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PubMed2003

Hepatitis delta virus RNA encoding the large delta antigen cannot sustain replication due to rapid accumulation of mutations associated with RNA editing.

Hepatitis delta virus (HDV) contains two RNA species (HDV-S and HDV-L), which encode the small and large forms of hepatitis delta antigens (S- and L-HDAg), respectively. HDV-L RNA is a result of an RNA editing event occurring at an amber/W site of HDV-S RNA. RNA editing must be regulated to prevent premature and excessive accumulation of HDV-L RNA in the viral life cycle. In this study, we used an RNA transfection procedure to study the replication abilities of HDV-L and HDV-S RNA. While HDV-S led to robust RNA replication, HDV-L could not replicate even after 6 days following transfection. The failure of HDV-L to replicate was not due to insufficient amounts of S-HDAg, as identical results were obtained in a cell line that stably overexpresses S-HDAg. Also, it was not due to possible inhibition by L-HDAg, as HDV-S RNA replication was not affected when both HDV-L and HDV-S RNA were cotransfected. Further, when L-HDAg expression from HDV-L RNA was abolished by site-directed mutagenesis, the mutant HDV-L RNA also failed to replicate. Unexpectedly, when the kinetics of RNA replication was examined daily, HDV-L was found to replicate at a low level at the early time points (1 to 2 days posttransfection) but then lose this capability at later time points. Sequence analysis of the replicated HDV-L RNA at day 1 posttransfection showed that it had undergone multiple nucleotide changes, particularly in the region near the putative promoter region of HDV RNA replication. In contrast, very few mutations were found in HDV-S RNA. These results suggest that the editing at the amber/W site triggers a series of additional mutations which rapidly reduce the replication efficiency of the resultant HDV genome and thus help regulate the amount of HDV-L RNA in infected cells. They also explain why L-HDAg is not produced early in HDV infection, despite the fact that HDV-L RNA is present in the virion.

Cell Line, Tumor↗

PubMed2004

The binding selectivity of ADAR2's dsRBMs contributes to RNA-editing selectivity.

ADAR2 is an RNA editing enzyme that deaminates adenosines in certain duplex structures. Here, we describe the role of its RNA binding domain, consisting of two copies of a common dsRNA binding motif (dsRBM), in editing site selectivity. ADAR2's dsRBMs bind selectively on a duplex RNA that mimics the Q/R editing site in the glutamate receptor B-subunit pre-mRNA. This selectivity is different from that of PKR's dsRBM I, indicating that dsRBMs from different proteins possess intrinsic binding selectivity. Using directed hydroxyl radical cleavage data, molecular models were developed that predict important recognition surfaces on the RNA for identified dsRBM binding sites. Blocking these surfaces by benzyl modification of guanosine 2-amino groups impeded RNA-editing, demonstrating a correlation between deamination efficiency by ADAR2 and selective binding by its dsRBMs. In addition, the editing activity of a mutant of ADAR2 lacking dsRBM I on N(2)-benzylguanosine-modified RNA suggests the location of the dsRBM I binding site that leads to editing at the GluR-B Q/R site.

Adenosine Deaminase↗

PubMed1991

RNA editing in kinetoplastid protozoa.

RNA editing produces mature mRNAs by adding and removing uridines within the mitochondrial transcripts. The edited sequence appears to be specified by small complementary RNAs using a non-templated process that may have features resembling RNA splicing. The accumulation of edited mRNAs is developmentally regulated.

Animals↗

PubMed1996

RNA editing is required for efficient excision of tRNA(Phe) from precursors in plant mitochondria.

