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PubMed1995

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Adenosine Deaminase↗

PubMed2001

RNA editing-associated protein 1 is an RNA binding protein with specificity for preedited mRNA.

RNA editing in the mitochondria of kinetoplastids involves the addition and deletion of uridines at specific sites as directed by guide RNAs (gRNAs). Ample evidence shows that ribonucleoprotein (RNP) complexes carry out this posttranscriptional processing. One component of RNA editing complexes is REAP-1, a protein of previously unknown function found primarily in mRNA containing editing complexes. We now show that REAP-1 is an RNA binding protein and map the binding activity to the amino-terminal third of the protein. REAP-1 binds to poly(G) and single-stranded guanosine rich RNAs. Data presented here demonstrates that preedited RNAs are the preferred substrate for REAP-1. The results suggest a model in which the role of REAP-1 is to bring preedited mRNAs into the editing complex.

Animals↗

PubMed2004

Simple statistical models predict C-to-U edited sites in plant mitochondrial RNA.

BACKGROUND: RNA editing is the process whereby an RNA sequence is modified from the sequence of the corresponding DNA template. In the mitochondria of land plants, some cytidines are converted to uridines before translation. Despite substantial study, the molecular biological mechanism by which C-to-U RNA editing proceeds remains relatively obscure, although several experimental studies have implicated a role for cis-recognition. A highly non-random distribution of nucleotides is observed in the immediate vicinity of edited sites (within 20 nucleotides 5' and 3'), but no precise consensus motif has been identified. RESULTS: Data for analysis were derived from the the complete mitochondrial genomes of Arabidopsis thaliana, Brassica napus, and Oryza sativa; additionally, a combined data set of observations across all three genomes was generated. We selected datasets based on the 20 nucleotides 5' and the 20 nucleotides 3' of edited sites and an equivalently sized and appropriately constructed null-set of non-edited sites. We used tree-based statistical methods and random forests to generate models of C-to-U RNA editing based on the nucleotides surrounding the edited/non-edited sites and on the estimated folding energies of those regions. Tree-based statistical methods based on primary sequence data surrounding edited/non-edited sites and estimates of free energy of folding yield models with optimistic re-substitution-based estimates of approximately 0.71 accuracy, approximately 0.64 sensitivity, and approximately 0.88 specificity. Random forest analysis yielded better models and more exact performance estimates with approximately 0.74 accuracy, approximately 0.72 sensitivity, and approximately 0.81 specificity for the combined observations. CONCLUSIONS: Simple models do moderately well in predicting which cytidines will be edited to uridines, and provide the first quantitative predictive models for RNA edited sites in plant mitochondria. Our analysis shows that the identity of the nucleotide -1 to the edited C and the estimated free energy of folding for a 41 nt region surrounding the edited C are the most important variables that distinguish most edited from non-edited sites. However, the results suggest that primary sequence data and simple free energy of folding calculations alone are insufficient to make highly accurate predictions.

Arabidopsis↗

PubMed2001

Human RNA helicase II/Gu gene: genomic organization and promoter analysis.

Human RNA helicase II/Gu (hRH II/Gu) protein unwinds double-stranded RNA, folds single-stranded RNA, and may play important roles in ribosomal RNA biogenesis, RNA editing, RNA transport, and general transcription. The genomic organization and the regulation of expression of the hRH II/Gu gene were analyzed. The hRH II/Gu gene is over 29 kb in length, and includes 15 exons. All intron-exon junctions contain consensus GT-AG sequences. An 85 bp basic promoter was identified which lacks a functional TATA box. In LNCaP cells, the hRH II/Gu promoter was up to 50 times as active as pGL2-control vector which has an SV 40 early promoter. Mutation of a putative c-Myc/USF binding site in the region between -110 and -85 caused a decrease in the promoter activity by 2- to 3-fold. A negative regulatory region was also found between -324 and -264. Studies of transcriptional regulation of hRH II/Gu under different physiological and pathophysiological conditions may provide clues to its biological function.

Base Sequence↗

PubMed2002

The sugar beet mitochondrial nad4 gene: an intron loss and its phylogenetic implication in the Caryophyllales.

