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PubMed2006

Partially edited RNAs are intermediates of RNA editing in plant mitochondria.

RNA editing in flowering plant mitochondria addresses several hundred specific C nucleotides in individual sequence contexts in mRNAs and tRNAs. Many of the in vivo steady state RNAs are edited at some sites but not at others. It is still unclear whether such incompletely edited RNAs can either be completed or are aborted. To learn more about the dynamics of the substrate recognition process, we investigated in vitro RNA editing at a locus in the atp4 mRNA where three editing sites are clustered within four nucleotides. A single cis-element of about 20 nucleotides serves in the recognition of at least two sites. Competition with this sequence element suppresses in vitro editing. Surprisingly, unedited and edited competitors are equally effective. Experiments with partially pre-edited substrates indicate that indeed the editing status of a substrate RNA does not affect the binding affinity of the specificity factor(s). RNA molecules in which all editing sites are substituted by either A or G still compete, confirming that editing site recognition can occur independently of the actual editing site. These results show that incompletely edited mRNAs can be substrates for further rounds of RNA editing, resolving a long debated question.

Amino Acid Sequence↗

PubMed1993

On the evolution of RNA editing.

The term 'RNA editing' encompasses a variety of processes that change the primary nucleotide sequence of an RNA transcript from that of its encoding DNA. As in the case of certain other molecular genetic phenomena, for example RNA splicing, the discovery of RNA editing presented molecular biologists with an evolutionary puzzle, since the existence of RNA editing offers no obvious selective advantage. A three-step model for the evolution of RNA editing is proposed, based on the co-evolution of editing activity and editing sites, with genetic drift as an important component. The implications of this model for the known forms of RNA editing are discussed.

Animals↗

PubMed2002

Inhibition of hepatitis delta virus RNA editing by short inhibitory RNA-mediated knockdown of ADAR1 but not ADAR2 expression.

Hepatitis delta virus (HDV) requires host RNA editing at the viral RNA amber/W site. Of the two host genes responsible for RNA editing via deamination of adenosines in double-stranded RNAs, short inhibitory RNA-mediated knockdown of host ADAR1 expression but not that of ADAR2 led to decreased HDV amber/W editing and virus production. Despite substantial sequence and structural variation among the amber/W sites of the three HDV genotypes, ADAR1a was primarily responsible for editing all three. We conclude that ADAR1 is primarily responsible for editing HDV RNA at the amber/W site during HDV infection.

Adenosine Deaminase↗

PubMed1996

Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities.

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.

Animals↗

PubMed2002

Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes.

Trypanosome RNA editing is a unique U insertion and U deletion process that involves cycles of pre-mRNA cleavage, terminal U addition or U removal, and religation. This editing can occur at massive levels and is directed by base pairing of trans-acting guide RNAs. Both U insertion and U deletion cycles are catalyzed by a single protein complex that contains only seven major proteins, band I through band VII. However, little is known about their catalytic functions, except that band IV and band V are RNA ligases and genetic analysis indicates that the former is important in U deletion. Here we establish biochemical approaches to distinguish the individual roles of these ligases, based on their distinctive ATP and pyrophosphate utilization. These in vitro analyses revealed that both ligases serve in RNA editing. Band V is the RNA editing ligase that functions very selectively to seal in U insertion (IREL), while band IV is the RNA editing ligase needed to seal in U deletion (DREL). In combination with our earlier findings about the cleavage and the U-addition/U-removal steps of U deletion and U insertion, these results show that all three steps of these editing pathways exhibit major differences and suggest that the editing complex could have physically separate regions for U deletion and U insertion.

Adenosine Triphosphate↗

PubMed1999

Transfer of plastid RNA-editing activity to novel sites suggests a critical role for spacing in editing-site recognition.

RNA editing in higher plant plastids alters mRNA sequences by C-to-U conversions at highly specific sites through an unknown mechanism. To elucidate how the cytidine residues to be edited are specifically recognized and distinguished from other cytidines in close proximity, we have changed in vivo the distances of two plastid RNA-editing sites from their essential upstream cis-acting sequence element. Analysis of RNA editing in transgenic chloroplasts revealed that reduction of this distance by 1 nt entirely abolishes RNA editing. Surprisingly, deletions or combinations of deletional and point mutations that shift a heterologous cytidine residue in the same distance from the upstream cis-element as the editing site in the wild type result in transfer of the RNA-editing activity to the heterologous cytidine whereas the wild-type site remains unedited. Our results suggest that the molecular identity of at least some editing sites in the chloroplast genome is defined by their distance from an essential upstream sequence element.

Journal Article↗

PubMed2001

RNA-editing of the 5-HT(2C) receptor alters agonist-receptor-effector coupling specificity.