RNA editing corrects a 4C-A69 mismatch to a conventional 4T-A69 Watson-Crick base pair in the acceptor stem of the mitochondrially encoded tRNAPhe in plants. In vitro processing of edited and unedited Oenothera tRNA Phe precursor RNAs with pea mitochondrial protein extracts shows a significant effect of this RNA-editing event on the efficiency of 5' and 3' processing. While mature tRNA molecules are rapidly generated by in vitro processing from edited precursors, the formation of mature tRNAs from unedited pre-tRNAs is considerably reduced. Primer extension analyses of in vitro processing products show that processing at both 5' and 3' termini is governed by the RNA-editing event. Investigation of edited and unedited precursor RNAs by lead cleavage experiments reveals differences in the higher order structures of the pre-tRNAs. The differing conformations are most likely responsible for the altered processing efficiencies of edited and unedited precursor molecules. RNA editing of the tRNAPhe precursors is thus a prerequisite for efficient excision of the mature tRNAPhe in vitro. Hence RNA editing might be involved in regulating the amount of mature tRNAPhe in the steady state RNA pool of mitochondria in higher plants.

Amino Acid Sequence↗

PubMed1998

Identification and characterization of RNA editing events within the 5-HT2C receptor.

RNA editing is a post-transcriptional modification that generates an RNA transcript with a nucleotide sequence different from its gene. We have recently discovered RNA editing events, involving the conversion of adenosine bases to inosine residues, within the RNA encoding the serotonin 2C (5-HT2C) receptor. Editing events at four major positions, termed A, B, C and D, as well as one minor site termed C', are predicted to alter amino acids within the second intracellular loop of the G-protein coupled 5-HT2C receptor. Editing is mediated by at least two members of a family of adenosine deaminases and is contingent upon the presence of an extensive RNA duplex structure formed by exonic and intronic sequences of 5-HT2C receptor precursor messenger RNA (pre-mRNA). This critical secondary structure has been observed within brain pre-mRNA derived from four species; the isolation of edited 5-HT2C receptor transcripts from these samples further confirms the evolutionary conservation of this RNA processing event. Among members of the 5-HT2 receptor family, editing within second intracellular loop RNA is unique to the 5-HT2C receptor. Editing within the 5-HT2C receptor generates receptor isoforms that differ in their ability to interact with the phospholipase C signaling cascade in a transfected cell line, suggesting that this RNA processing event may contribute to the modulation of serotonergic neurotransmission in the central nervous system.

Amino Acid Sequence↗

PubMed1996

RNA editing: a mechanism for gRNA-specified uridylate insertion into precursor mRNA.

In the mitochondria of trypanosomatid protozoa the precursors of messenger RNAs (pre-mRNAs) have their coding information remodeled by the site-specific insertion and deletion of uridylate (U) residues. Small trans-acting guide RNAs (gRNAs) supply the genetic information for this RNA editing. An in vitro system was developed to study the mechanism of U insertion into pre-mRNA. U-insertion editing occurs through a series of enzymatic steps that begin with gRNA-directed pre-mRNA cleavage. Inserted U's are derived from free uridine triphosphate and are added to the 3' terminus of a 5' pre-mRNA cleavage product. gRNA specifies edited RNA sequence at the subsequent ligation step by base pairing-mediated juxtaposition of the 3' cleavage product and the processed 5' cleavage product. gRNA/pre-mRNA chimeras, purported intermediates, seem to be abortive end products of the same reaction.

Animals↗

PubMed2002

Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein.

Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3'-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

Amino Acid Sequence↗

PubMed2006

RNA editing site recognition in heterologous plant mitochondria.

RNA editing is a process that modifies the information content of mitochondrial messenger RNAs in flowering plants changing specific cytosine residues into uridine. To gain insight into editing site recognition, we used electroporation to introduce engineered wheat (Triticum aestivum) or potato (Solanum tuberosum) mitochondrial cox2 genes, and an atp9-containing chimeric gene, into non-cognate mitochondria, and observed the efficiency of editing in these contexts. Both wheat and potato mitochondria were able to express "foreign" constructs, and their products were properly spliced. Seventeen and twelve editing sites are present in the coding regions of wheat and potato cox2 transcripts, respectively. Eight are common to both plants, whereas nine are specific to wheat, and four to potato. An analogous situation is found for the atp9 mRNA coding regions from these species. We found that both mitochondria were able to recognize sites that are already present as T at the genomic level, making RNA editing unnecessary for that specific residue in the cognate organelle. Our results demonstrate that non-cognate mitochondria are able to edit residues that are not edited in their own transcripts, and support the hypothesis that the same trans-acting factor may recognize several editing sites.

