonist-dependent conductance properties reminiscent of those found for a subset of AMPA receptors in cultured cerebellar granule cells. These results provide a description of the single-channel properties of certain recombinant AMPA receptors and suggest that the single-channel conductance may be determined by the expression of edited GluR2 subunits in neurons.

Cell Line↗

PubMed2005

Letter from the editor: Adenosine-to-inosine RNA editing in Alu repeats in the human genome.

Adenosine-to-inosine (A-to-I) RNA editing increases the complexity of the human transcriptome and is essential for maintenance of normal life in mammals. Most A-to-I substitutions occur within repetitive elements in the genome, mainly in Alu repeats. The phenomenon of A-to-I editing is far less abundant in mice, rats, chickens and flies than in humans, which correlates with the relative under-representation of Alu repeats in these non-primate genomes. Here, we review the recent results of bioinformatic and laboratory approaches that have estimated the extent of the editing phenomenon. We discuss the possible biological relevance of the editing pathway, its possible interaction with other cellular pathways that respond to double-stranded RNA and its possible contribution to the accelerated evolution of primates.

Adenosine↗