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PubMed1991

Expression of the wheat mitochondrial nad3-rps12 transcription unit: correlation between editing and mRNA maturation.

In plant mitochondria, RNA editing involves the conversion of cytidines in the genomic DNA into uridines in the corresponding RNA. Analysis of cDNAs prepared by reverse transcription of mitochondrial RNAs has shown that partially edited RNAs are present in wheat mitochondria. The extent of this partial editing as well as its potential influence on the corresponding protein sequence were studied along with the expression of a wheat mitochondrial locus. The sequence, expression, and RNA editing of the wheat mitochondrial transcription unit containing four open reading frames (nad3, rps12, orf299, orf156), all cotranscribed into a same predominant precursor RNA, have been studied. The product of orf156 is an 18-kD mitochondrial membrane protein of unknown function, whereas the product of orf299 could not be detected and this sequence seems to be a pseudogene. Sequences of cDNA clones derived by the polymerase chain reaction technique show that nad3, rps12, and orf156 transcripts are edited, whereas orf299 is not edited, except for a sequence identical to part of the coxII gene. Analysis of cDNA clones obtained from the precursor RNA shows the presence of a large number of partially edited nad3-rps12 transcripts with no evident polarity for the editing process. This shows that RNA editing is a post-transcriptional event. In addition, study of partial editing at the level of precursor, mature, and polysomal transcripts shows that mainly mature, completely edited sequences are used for translation. Deletions of a nucleotide at editing sites were observed in a number of cDNA clones, suggesting that C----U RNA editing in plant mitochondria would be achieved by nucleotide replacement.

Amino Acid Sequence↗

PubMed1994

The NADH dehydrogenase subunit 7 gene is interrupted by four group II introns in the wheat mitochondrial genome.

We have characterized a wheat mitochondrial gene, designated nad7, capable of encoding a 394-amino acid subunit of the respiratory chain NADH dehydrogenase complex. It contains four introns possessing group II features and their positions differ from those in both the liverwort mitochondrial nad7 pseudogene and the nuclear gene encoding the homologous 49 kDa subunit of complex I in Neurospora. The derived amino acid sequence of the wheat nad7 gene is strongly conserved relative to its nuclear or organellar counterparts in other organisms. C-to-U type RNA editing, which is observed at 32 positions within the coding region of wheat nad7 transcripts, strengthens protein sequence similarity. RNA editing is also predicted to improve base-pairing within the domain V/VI regions of all four introns.

Amino Acid Sequence↗

PubMed2006

The effect of down-regulation of mitochondrial RNA-binding proteins MRP1 and MRP2 on respiratory complexes in procyclic Trypanosoma brucei.

MRP1 and MRP2 are multifunctional mitochondrial RNA-binding proteins with a regulatory role in RNA editing and putative role(s) in RNA processing in Trypanosoma brucei. Silencing of MRP1 and/or MRP2 by RNA interference affected the assembly and functionality of respiratory complexes. The absence of several subunits of complexes I, III and IV resulted in their disintegration and subsequent decrease of specific activities and also caused a significant decrease of membrane potential. The overall respiration in the interfered cells decreased by only about 20%, since the trypanosome alternative oxidase effectively replaced the missing cytochromes and became the principal terminal oxidase.

Animals↗

PubMed2001

ADAR1 is involved in the development of microvascular lung injury.

Deamination of adenosine on pre-mRNA to inosine is a recently discovered process of posttranscription modification of pre-mRNA, termed A-to-I RNA editing, which results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the development of microvascular lung injury. To that end, the pulmonary expression and activity of the RNA editase ADAR1 were evaluated in a mouse model of endotoxin (15 mg/kg IP)-induced microvascular lung injury (n=5) as well as in cultured alveolar macrophages stimulated with endotoxin, live bacteria, or interferon. ADAR1 expression and activity were identified in sham lungs that were upregulated in lungs from endotoxin-treated mice (at 2 hours). Expression was localized to polymorphonuclear and monocytic cells. These events preceded the development of pulmonary edema and leukocyte accumulation in lung tissue and followed the local production of interferon-gamma, a known inducer of ADAR1 in other cell systems. ADAR1 was found to be upregulated in alveolar macrophages (MH-S cells) stimulated with endotoxin (1 to 100 microg/mL), live Escherichia coli (5x10(7) colony-forming units), or interferon-gamma (1000 U/mL). Taken together, these data suggest that ADAR1 may play a role in the pathogenesis of microvascular lung injury possibly through induction by interferon.

