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PubMed2001

The two RNA ligases of the Trypanosoma brucei RNA editing complex: cloning the essential band IV gene and identifying the band V gene.

Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified from Trypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of approximately 57 kDa (band IV) and approximately 50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

Alternative Splicing↗

PubMed2003

Requirement of dimerization for RNA editing activity of adenosine deaminases acting on RNA.

Adenosine deaminases acting on RNA (ADAR) convert adenosine residues into inosines in double-stranded RNA. Three vertebrate ADAR gene family members, ADAR1, ADAR2, and ADAR3, have been identified. The catalytic domain of all three ADAR gene family members is very similar to that of Escherichia coli cytidine deaminase and APOBEC-1. Homodimerization is essential for the enzyme activity of those cytidine deaminases. In this study, we investigated the formation of complexes between differentially epitope-tagged ADAR monomers by sequential affinity chromatography and size exclusion column chromatography. Both ADAR1 and ADAR2 form a stable enzymatically active homodimer complex, whereas ADAR3 remains as a monomeric, enzymatically inactive form. No heterodimer complex formation among different ADAR gene family members was detected. Analysis of HeLa and mouse brain nuclear extracts suggested that endogenous ADAR1 and ADAR2 both form a homodimer complex. Interestingly, endogenous ADAR3 also appears to form a homodimer complex, indicating the presence of a brain-specific mechanism for ADAR3 dimerization. Homodimer formation may be necessary for ADAR to act as active deaminases. Analysis of dimer complexes consisting of one wild-type and one mutant monomer suggests functional interactions between the two subunits during site-selective RNA editing.

Adenosine Deaminase↗

PubMed2003

Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene.

One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR edits also non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Here we investigated phenotypes of mice homozygous for ADAR1 null mutation. Although live ADAR1-/- embryos with normal gross appearance could be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos were also prone to apoptosis induced by serum deprivation. Our results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis.

Adenosine Deaminase↗

PubMed2004

Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA editing.

The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K(D) in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

Animals↗

PubMed2002

RNA editing of neurotransmitter receptors in the mammalian brain.

RNA editing refers to various posttranscriptional mechanisms that alter the nucleotide sequence of RNA. In the mammalian brain, RNA editing results in significant changes in the functional properties of receptors for the important neurotransmitters glutamate and serotonin. These changes result from site-specific deamination of single adenosines in the pre-messenger RNA encoding these receptors. Here, we review what is known about the mechanisms underlying this editing, the consequences of RNA editing for glutamate and serotonin receptor function, and recent studies on transgenic mice and human post-mortem tissue that have begun to elucidate the role of RNA editing in the intact mammalian brain.

Animals↗

PubMed2006

5-HT(2C) receptor RNA editing in the amygdala of C57BL/6J, DBA/2J, and BALB/cJ mice.

Post-transcriptional RNA editing of the G-protein coupled 5-hydroxytryptamine-2C (5-HT(2C)) receptor predicts an array of 24 receptor isoforms, some of which are characterized by reduced constitutive activity and potency to initiate intracellular signaling. The amygdala is integral to anxiety, fear, and related psychiatric diseases. Activation of 5-HT(2C) receptors within the amygdala is anxiogenic. Here, we describe the RNA editing profiles from amygdala of two inbred mouse strains (BALB/cJ and DBA/2J) known to be more anxious than a third (C57BL/6J). We confirmed the strain anxiety differences using light<-->dark exploration, and we discovered that BALB/cJ and DBA/2J are each characterized by a higher functioning RNA editing profile than C57BL/6J. BALB/cJ and DBA/2J exhibit a roughly two-fold reduction in C site editing, and a corresponding two-fold reduction in the edited isoform VSV. C57BL/6J is characterized by a relative decrease in the unedited highly functional isoform INI. We estimated the heritability of editing at the C site to be approximately 40%. By sequencing genomic DNA, we found complete conservation between C57BL/6J, BALB/cJ, DBA/2J and 37 other inbred strains for the RNA edited region of Htr2c, suggesting Htr2c DNA sequence does not influence variation in Htr2c RNA editing between inbred strains of mice. We did, however, discover that serotonin turnover is reduced in BALB/cJ and DBA/2J, consistent with emerging evidence that synaptic serotonin levels regulate RNA editing. These results encourage further study of the causes and consequences of 5-HT(2C) receptor RNA editing in the amygdala of mice.

Amygdala↗

PubMed2004

Rapid evolution of RNA editing sites in a small non-essential plastid gene.