1. The serotonin(2C) (5-HT(2C)) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A(2) (PLA(2))-arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist-directed trafficking of receptor stimulus) perhaps due to agonist-specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5-HT(2C) receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC-IP and PLA(2)-AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT(2C-VSV), 5-HT(2C-VGV)) of the h5-HT(2C) receptor. 2. 5-HT increased AA release and IP accumulation in both 5-HT(2C-VSV) and 5-HT(2C-VGV) expressing cells. As expected, the potency of 5-HT for both RNA-edited isoforms for both responses was 10 fold lower relative to that of the non-edited receptor (5-HT(2C-INI)) when receptors were expressed at similar levels. 3. Consistent with our previous report, the efficacy order of two 5-HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non-edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA-edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5-HT(2C) agonists to traffic receptor stimulus is lost as a result of RNA editing. 4. These results suggest an important role for the second intracellular loop in transmitting agonist-specific information to signalling molecules.

Amphetamines↗

PubMed2001

A single alteration 20 nt 5' to an editing target inhibits chloroplast RNA editing in vivo.

Transcripts of typical dicot plant plastid genes undergo C-->U RNA editing at approximately 30 locations, but there is no consensus sequence surrounding the C targets of editing. The cis-acting elements required for editing of the C located at tobacco rpoB editing site II were investigated by introducing translatable chimeric minigenes containing sequence -20 to +6 surrounding the C target of editing. When the -20 to +6 sequence specified by the homologous region present in the black pine chloroplast genome was incorporated, virtually no editing of the transcripts occurred in transgenic tobacco plastids. Nucleotides that differ between the black pine and tobacco sequence were tested for their role in C-->U editing by designing chimeric genes containing one or more of these divergent nucleotides. Surprisingly, the divergent nucleotide that had the strongest negative effect on editing of the minigene transcript was located -20 nt 5' to the C target of editing. Expression of transgene transcripts carrying the 27 nt sequence did not affect the editing extent of the endogenous rpoB transcripts, even though the chimeric transcripts were much more abundant than those of the endogenous gene. In plants carrying a 93 nt rpoB editing site sequence, transgene transcripts accumulated to a level three times greater than transgene transcripts in the plants carrying the 27 nt rpoB editing sites and resulted in editing of the endogenous transcripts from 100 to 50%. Both a lower affinity of the 27 nt site for a trans-acting factor and lower abundance of the transcript could explain why expression of minigene transcripts containing the 27 nt sequence did not affect endogenous editing.

Base Sequence↗

PubMed2004

RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei.

Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth inhibition. It also resulted in the loss of both proteins, even when only one of the MRP mRNAs was reduced, indicating a mutual dependence for stability. Elimination of the MRPs gave rise to substantially reduced levels of edited CyB and RPS12 mRNAs but little or no reduction of the level of edited Cox2, Cox3, and A6 mRNAs as measured by poisoned primer extension analyses. In contrast, edited NADH-dehydrogenase (ND) subunit 7 mRNA was increased 5-fold in MRP1+2 double knock-down cells. Furthermore, MRP elimination resulted in reduced levels of Cox1, ND4, and ND5 mRNAs, which are never edited, whereas mitoribosomal 12 S rRNA levels were not affected. These data indicate that MRP1 and MRP2 are not essential for RNA editing per se but, rather, play a regulatory role in the editing of specific transcripts and other RNA processing activities.

Animals↗

PubMed1990

RNA editing involves indiscriminate U changes throughout precisely defined editing domains.

RNA editing is the specific posttranscriptional insertion/deletion of U residues within trypanosomatid mitochondrial transcripts. We used a novel cDNA cloning scheme and analyzed partially edited COIII and CYb RNAs. Our major unanticipated findings are: First, editing appears strikingly indiscriminate within editing domains, inserting and deleting variable numbers of U residues at all sites, both those requiring editing and those that do not. Second, this incomplete editing occurs in precise domains, not in adjacent regions unedited in mature mRNA. Third, editing appears to commence prior to polyadenylation. We propose that within a region being edited, there are cycles of indiscriminate cleavage, U addition or deletion, and religation; sites that become correctly edited would be protected from further modification by duplexing with short RNAs complementary to the edited sequence.

Animals↗

PubMed2002

Recognition of RNA editing sites is directed by unique proteins in chloroplasts: biochemical identification of cis-acting elements and trans-acting factors involved in RNA editing in tobacco and pea chloroplasts.

RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants.

5' Untranslated Regions↗

PubMed1996

Extensive RNA editing of U to C in addition to C to U substitution in the rbcL transcripts of hornwort chloroplasts and the origin of RNA editing in green plants.