Adenosine↗

PubMed2005

DNA cleavage in immunoglobulin somatic hypermutation depends on de novo protein synthesis but not on uracil DNA glycosylase.

Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step of Ig somatic hypermutation (SHM). However, its molecular mechanism is controversial. The RNA editing hypothesis postulates that AID deaminates cytosine in an unknown mRNA to generate a new mRNA encoding SHM endonuclease. On the other hand, the DNA deamination hypothesis explains DNA cleavage by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase (UNG). By using the protein synthesis inhibitor cycloheximide, we showed that SHM requires de novo protein synthesis in accord with predictions by the RNA editing hypothesis. In addition, we found that cycloheximide but not Ugi (the specific inhibitor of UNG) inhibited AID-dependent DNA cleavage in the Ig gene during SHM, by using histone H2AX focus formation as a marker of DNA cleavage. The results indicate the following order of events: AID expression, protein synthesis, DNA cleavage, and SHM. The requirement of protein synthesis but not of UNG for the DNA cleavage step of SHM forces us to reconsider the DNA deamination hypothesis and strengthens the RNA editing hypothesis.

Animals↗

PubMed1993

Editing of transfer RNAs in Acanthamoeba castellanii mitochondria.

With the discovery of RNA editing, a process whereby the primary sequence of RNA is altered after transcription, traditional concepts of genetic information transfer had to be revised. The known RNA editing systems act mainly on messenger RNAs, introducing sequence changes that alter their coding properties. An editing system that acts on transfer RNAs is described here. In the mitochondria of Acanthamoeba castellanii, an amoeboid protozoan, certain transfer RNAs differ in sequence from the genes that encode them. The changes consist of single-nucleotide conversions (U to A, U to G, and A to G) that appear to arise posttranscriptionally, are localized in the acceptor stem, and have the effect of correcting mismatched base pairs. Editing thus restores the base pairing expected of a normal transfer RNA in this region.

Acanthamoeba↗

PubMed2000

Wilms' tumor suppressor gene WT1: from structure to renal pathophysiologic features.

Normal development of the kidney is a highly complex process that requires precise orchestration of proliferation, differentiation, and apoptosis. In the past few years, a number of genes that regulate these processes, and hence play pivotal roles in kidney development, have been identified. The Wilms' tumor suppressor gene WT1 has been shown to be one of these essential regulators of kidney development, and mutations in this gene result in the formation of tumors and developmental abnormalities such as the Denys-Drash and Frasier syndromes. A fascinating aspect of the WT1 gene is the multitude of isoforms produced from its genomic locus. In this review, our current understanding of the structural features of WT1, how they modulate the transcriptional and post-transcriptional activities of the protein, and how mutations affecting individual isoforms can lead to diseased kidneys is summarized. In addition, results from transgenic experiments, which have yielded important findings regarding the function of WT1 in vivo, are discussed. Finally, data on the unusual feature of RNA editing of WT1 transcripts are presented, and the relevance of RNA editing for the normal functioning of the WT1 protein in the kidney is discussed.

Animals↗

PubMed2005

[Death of motor neurons and molecular change of glutamate receptors in ALS].

AMPA receptor-mediated neuronal death is initiated by exaggerated Ca2+ influx through AMPA receptor channels, and the Ca2+ permeability of the AMPA receptor ion channel depends strongly upon the presence or absence in its composition of an edited GluR2 subunit whose glutamine (Q) residue is substituted by arginine (R) at the Q/R site due to RNA editing. The pivotal role of the RNA editing at the GluR2 Q/R site in neuronal death has been clearly demonstrated in animal experiments and its deficiency is a direct cause of neuronal death. We demonstrated that the editing efficiency at the GluR2 mRNA Q/R site varied greatly, from 0% to 100%, among the single motoneurons of each individual with ALS, whereas it remained 100% among those of normal controls. In addition, the editing efficiency was more than 99% in the cerebellar Purkinje cells of ALS, spinocerebellar degeneration and normal control groups. By contrast, there was no significant difference as to both the amount and the proportion to total GluRs mRNA of GluR2 mRNA between normal and ALS motoneurons. Thus, marked GluR2 underediting in ALS motoneurons occurs in a disease specific and region selective manner, and may be closely relevant to ALS etiology.