The sugar beet mitochondrial gene for subunit IV of NADH dehydrogenase ( nad4) has been characterized. Unlike the corresponding genes in wheat and turnip, sugar beet nad4 lacks the second intron ( nad4-i2). Northern-blot analysis demonstrates transcription of the gene. A total of 19 RNA editing sites were identified in the sugar beet nad4 transcripts; interestingly, there is no editing in the region which flanks the lost intron. This observation is in favour of intron loss via homologous recombination of an edited RNA intermediate. We also found that the nad4-i2 intron is absent from the mitochondrial genomes of all examined members of the Caryophyllales, but present in the closely related orders, Polygonales and Plumbaginales, which suggests that the intron was lost in the common ancestor of the Caryophyllales.

Journal Article↗

PubMedDate not supplied

Participation of nuclear genes in chloroplast gene expression.

The expression of the plastid genome is dependent on a large number of nucleus-encoded factors. Some of these factors have been identified through biochemical assays, and many others by genetic screens in Arabidopsis, Chlamydomonas and maize. Nucleus-encoded factors function in each step in plastid gene expression, including transcription, RNA editing, RNA splicing, RNA processing, RNA degradation, and translation. Many of the factors discovered via biochemical approaches play general roles as components of the basic gene expression machinery, whereas the majority of those identified by genetic approaches are specifically required for the expression of small subsets of chloroplast genes and are involved in post-transcriptional steps. Some of the nucleus-encoded factors may play regulatory roles and modulate chloroplast gene expression in response to developmental or environmental cues. They may also serve to couple chloroplast gene expression with the assembly of the protein products into the large complexes of the photosynthetic apparatus. The convergence of biochemical approaches with those of classical and reverse genetics, and the contributions from large scale genomic sequencing should result in rapid advances in our understanding of the regulatory interactions that govern plastid gene expression.

Cell Nucleus↗

PubMed2006

Small dsRNAs induce transcriptional activation in human cells.

Recent studies have shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi. To further study the regulatory role of small RNAs at the transcriptional level, we designed and synthesized 21-nt dsRNAs targeting selected promoter regions of human genes E-cadherin, p21(WAF1/CIP1) (p21), and VEGF. Surprisingly, transfection of these dsRNAs into human cell lines caused long-lasting and sequence-specific induction of targeted genes. dsRNA mutation studies reveal that the 5' end of the antisense strand, or "seed" sequence, is critical for activity. Mechanistically, the dsRNA-induced gene activation requires the Argonaute 2 (Ago2) protein and is associated with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites. In conclusion, we have identified several dsRNAs that activate gene expression by targeting noncoding regulatory regions in gene promoters. These findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized and identify a potential therapeutic use for dsRNA in targeted gene activation.

Argonaute Proteins↗

PubMed1999

The involvement of gRNA-binding protein gBP21 in RNA editing-an in vitro and in vivo analysis.

RNA editing in the parasitic organism Trypanosoma brucei is characterised by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The processing reaction is a required pathway for the expression of most mitochondrial genes and proceeds by a cascade of enzyme-catalysed steps. RNA editing involves one or more macromolecular ribonucleoprotein complexes which are likely to interact with additional components as the reaction proceeds. Here we examined the involvement of the gRNA-binding polypeptide gBP21, a protein which has been demonstrated to be associated with active RNA editing complexes. We show that in vitro RNA editing can be suppressed by the addition of a gBP21-specific antibody or by immunodepletion of the protein. By creating a gBP21 knockout mutant we analysed the requirement for the protein in vivo. gBP21(-) trypanosomes are viable as bloodstream stage cells and contain edited mRNAs. However, the knockout mutant is not capable of differentiating from the bloodstream to the insect life cycle stage in vitro. Moreover, mutant cells are characterised by a low mitochondrial transcript abundance. Together, these data establish that gBP21 contributes a non-essential function to the RNA editing reaction and further suggest that the protein is involved in additional mitochondrial processes which impact a larger pool of mitochondrial transcripts.