Arabidopsis Proteins↗

PubMed2005

Effects of conserved RNA secondary structures on hepatitis delta virus genotype I RNA editing, replication, and virus production.

RNA editing of the hepatitis delta virus (HDV) antigenome at the amber/W site by the host RNA adenosine deaminase ADAR1 is a critical step in the HDV replication cycle. Editing is required for production of the viral protein hepatitis delta antigen long form (HDAg-L), which is necessary for viral particle production but can inhibit HDV RNA replication. The RNA secondary structural features in ADAR1 substrates are not completely defined, but base pairing in the 20-nucleotide (nt) region 3' of editing sites is thought to be important. The 25-nt region 3' of the HDV amber/W site in HDV genotype I RNA consists of a conserved secondary structure that is mostly base paired but also has asymmetric internal loops and single-base bulges. To understand the effect of this 3' region on the HDV replication cycle, mutations that either increase or decrease base pairing in this region were created and the effects of these changes on amber/W site editing, RNA replication, and virus production were studied. Increased base pairing, particularly in the region 15 to 25 nt 3' of the editing site, significantly increased editing; disruption of base pairing in this region had little effect. Increased editing resulted in a dramatic inhibition of HDV RNA synthesis, mostly due to excess HDAg-L production. Although virus production at early times was unaffected by this reduced RNA replication, at later times it was significantly reduced. Therefore, it appears that the conserved RNA secondary structure around the HDV genotype I amber/W site has been selected not for the highest editing efficiency but for optimal viral replication and secretion.

Adenosine Deaminase↗

PubMed1997

Insertional and deletional RNA editing in trypanosome mitochondria.

The mitochondrial mRNAs of trypanosomes are often post-transcriptionally modified by an RNA processing event, termed RNA editing, which results in the insertion or deletion of uridylate (U) residues in mRNAs. RNA editing is necessary for the formation of complete coding sequences for several essential mitochondrial proteins. The number and site of U addition and deletion is directed by small guide RNAs (gRNAs). Recent studies indicate that the mechanism of RNA editing in trypanosomes involves a series of enzymatic steps. We show that the initial step in this enzymatic cascade requires the formation of a binary RNA complex between the gRNA and its cognate pre-mRNA. Depletion of specific gRNAs inhibits cleavage of the pre-mRNA by an editing site specific endoribonuclease. Addition of synthetic gRNAs reverses this inhibition. All of the activities needed for RNA editing in vitro are present within a 19S ribonucleo-protein complex (RNP) composed of gRNAs, the editing site specific endonuclease, an RNA ligase, a terminal uridylate transferase (TUTase) and approximately 15 other unidentified proteins. We have recently identified and cloned the gene for a 45kDa protein, the RNA Editing Associated Protein-1 (REAP-1), which is a component of trypanosome editing complexes. REAP-1 co-purifies with RNA ligase and TUTase activities and is part of a > 700 kDa RNP containing gRNAs. Antibodies against REAP-1 inhibit in vitro RNA editing reactions confirming its role in RNA editing.

Animals↗

PubMed2005

RNA editing sites in plant mitochondria can share cis-elements.

RNA editing in flowering plant mitochondria alters numerous C nucleotides in a given mRNA molecule to U residues. To investigate whether neighbouring editing sites can influence each other we analyzed in vitro RNA editing of two sites spaced 30 nt apart. Deletion and competition experiments show that these two sites carry independent essential specificity determinants in the respective upstream 20-30 nucleotides. However, deletion of a an upstream sequence region promoting editing of the upstream site concomitantly decreases RNA editing of the second site 50-70 nucleotides downstream. This result suggests that supporting cis-/trans-interactions can be effective over larger distances and can affect more than one editing event.