Amyotrophic Lateral Sclerosis↗

PubMed2001

Underediting of glutamate receptor GluR-B mRNA in malignant gliomas.

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.

Adenosine Deaminase↗

PubMed2002

The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: frequent DNA sequence acquisition and loss during the evolution of flowering plants.

The entire mitochondrial genome of rice (Oryza sativa L.), a monocot plant, has been sequenced. It was found to comprise 490,520 bp, with an average G+C content of 43.8%. Three rRNA genes, 17 tRNA genes and five pseudo tRNA sequences were identified. In addition, eleven ribosomal protein genes and two pseudo ribosomal protein genes were found, which are homologous to 13 of the 16 genes for ribosomal proteins in the mitochondrial genome of the liverwort (Marchantia polymorpha). A greater degree of variation in terms of presence/absence and integrity of genes was observed among the ribosomal protein genes and tRNA genes of rice, Arabidopsis and sugar beet. Transcription and post-transcriptional modification (RNA editing) in the rice mitochondrial sequence were also examined. In all, 491 Cs in the genomic DNA were converted to Ts in cDNA. The frequency of RNA editing differed markedly depending upon the ORF considered. Sequences derived from plastid and nuclear genomes make up 6.3% and 13.4% of the mitochondrial genome, respectively. The degree of conservation of plastid sequences in the mitochondrial genome ranged from 61% to 100%, suggesting that sequence migration has occurred very frequently. Three plastid DNA fragments that were incorporated into the mitochondrial genome were subsequently transferred to the nuclear genome. Nineteen fragments that were similar to transposon or retrotransposon sequences, but different from those found in the mitochondrial genomes of dicots, were identified. The results indicate frequent and independent DNA sequence flow to and from the mitochondrial genome during the evolution of flowering plants, and this may account for the range of genetic variation observed between the mitochondrial genomes of higher plants.

Biological Evolution↗

PubMed2002

Transcript abundance supercedes editing efficiency as a factor in developmental variation of chloroplast gene expression.

In maize plastids, transcripts are known to be modified at 27 C-to-U RNA editing sites, affecting the expression-of 15 different genes. The relative contribution of editing efficiency versus transcript abundance in regulation of chloroplast gene expression has previously been analyzed for only a few genes. We undertook a comprehensive analysis of the editing efficiency of each of the 27 maize editing sites in 10 different maize tissues, which contain a range of plastid types including chloroplasts, etioplasts, and amyloplasts. Using a reproducible poisoned primer extension assay, we detected variation between RNA editing extent of different sites in the same transcript in the same tissue, and between the same site in different tissues. The most striking editing deficiency is in an editing site in ndhB that is edited at only 8% and 1% in roots and callus plastids respectively, whereas green leaf chloroplasts edit this site at 100%. Editing efficiencies of some sites are not affected by the developmental stages we examined and are always edited close to 80-100%. The relative amounts of transcripts of each of the 10 genes that exhibited variable editing extents were determined by real-time PCR. Seven genes exhibited over 100 times lower transcript abundance in either roots or tissue-cultured cells relative to green leaf tissue. The quantitative analysis indicates that a particular editing site can be efficiently edited over a large range of transcript abundance, resulting in no general correlation of transcript abundance and editing extent. The independent variation of editing efficiency of different sites within the same transcript fits with a model that postulates individual trans-acting factors specific to each editing site. Because tissues where editing efficiency at certain sites is low invariably also exhibited greatly decreased abundance of the transcripts carrying those sites, decrease in the amounts of particular RNAs rather than a lack of editing is predicted to have the most significant impact on gene expression under steady-state conditions. Our data is consistent with the hypothesis that the role of editing in angiosperm plastids is to correct otherwise detrimental mutations rather than to generate significant protein diversity.

Amino Acid Sequence↗

PubMed1997

Disruption of a gene encoding a novel mitochondrial DEAD-box protein in Trypanosoma brucei affects edited mRNAs.