Animals↗

PubMed1995

RNA ligase and its involvement in guide RNA/mRNA chimera formation. Evidence for a cleavage-ligation mechanism of Trypanosoma brucei mRNA editing.

RNA editing in Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. Editing is thought to be directed by guide RNAs and may proceed via a chimeric guide RNA/mRNA intermediate. We have previously shown that chimera-forming activity sediments with 19 S and 35-40 S mitochondrial ribonucleoprotein particles (RNPs). In this report we examine the involvement of RNA ligase in the production of chimeric molecules in vitro. Two adenylylated proteins of 50 and 57 kDa co-sediment on glycerol gradients with RNA ligase activity as components of the ribonucleoprotein particles. The two adenylylated proteins differ in sequence and contain AMP linked via a phosphoamide bond. Both proteins are deadenylylated by the addition of ligatable RNA substrate with the concomitant release of AMP and by the addition of pyrophosphate to yield ATP. Incubation with nonligatable RNA substrate results in an accumulation of the adenylylated RNA intermediate. These experiments identify the adenylylated proteins as RNA ligases. AMP release from the mitochondrial RNA ligase is also concomitant with chimera formation. Inhibition by nonhydrolyzable analogs indicates that both RNA ligase and chimera-forming activities require alpha-beta bond hydrolysis of ATP. Deadenylylation of the ligase inhibits chimera formation. These results strongly suggest the involvement of RNA ligase in in vitro chimera formation and support the cleavage-ligation mechanism for kinetoplastid RNA editing.

Adenosine Monophosphate↗

PubMed1996

A promiscuous chloroplast DNA fragment is transcribed in plant mitochondria but the encoded RNA is not edited.

The RNA editing processes in chloroplasts and mitochondira of higher plants show several similarities which are suggestive of common components and/or biochemical steps between the two plant organelles. The existence of various promiscuous DNA fragments of chloroplast origin in plant mitochondrial genomes allowed us to test the possibility that chloroplast sequences are also edited in mitochondria. An rpoB fragment transferred from chloroplasts to mitochondria in rice was chosen as it contains several editing sites, two of which match sequence motifs surrounding even non-homologous editing sites in both chloroplast and mitochondrial transcripts. Rice chloroplast and mitochondrial rpoB DNA and cDNA sequences were selectively amplified and the editing status of the cDNA sequences was determined. Three of the four potential rpoB editing sites previously detected in maize were found to be edited in the rice chloroplast rpoB transcript, whereas the fourth was found to remain unedited. In mitochondria, however, all four editing sites remain unmodified at the cDNA level. This indicates that the editing processes of higher plant mitochondria and chloroplasts are not identical and that organelle-specific factors are required for eliciting the respective editing events.

Amino Acid Sequence↗

PubMed2005

Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase.

The approximately 20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains approximately 16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.

Animals↗

PubMed2003

Molecular regulation, evolutionary, and functional adaptations associated with C to U editing of mammalian apolipoproteinB mRNA.

RNA editing encompasses an important class of co- or posttranscriptional nucleic acid modification that has expanded our understanding of the range of mechanisms that facilitate genetic plasticity. Since the initial description of RNA editing in trypanosome mitochondria, a model of gene regulation has emerged that now encompasses a diverse range of biochemical and genetic mechanisms by which nuclear, mitochondrial, and t-RNA sequences are modified from templated versions encoded in the genome. RNA editing is genetically and biochemically distinct from other RNA modifications such as splicing, capping, and polyadenylation although, as discussed in Section I, these modifications may have relevance to the regulation of certain types of mammalian RNA editing. This review will focus on C to U RNA editing, in particular, the biochemical and genetic mechanisms that regulate this process in mammals. These mechanisms will be examined in the context of the prototype model of C to U RNA editing, namely the posttranscriptional cytidine deamination targeting a single nucleotide in mammalian apolipoproteinB (apoB). Other examples of C to U RNA editing will be discussed and the molecular mechanisms--where known--contrasted with those regulating apoB RNA editing.

Animals↗

PubMed2000

The tobacco apocytochrome b gene predicts sensitivity to the respiratory inhibitors antimycin A and myxothiazol.