Brassica↗

PubMed2005

RNA editing: a molecular mechanism for the fine modulation of neuronal transmission.

The term "RNA editing" is used to identify any mechanism responsible for producing mRNA molecules with sequence information not specifically encoded in the DNA. RNA editing is therefore an important event in gene modification, which takes place at a post-transcriptional level. The molecular mechanism of RNA editing involves site-selective deamination of adenosine to inosine in pre-mRNA, which leads to altering translation codons and splicing in nuclear transcripts, whereby functionally distinct proteins can be produced from a single gene. The mammalian editing enzymes ADARs (adenosine deaminases acting on RNA) are widely expressed in brain and other tissues: however, up until now their substrates have mainly been found in the Central Nervous System (CNS). Of particular relevance in the CNS is the editing occurring at the ionotropic glutamate receptors (GluRs) level. Three AMPA and two Kainate receptors are subject to RNA editing. The consequence of this process is the substitution of specific amino acids in functionally critical positions of the receptors. Depending on the GluR involved, the consequences of editing will involve: activation and/or inhibition of splicing sites; modulation of the trafficking of the receptor to the plasma membrane; the process of tetramerization of the receptor subunits; modification of the ions passage through the receptor channel; modulation of the desensitization and action potential recovery times. All these events are specific to the different GluRs and are genetically and developmentally controlled. RNA editing is therefore a crucial event involved in controlling transmission of the action potential at the postsynaptic level. This modulation involves the transmission of all sensory stimuli to the CNS and gives rise to the "Sensotype". The Sensotype therefore defines the "way" in which the information acquired from the environment by the sensory systems is transmitted to the brain. The signals and inputs deriving from the Sensotype are transmitted to the brain, which processes and stores these signals thus generating the "Brainotype". Brainotype and Sensotype are genetically and environmentally determined; they are individually unique and specific to every living organism with a nervous system. Their characteristics are, at least in part, dependent on the modulation of the "RNA editing" process since glutamate receptors represent the main neurotransmitter system in the CNS.

Animals↗

PubMed2000

Chloroplast RNA editing required for functional acetyl-CoA carboxylase in plants.

RNA editing is an important post-transcriptional process in chloroplasts and is thought to be functionally significant. Here we show a requirement of RNA editing for a functional enzyme. In peas, acetyl-CoA carboxylase (ACCase), a key enzyme of fatty acid synthesis, is composed of biotin carboxylase with the biotin carboxyl carrier protein and carboxyltransferase (CT). CT is composed of the nuclear-encoded alpha polypeptide and the chloroplast-encoded beta polypeptide in peas. One nucleotide of the beta polypeptide mRNA, which is edited in pea chloroplasts, converts the serine codon to the leucine codon. We show that this RNA editing is required for functional CT by comparing the unedited and edited recombinant enzymes. In plants not having a leucine codon at the same position, editing was shown to take place so as to create the leucine codon, indicating that editing is needed for in vivo CT activity and therefore for ACCase. To our knowledge, ACCase is an essential enzyme, suggesting that the chloroplast RNA editing is necessary for these plants.

Acetyl-CoA Carboxylase↗

PubMed2006

Extensive RNA editing in transcripts from the PsbB operon and RpoA gene of plastids from the enigmatic moss Takakia lepidozioides.

RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.

Base Sequence↗

PubMed2006

Mono- and dicotyledonous plant-specific RNA editing sites are correctly edited in both in organello systems.

We set out to analyse the phylogenetic distribution of cox2 RNA editing sites. Database searches have revealed the presence of mono- and dicotyledonous-specific RNA editing sites. Therefore, to better understand tRNA editing system in plants, we developed a new dicotyledonous in organello RNA editing system using cauliflower mitochondria and analysed the transcription of the cox2 gene for both maize and Arabidopsis. These results were compared with those obtained from a maize mitochondrial in organello system. Surprisingly, both the mono- and dicotyledonous cox2 transcripts were efficiently edited in the mitochondrial cauliflower and maize in organello systems, respectively, even for RNA editing sites not present in the endogenous cox2 sequences. Taken together, our observations support a self-guiding-transcript model for RNA editing in higher plants.