The majority of mitochondrial pre-mRNAs in kinetoplastid protozoa such as Trypanosoma, Leishmania, and Crithidia are substrates of a posttranscriptional processing reaction referred to as RNA editing. The process results in the insertion and, to a lesser extent, deletion of uridylates, thereby completing the informational content of the mRNAs. The specificity of the RNA editing reaction is provided by guide RNAs (gRNAs), which serve as templates for the editing apparatus. In addition, the process relies on mitochondrial proteins, presumably acting within a high-molecular-mass ribonucleoprotein complex. Although several enzymatic activities have been implicated in the editing process, no protein has been identified to date. Here we report the identification of a novel mitochondrial DEAD-box protein, which we termed mHel61p. Disruption of the mHEL61 alleles in insect-stage Trypanosoma brucei cells resulted in a reduced growth rate phenotype. On a molecular level, the null mutant showed significantly reduced amounts of edited mRNAs, whereas never-edited and nuclear mRNAs were unaffected. Reexpression of mHel61p in the knockout cell line restored the ability to efficiently synthesize edited mRNAs. The results suggest an involvement of mHel61p in the control of the abundance of edited mRNAs and thus reveal a novel function for DEAD-box proteins.

Alleles↗

PubMed2002

Composition of the editing complex of Trypanosoma brucei.

The RNA editing that produces most functional mRNAs in trypanosomes is catalysed by a multiprotein complex. This complex catalyses the endoribonucleolytic cleavage, uridylate addition and removal, and RNA ligation steps of the editing process. Enzymatic and in vitro editing analyses reveal that each catalytic step contributes to the specificity of the editing and, together with the interaction between gRNA and the mRNA, results in precisely edited mRNAs. Tandem mass spectrometric analysis was used to identify the genes for several components of biochemically purified editing complexes. Their identity and presence in the editing complex were confirmed using immunochemical analyses utilizing mAbs specific to the editing complex components. The genes for two RNA ligases were identified. Genetic studies show that some, but not all, of the components of the complex are essential for editing. The TbMP52 RNA ligase is essential for editing while the TbMP48 RNA ligase is not. Editing was found to be essential in bloodstream form trypanosomes. This is surprising because mutants devoid of genes encoding RNAs that become edited survive as bloodstream forms but encouraging since editing complex components may be targets for chemotherapy.

Amino Acid Sequence↗

PubMed2001

Unique aspects of mitochondrial biogenesis in trypanosomatids.

Mitochondrial biogenesis consists of the sum of all processes required for the formation of the mitochondrial membranes as well as the soluble compartments they contain. Furthermore, it includes the replication of the mitochondrial genome and correct segregation of the organelles during cell division. Mitochondrial proteins come from two sources, a limited but essential set of inner membrane proteins is encoded by the mitochondrial genome, whereas the large majority (90-95%) is derived from nucleus-encoded genes and are posttranslationally imported into the organelle. Trypanosomatids belong to the earliest diverging branches of the eukaryotic evolutionary tree which have mitochondria. This is reflected in the organisation of their mitochondrial DNA that consists of a network of two classes of topologically interlocked circular DNA molecules as well as many unique features in their mitochondrial biogenesis. The proteins encoded on the mitochondrial genome are conventional for a mitochondrial genome, their expression, however, involves a complex series of processes. Many genes represent incomplete open reading frames and their primary transcripts have to remodelled by RNA editing to convert them into translatable mRNAs. RNA editing is mediated by small mitochondria-encoded transcripts, the guide RNAs, and is in that form specific for trypanosomatids and closely related organisms. Mitochondrial translation is also unconventional. No tRNA genes are encoded on the mitochondrial genome. Instead, mitochondrial protein synthesis functions exclusively with imported cytosolic, eukaryotic-type tRNAs. The composition of mitochondrial ribosomes is also unusual in that they contain the smallest known rRNAs. They are about 30% shorter than the already much reduced rRNAs in human mitochondria. Furthermore, the topological organisation of the mitochondrial genome requires an elaborate replication machinery involving topoisomerases. Finally, some trypanosomatids have life cycle stages exhibiting very different mitochondrial activities and can therefore serve as a model system for the regulation of mitochondrial biogenesis.

Animals↗

PubMed1995

Double-stranded RNA adenosine deaminase binds Z-DNA in vitro.