The potential for use of the cytochrome-pathway electron-transfer inhibitors antimycin A and myxothiazol in the selection of plant mitochondrial genome transformants was investigated. The net growth of Nicotiana tabacum L. (tobacco) suspension-culture cells was reduced by these inhibitors, but complete repression of cell growth occurred only in the presence of both cytochrome and alternative electron-transfer-pathway inhibitors. Antimycin A and myxothiazol bind to and block electron transfer through different sites in the cytochrome b (COB) subunit of the mitochondrial bc1 respiratory complex (complex III). The nucleotide sequence of the tobacco cob gene was determined and found to predict highly conserved glycine and phenylalanine residues that are associated with sensitivity to antimycin A and myxothiazol, respectively. These residues are altered by mutations that confer resistance to antimycin A or myxothiazol in diverse organisms. Tobacco cob cDNA clones were constructed and sequenced, revealing eight full and 11 partial RNA-editing sites. RNA editing did not, however, alter codons for the conserved glycine and phenylalanine residues associated with sensitivity to the respiratory inhibitors. Antimycin A or myxothiazol, in conjunction with a modified cob gene, may therefore be useful in the selection of tobacco cells carrying a genetically transformed mitochondrial genome.

Amino Acid Sequence↗

PubMed1994

Requirements for editing in the genomic RNA of hepatitis delta virus.

Hepatitis delta virus is a satellite of the hepatitis B virus which provides the surface antigen for the viral coat. The genome of the hepatitis delta virus consists of a single-stranded, circular RNA of 1679 nucleotides which forms a rod structure due to a high extent of self homology and which replicates via synthesis of an antigenomic RNA in a rolling circle mechanism similar to plant viroids. The antigenomic RNA contains the open reading frame for the delta-antigen which exists in two isoforms, p24 and p27. The formation of these two isoforms is explained by RNA editing at nucleotide 1012 which changes the stop translation codon UAG at amino acid residue 196 into the codon UGG for tryptophan and extends the open reading frame for the synthesis of p27. In order to investigate whether the editing occurs cotranscriptionally during RNA replication or is a posttranscriptional base modification in the genomic or antigenomic RNA, replication defective deletion mutants of the HDV genome were constructed and expressed in COS-7 cells. Editing was demonstrated in non-replicating fragments of genomic HDV RNA but not in antigenomic HDV RNA fragments. The sequences from nucleotide position 337-1200 of the genomic RNA were sufficient to enable low levels of editing. Editing at position 1012 required the opposite strand of the RNA rod from nucleotide position 337-783. Replicating circular HDV RNA was much more efficiently edited than non-replicating full length genomic HDV RNA. Expression of delta-antigen in trans did not complement the low editing efficiency of replication defective genomic HDV RNA. These results demonstrate posttranscriptional U to C editing in the genomic HDV RNA and exclude misincorporation during HDV RNA replication as the editing mechanism. The minimal structural requirements for HDV RNA editing reside between nucleotide position 337-1200.

Animals↗

PubMed2001

Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system.

RNA editing in higher plant chloroplasts involves C-->U conversion at approximately 30 specific sites. An in vitro system supporting accurate editing has been developed from tobacco chloroplasts. Mutational analysis of substrate mRNAs derived from tobacco chloroplast psbL and ndhB mRNAs confirmed the participation of cis-acting elements that had previously been identified in vivo. Competition analysis revealed the existence of site-specific trans-acting factors interacting with the corresponding upstream cis-elements. A chloroplast protein of 25 kDa was found to be specifically associated with the cis-element involved in psbL mRNA editing. Immunological analyses revealed that an additional factor, the chloroplast RNA-binding protein cp31, is also required for RNA editing at multiple sites. This combination of site-specific and common RNA-binding proteins recognizes editing sites in chloroplasts.

Base Sequence↗

PubMed2001

C-->U editing of neurofibromatosis 1 mRNA occurs in tumors that express both the type II transcript and apobec-1, the catalytic subunit of the apolipoprotein B mRNA-editing enzyme.