We cloned and sequenced a portion of chloroplast DNA from the hornwort Anthoceros formosae. A nucleotide sequence of 7556 bp contained structures similar to those of ndhK, ndhC, trnV, trnM, atpE, atpB, rbcL, trnR and accD. The arrangement of these was the same as that of other chloroplast DNA. However, two nonsense codons were located within the putative coding region of rbcL, although they were used as putative termination codons of the genes. RNA was extensively edited in the transcripts of rbcL when cDNA sequences were analyzed. The unusual nonsense codons of TGA and TAA became CGA and CAA respectively. These are examples of U to C type RNA editing, which was never been found before in chloroplast mRNA. In general, 13 Cs of genomic DNA were found as Ts in the cDNA sequence and seven Ts were found as Cs. This is the first finding of RNA editing on the transcripts of rbcL and also in bryophytes. This event had been thought to arise in land plants after the split of bryophytes. The origin of RNA editing is discussed in relation to the landing of green plants.

Amino Acid Sequence↗

PubMed1990

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants.

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.

Amino Acid Sequence↗

PubMed2000

Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2.

RNA editing by site-selective deamination of adenosine to inosine alters codons and splicing in nuclear transcripts, and therefore protein function. ADAR2 (refs 7, 8) is a candidate mammalian editing enzyme that is widely expressed in brain and other tissues, but its RNA substrates are unknown. Here we have studied ADAR2-mediated RNA editing by generating mice that are homozygous for a targeted functional null allele. Editing in ADAR2-/- mice was substantially reduced at most of 25 positions in diverse transcripts; the mutant mice became prone to seizures and died young. The impaired phenotype appeared to result entirely from a single underedited position, as it reverted to normal when both alleles for the underedited transcript were substituted with alleles encoding the edited version exonically. The critical position specifies an ion channel determinant, the Q/R site, in AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor GluR-B pre-messenger RNA. We conclude that this transcript is the physiologically most important substrate of ADAR2.

Adenosine Deaminase↗

PubMed2006

Genetic algorithm learning as a robust approach to RNA editing site prediction.

BACKGROUND: RNA editing is one of several post-transcriptional modifications that may contribute to organismal complexity in the face of limited gene complement in a genome. One form, known as C --> U editing, appears to exist in a wide range of organisms, but most instances of this form of RNA editing have been discovered serendipitously. With the large amount of genomic and transcriptomic data now available, a computational analysis could provide a more rapid means of identifying novel sites of C --> U RNA editing. Previous efforts have had some success but also some limitations. We present a computational method for identifying C --> U RNA editing sites in genomic sequences that is both robust and generalizable. We evaluate its potential use on the best data set available for these purposes: C --> U editing sites in plant mitochondrial genomes. RESULTS: Our method is derived from a machine learning approach known as a genetic algorithm. REGAL (RNA Editing site prediction by Genetic Algorithm Learning) is 87% accurate when tested on three mitochondrial genomes, with an overall sensitivity of 82% and an overall specificity of 91%. REGAL's performance significantly improves on other ab initio approaches to predicting RNA editing sites in this data set. REGAL has a comparable sensitivity and higher specificity than approaches which rely on sequence homology, and it has the advantage that strong sequence conservation is not required for reliable prediction of edit sites. CONCLUSION: Our results suggest that ab initio methods can generate robust classifiers of putative edit sites, and we highlight the value of combinatorial approaches as embodied by genetic algorithms. We present REGAL as one approach with the potential to be generalized to other organisms exhibiting C --> U RNA editing.

Algorithms↗

PubMed2006

REDIdb: the RNA editing database.

The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at http://biologia.unical.it/py_script/search.html.

DNA, Complementary↗

PubMed2004

Complete cycles of bloodstream trypanosome RNA editing in vitro.

RNA editing in kinetoplastid protists is required for the maturation of mitochondrial pre-mRNAs and occurs by protein-catalyzed cycles of uridylate insertion and deletion. During the complex life cycle of Trypanosoma brucei this process is differentially regulated in the mammalian bloodstream and insect procyclic stages. Complementary guide RNAs (gRNAs) direct editing, but the abundance of these transcripts is not developmentally controlled. The establishment of in vitro systems that recreate efficient RNA editing in bloodstream T. brucei would be valuable for mechanistic studies of regulation. Here we describe a robust in vitro system that reconstitutes full cycles of both U insertion and U deletion in bloodstream trypanosomes, and the first direct comparisons of the in vitro systems for strains of mammalian and insect stages.

Animals↗

PubMed1992

The evolution of RNA editing in kinetoplastid protozoa.

RNA editing alters messenger RNA transcripts by the addition and deletion of uridine residues. This remarkable process corrects frameshift mutations and replaces AUG initiation codons in most trypanosomatid mitochondrial RNA transcripts. Sometimes, however, RNA editing results in open reading frames whose sequences differ by greater than 50% from the original RNA sequences. In this paper, we will explore two models for the evolution of RNA editing in kinetoplastid protoza.

Animals↗