A Z-DNA binding protein of 140,000 M(r) has been purified from chicken lungs by sedimentation through 40%(w/w) sucrose and Z-DNA affinity chromatography. Specificity of the protein for Z-DNA was confirmed by competition with polyd(CG) that had been stabilized in the Z-DNA conformer by chemical bromination and also with a supercoiled plasmid that contains a Z-DNA-forming insert. In addition to a Z-DNA binding site, the protein also has a separate binding site for double-stranded RNA. Peptide sequence of the protein shows that it has high similarity to the RNA editing enzyme double-stranded RNA adenosine deaminase (dsRAD), which deaminates adenosine in dsRNA to form inosine. The Z-DNA binding protein has this enzymatic activity, confirming its identity to dsRAD. Recombinant human dsRAD also binds to Z-DNA. Z-DNA is stabilized in a sequence-dependent manner by negative supercoiling, which occurs in actively transcribed genes upstream to RNA polymerase. It is proposed that Z-DNA links editing to transcription by localizing dsRAD to a particular region of a gene and thus determines the efficiency with which an RNA is edited. The presence of Z-DNA forming elements in many genes raises the possibility that RNA editing by dsRAD is far more prevalent than is currently thought.

Adenosine Deaminase↗

PubMed2002

Extensive editing of mRNAs for the squid delayed rectifier K+ channel regulates subunit tetramerization.

We report the extensive editing of mRNAs that encode the classical delayed rectifier K+ channel (SqK(v)1.1A) in the squid giant axon. Using a quantitative RNA editing assay, 14 adenosine to guanine transitions were identified, and editing efficiency varied tremendously between positions. Interestingly, half of the sites are targeted to the T1 domain, important for subunit assembly. Other sites occur in the channel's transmembrane spans. The effects of editing on K+ channel function are elaborate. Edited codons affect channel gating, and several T1 sites regulate functional expression as well. In particular, the edit R87G, a phylogenetically conserved position, reduces expression close to 50-fold by regulating the channel's ability to form tetramers. These data suggest that RNA editing plays a dynamic role in regulating action potential repolarization in the giant axon.

Action Potentials↗

PubMed1997

Trypanosoma brucei gBP21. An arginine-rich mitochondrial protein that binds to guide RNA with high affinity.

RNA editing in Trypanosoma brucei is a mitochondrial RNA processing reaction that results in the insertion and deletion of uridylate residues into otherwise untranslatable mRNAs. The process is directed by guide RNAs which function to specify the edited sequence. RNA editing in vitro requires mitochondrial protein extracts and guide RNAs have been identified as part of high molecular weight ribonucleoprotein complexes. Within the complexes, the RNAs are in close contact with several mitochondrial proteins and here we describe the isolation and cloning of a gRNA-interacting polypeptide from Trypanosoma brucei. The protein was named gBP21 for guide RNA-binding protein of 21 kDa. gBP21 shows no homology to proteins in other organisms, it is arginine-rich and binds to gRNA molecules with a dissociation constant in the nanomolar range. The protein does not discriminate for differences in the primary structures of gRNAs and thus likely binds to higher order structural features common to all gRNA molecules. gBP21 binding does not perturb the overall structure of gRNAs but the gRNA/gBP21 ribonucleoprotein complex is more stable than naked guide RNAs. Although the protein is arginine-rich, the free amino acid or an arginine-rich peptide were not able to inhibit the association to the RNAs. In contrast, the gRNA-gBP21 complex formation was sensitive to potassium and ammonium cations, thus indicating a contribution of ionic contacts to the binding.

Amino Acid Sequence↗

PubMed2005

Underediting of GluR2 mRNA, a neuronal death inducing molecular change in sporadic ALS, does not occur in motor neurons in ALS1 or SBMA.

Deficient RNA editing of the AMPA receptor subunit GluR2 at the Q/R site is a primary cause of neuronal death and recently has been reported to be a tightly linked etiological cause of motor neuron death in sporadic amyotrophic lateral sclerosis (ALS). We quantified the RNA editing efficiency of the GluR2 Q/R site in single motor neurons of rats transgenic for mutant human Cu/Zn-superoxide dismutase (SOD1) as well as patients with spinal and bulbar muscular atrophy (SBMA), and found that GluR2 mRNA was completely edited in all the motor neurons examined. It seems likely that the death cascade is different among the dying motor neurons in sporadic ALS, familial ALS with mutant SOD1 and SBMA.

Aged↗