Arabidopsis↗

PubMed1999

RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs.

On the basis of the sequence of the mitochondrial genome in the flowering plant Arabidopsis thaliana, RNA editing events were systematically investigated in the respective RNA population. A total of 456 C to U, but no U to C, conversions were identified exclusively in mRNAs, 441 in ORFs, 8 in introns, and 7 in leader and trailer sequences. No RNA editing was seen in any of the rRNAs or in several tRNAs investigated for potential mismatch corrections. RNA editing affects individual coding regions with frequencies varying between 0 and 18.9% of the codons. The predominance of RNA editing events in the first two codon positions is not related to translational decoding, because it is not correlated with codon usage. As a general effect, RNA editing increases the hydrophobicity of the coded mitochondrial proteins. Concerning the selection of RNA editing sites, little significant nucleotide preference is observed in their vicinity in comparison to unedited C residues. This sequence bias is, per se, not sufficient to specify individual C nucleotides in the total RNA population in Arabidopsis mitochondria.

Arabidopsis↗

PubMed1991

Distribution of RNA editing sites in Oenothera mitochondrial mRNAs and rRNAs.

To investigate whether RNA editing in plant mitochondria modifies structural RNAs as well as protein-coding RNAs we compared the genomic-encoded information with the respective transcripts of several genes in Oenothera. The genes analysed are the 5S, 18S and 26 S rRNAs, the alpha-subunit of ATPase (atpA), cytochrome b (cytb), orfB, which is located upstream of cytochrome oxidase subunit III, and the respective leader, trailer and spacer sequences. All open reading frames were found to be edited to some degree. The atpA coding region has the least edited mRNA in Oenothera mitochondria, with only four nucleotides altered in the 1533 nucleotide open reading frame. From this analysis we conclude that frequent RNA editing is indicative of functional protein coding regions in plant mitochondria. The extensive editing in orfB, for example, suggests that this orf codes for a mitochondrial protein. No RNA editing event was found in the 5S rRNA or in the 1824 nucleotides analysed of the 18S rRNA, but two nucleotides were found to be altered in the 1970 nucleotides compared for the 26S rRNA. One nucleotide alteration has changed C to U, the other in reverse U to C. However, only one of five cDNA clones covering this region shows the modifications, similar to many silent editing events in open reading frames. RNA editing in the structural RNAs thus does not seem to be essential for their function in the mitochondrial ribosome.

Adenosine Triphosphatases↗

PubMed2001

RNA editing of the human serotonin 5-HT2C receptor. alterations in suicide and implications for serotonergic pharmacotherapy.

RNA encoding the human serotonin 5-HT2C receptor (5-HT(2C)R) undergoes adenosine-to-inosine RNA editing events at five positions, resulting in an alteration of amino acids in the second intracellular loop. Several edited 5-HT(2C)Rs possess a reduced G-protein coupling efficiency compared to the completely non-edited isoform. The current studies show that the efficacy of the hallucinogenic drug lysergic acid diethylamide and of antipsychotic drugs is regulated by RNA editing, suggesting that alterations in editing efficiencies or patterns might result in the generation of a 5-HT(2C)R population differentially responsive to serotonergic drugs. An examination of the efficiencies of RNA editing of the 5-HT(2C)R in prefrontal cortex of control individuals vs. subjects diagnosed with schizophrenia or major depressive disorder revealed no significant differences in RNA editing among the three populations. However, subjects who had committed suicide (regardless of diagnosis) exhibited a statistically significant elevation of editing at the A-site, which is predicted to change the amino acid sequence in the second intracellular loop of the 5-HT(2C)R. These findings suggest that alterations in RNA editing may contribute to or complicate therapy in certain psychiatric disorders.

Adult↗