C-->U RNA editing of neurofibromatosis 1 (NF1) mRNA changes an arginine (CGA) to a UGA translational stop codon, predicted to result in translational termination of the edited mRNA. Previous studies demonstrated varying degrees of C-->U RNA editing in peripheral nerve-sheath tumor samples (PNSTs) from patients with NF1, but the basis for this heterogeneity was unexplained. In addition, the role, if any, of apobec-1, the catalytic deaminase that mediates C-->U editing of mammalian apolipoprotein B (apoB) RNA, was unresolved. We have examined these questions in PNSTs from patients with NF1 and demonstrate that a subset (8/34) manifest C-->U editing of RNA. Two distinguishing characteristics were found in the PNSTs that demonstrated editing of NF1 RNA. First, these tumors express apobec-1 mRNA, the first demonstration, in humans, of its expression beyond the luminal gastrointestinal tract. Second, PNSTs with C-->U editing of RNA manifest increased proportions of an alternatively spliced exon, 23A, downstream of the edited base. C-->U editing of RNA in these PNSTs was observed preferentially in transcripts containing exon 23A. These findings were complemented by in vitro studies using synthetic RNA templates incubated in the presence of recombinant apobec-1, which again confirmed preferential editing of transcripts containing exon 23A. Finally, adenovirus-mediated transfection of HepG2 cells revealed induction of editing of apoB RNA, along with preferential editing of NF1 transcripts containing exon 23A. Taken together, the data support the hypothesis that C-->U RNA editing of the NF1 transcript occurs both in a subset of PNSTs and in an alternatively spliced form containing a downstream exon, presumably an optimal configuration for enzymatic deamination by apobec-1.

APOBEC-1 Deaminase↗

PubMed1990

Uncoupling of the synthesis of edited and unedited COIII RNA in Trypanosoma brucei.

RNA editing, a novel and unexpected type of information processing, was first demonstrated in the kinetoplasts of certain protozoans. It is a remarkable phenomenon: certain species of messenger RNA have nucleotide sequences that differ greatly from the sequences of the genes from which they are presumably transcribed. The differences are usually due to addition of uridylate residues, but occasionally also to their deletion. The most spectacular case of editing known occurs in the mRNA of the mitochondrial gene for subunit III of cytochrome oxidase (COIII) in Trypanosoma brucei. This mRNA is twice the length of its gene owing to the addition of several hundred uridylate residues and a few deletions, spread over the entire length of the RNA molecule. Whereas unedited RNA molecules, the nucleotide sequences of which correspond to the genomic sequence, have been isolated, no DNA template corresponding to the edited RNA sequence has been detected in either the mitochondrial or nuclear genome. It was suggested, therefore, that unedited mRNAs are transcribed from mitochondrial DNA and then edited post-transcriptionally by endonucleolytic cleavage of the primary transcript at specific sites, followed by insertion or deletion of uridylate residues and religation. We have now examined the general nature of the RNA editing process to determine whether it involves the insertion of nucleotide residues into pre-existing molecules or the continuous de novo synthesis of edited mRNA. The results of our experiments rule out an insertion mechanism and strongly indicate that edited mRNA is synthesized as a unit.

Animals↗

PubMed2006

RNA regulation and cancer development.

Cancer is viewed as a genetic disease. According to the currently accepted model of carcinogenesis, several consequential mutations in oncogenes or tumor suppressor genes are necessary for cancer development. In this model, mutated DNA sequence is transcribed to mRNA that is finally translated into functionally aberrant protein. mRNA is viewed solely as an intermediate between DNA (with 'coding' potential) and protein (with 'executive' function). However, recent findings suggest that (m)RNA is actively regulated by a variety of processes including nonsense-mediated decay, alternative splicing, RNA editing or RNA interference. Moreover, RNA molecules can regulate a variety of cellular functions through interactions with RNA, DNA as well as protein molecules. Although, the precise contribution of RNA molecules by themselves and RNA-regulated processes on cancer development is currently unknown, recent data suggest their important role in carcinogenesis. Here, we summarize recent knowledge on RNA-related processes and discuss their potential role in cancer development.

Alternative Splicing↗