Chloroplast RNA editing proceeds by C-to-U transitions at highly specific sites. Here, we provide a phylogenetic analysis of RNA editing in a small plastid gene, petL, encoding subunit VI of the cytochrome b6f complex. Analyzing representatives from most major groups of seed plants, we find an unexpectedly high frequency and dynamics of RNA editing. High-frequency editing has previously been observed in plastid ndh genes, which are remarkable in that their mutational inactivation does not produce an obvious mutant phenotype. In order to test the idea that reduced functional constraints allow for more flexible evolution of RNA editing sites, we have created petL knockout plants by tobacco chloroplast transformation. We find that, in the higher plant tobacco, targeted inactivation of petL does not impair plant growth under a variety of conditions markedly contrasting the important role of petL in photosynthesis in the green alga Chlamydomonas reinhardtii. Together with a low number of editing sites in plastid genes that are essential to gene expression and photosynthetic activity, these data suggest that RNA editing sites may evolve more readily in those genes whose transitory loss of function can be tolerated. Accumulated evidence for this 'relative neutrality hypothesis for the evolution of plastid editing sites' is discussed.

Base Sequence↗

PubMed1997

No editing of mitochondrial plasmid transcripts in mitochondria of Physarum that have an RNA-editing system.

Two types of RNA editing have been reported in the mitochondria of Physarum; extensive insertions of nucleotides and single-base substitutions. In the Ng strain of P. polycephalum and its derivatives, mitochondria have a specific plasmid (mF) that promotes fusion of mitochondria. We examined the editing of transcripts derived from the mF plasmid. For analysis, we selected three regions of the plasmid, including a DNA fragment that corresponded to missing conserved domains of the RNA polymerase. In contrast to the mitochondrial DNA (mtDNA), no RNA editing of the transcripts of the mF plasmid was detected. Our results suggest that the mechanism of transcription of the mitochondrial plasmid is independent of that of mtDNA, indicating that the plasmid has a different evolutionary origin from the mtDNA.

Animals↗

PubMed1994

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria.

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.

Animals↗

PubMed1999

Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1). structure-function relationships of RNA editing and dimerization.

APOBEC1 is the catalytic subunit of an enzyme complex that mediates apolipoprotein (apo) B mRNA editing. It dimerizes in vitro and requires complementation factor(s) for its editing activity. We have performed a systematic analysis of the structure-functional relationship of APOBEC1 by targeted mutagenesis of various sequence motifs within the protein. Using in vitro RNA editing assay, we found that basic amino acid clusters at the amino-terminal region R15R16R17 and R33K34, are essential for apoB mRNA editing. Mutation of R15R16R17 to K15K16K17 and mutation of R33K34 simultaneously to A33A34 almost completely abolished in vitro editing activity. The carboxy-terminal region of APOBEC1 contains a leucine-rich motif. Deletion analysis of this region indicates that residues 181 to 210 are important for in vitro apoB mRNA editing. Single amino acid substitutions demonstrate that L182, I185, and L189 are important residues required for normal editing function. Furthermore, the double mutant P190A/P191A also lost >90% of editing activity which suggests that a beta turn in this region of the molecule may be essential for proper functioning of APOBEC1. It was suggested that dimerization of APOBEC1 creates an active structure for deamination of apoB mRNA. When we examined the dimerization potential of truncated APOBEC1s using both amino and carboxy termini deletion mutants, we found that amino-terminal deletions up to residue A117 did not impair dimerization activity whereas carboxy-terminal deletions showed diminished dimerization. The systematic and extensive mutagenesis experiments in this study provide information on the role of various sequence motifs identified in APOBEC1 in enzyme catalysis and dimerization.

APOBEC-1 Deaminase↗

PubMed2004

Computational prediction of RNA editing sites.

MOTIVATION: Some organisms edit their messenger RNA resulting in differences between the genomic sequence for a gene and the corresponding messenger RNA sequence. This difference complicates experimental and computational attempts to find and study genes in organisms with RNA editing even if the full genomic sequence is known. Nevertheless, knowledge of these editing sites is crucial for understanding the editing machinery of these organisms. RESULTS: We present a computational technique that predicts the position of editing sites in the genomic sequence. It uses a statistical approach drawing on the protein sequences of related genes and general features of editing sites of the organism. We apply the method to the mitochondrion of the slime mold Physarum polycephalum. It correctly predicts over 90% of the amino acids and over 70% of the editing sites.

Algorithms↗

PubMed2025

Obstacles in quantifying A-to-I RNA editing by Sanger sequencing.

Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent type of RNA editing, in which adenosine within a completely or largely double-stranded RNA (dsRNA) is converted to inosine by deamination. RNA editing was shown to be involved in many neurological diseases and cancer; therefore, detection of A-to-I RNA editing and quantitation of editing levels are necessary for both basic and clinical biomedical research. While high-throughput sequencing (HTS) is widely used for global detection of editing events, Sanger sequencing is the method of choice for precise characterization of editing site clusters (hyper-editing) and for comparing levels of editing at a particular site under different environmental conditions, developmental stages, genetic backgrounds, or disease states. To detect A-to-I editing events and quantify them using Sanger sequencing, RNA samples are reverse transcribed, cDNA is amplified using gene-specific primers, and then sequenced. The chromatogram outputs are then compared to the genomic DNA sequence. As editing occurs in the context of dsRNA, the reverse transcription step is performed at a temperature as high as 65 °C, using thermostable reverse transcriptase to open double-stranded structures. However, this measure alone is insufficient for transcripts possessing long stems comprised of hundreds of nucleotide pairs. Consequently, the editing levels detected by Sanger sequencing are significantly lower than those obtained by HTS, and the amplification yield is low. We suggest that the reverse transcription is biased towards unedited transcripts, and the severity of the bias is dependent on the transcript's secondary structure. Here, we show how this bias can be significantly reduced to allow reliable detection of editing levels and sufficient product yield.

RNA Editing↗

PubMed1994

Differential RNA editing efficiency of AMPA receptor subunit GluR-2 in human brain.

RNA editing in rat brain has been found to control a determinant of cation flow in alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA)-gated channels. Here we provide the first evidence that this RNA editing phenomenon occurs in human brain and is differentially regulated. Sequence analysis of human genomic DNA revealed a Q codon (CAG) in the putative channel-forming segment of human GluR-2, whereas in the majority of cDNA clones an R codon (CGG) was found. Examination of editing in various brain tissues revealed differences in the efficiency of this process. The hippocampus, cerebellum and temporal cortex harbour 100% edited GluR-2, whereas only 72% of substantia nigra, 89% of corpus striatum and 96% of fetal cDNAs have been found to be edited. This new discovery of differential efficiency of RNA editing has important implications in AMPA receptor channel-mediated calcium influx. AMPA receptors are thought to mediate the majority of the fast excitatory synaptic neurotransmission; the RNA editing process may therefore play a critical role in normal brain function and development. Dysfunction of this RNA editing process may have neuropathological consequences and could be related to certain neurodegenerative diseases.

Animals↗

PubMed2006

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins.

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.

Amino Acid Sequence↗

PubMed2002

Rewriting the information in DNA: RNA editing in kinetoplastids and myxomycetes.

RNA editing has a major impact on the genes and genomes that it modifies. Editing by insertion, deletion and base conversion exists in nuclear, mitochondrial and viral genomes throughout the eukaryotic lineage. Editing was first discovered in kinetoplastids, and recent work has resulted in the characterization of some components of the editing machinery. Two proteins with ligase activity have been identified in Trypanosoma brucei, and other proteins in the editosome complex are yielding to the probe of research. A second group of protists, myxomycetes, are unique in their use of four different types of editing within a single transcript. Phylogenetic analysis of editing in representative myxomycetes revealed a different history of the four types of editing in this lineage. Development of a soluble in vitro editing system has provided further support for the co-transcriptional nature of editing in Physarum polycephalum, and will certainly provide future opportunities for understanding this mysterious process.

Cytidine↗

PubMed2005

Estimating RNA editing efficiency of five editing sites in the serotonin 2C receptor by pyrosequencing.

Accumulating evidence suggests that altered RNA editing of the serotonin 2C receptor (HTR2C) is involved in the pathophysiology of mental disorders and the action of antidepressants. Estimating RNA editing of HTR2C in various samples is a first step to understanding its pathophysiological roles. Here, we developed a high-throughput quantification method of RNA editing efficiency by pyrosequencing. By optimizing the dispensation order, the RNA editing efficiency of all five RNA editing sites including consecutively ordered sites in HTR2C was obtained. More importantly, our method made it possible to determine the content of partial HTR2C isoforms, which enabled us to monitor possible functional changes of HTR2C. This method was validated in both oligonucleotide and RT-PCR product templates, and showed good correlation with conventional cloning-sequencing analysis. Our method could be a valuable tool in the rapid assessment of RNA editing status, including assessment of natural variations, alterations in disease tissues, and responses to drugs.

Animals↗

PubMed2005

Evolutionarily conserved human targets of adenosine to inosine RNA editing.

A-to-I RNA editing by ADARs is a post-transcriptional mechanism for expanding the proteomic repertoire. Genetic recoding by editing was so far observed for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using comparative genomics and expressed sequence analysis, we identified and experimentally verified four additional candidate human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. Interestingly, none of these substrates encodes a receptor protein but two of them are strongly expressed in the CNS and seem important for proper nervous system function. The editing pattern observed suggests that some of the affected proteins might have altered physiological properties leaving the possibility that they can be related to the phenotypes of ADAR1 knockout mice.

Adenosine↗

PubMed1996

An auxiliary factor containing a 240-kDa protein complex is involved in apolipoprotein B RNA editing.

A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.

Animals↗