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PubMed2000

Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.

Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma-globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.

APOBEC-1 Deaminase↗
PubMed1998

Human apolipoprotein B RNA editing deaminase gene (APOBEC1).

Genomic clones encoding the human APOBEC1 gene and its 5' flanking region have been isolated and characterized. The human gene contains five coding exons. The introns dividing these exons correspond exactly to those found in the mouse gene. The translation initiation site, ATG, is located in exon 2 at the same site as in the mouse. The 5' flanking sequence contains two Alu repeats of the Sq family. Primer extension analysis demonstrated the presence of two major transcription initiation sites. The first transcription initiation site delineates the beginning of a noncoding first exon and resides downstream of the first Alu sequence. The second transcription initiation site is within the second Alu repeat. This Alu repeat resides within the first intron, which is spliced out of the transcript from the first start site. Neither transcription initiation site has a TATA or CCAT box. Comparison with the mouse gene suggests that the Alu sequence insertion split the intestinal promoter and that subsequently the down-stream Alu sequence took on a promoter function. No evidence was found for a far upstream non-tissue-specific promoter similar to that demonstrated in the mouse gene. Rather, consideration of results from the marsupial APOBEC-1 gene suggests that this upstream mouse promoter may have had a later evolutionary origin.

APOBEC-1 Deaminase↗
PubMed1996

Direct visualization of uridylate deletion in vitro suggests a mechanism for kinetoplastid RNA editing.

Deletion of uridylates from the 3'-most editing site of synthetic ATPase 6 pre-mRNA can be visualized directly by coincubation of a radiolabeled substrate RNA and a synthetic gRNA in 20S fractions of T.brucie mitochondrial lysates. Substrate RNA cleavage is gRNA directed and occurs 3' to the uridylates to be deleted. U residues appear to be sequentially removed from the 3' end of the 5' cleavage product prior to religation of the two pre-mRNA halves. gRNA/mRNA chimeric molecules are also produced. Time course experiments indicate that chimeras appear after cleavage intermediates and edited product. Furthermore, a mutant gRNA promotes formation of edited product but not detectable chimeras. Our results suggest a model for kinetoplastid RNA editing in which chimeric molecules are nonproductive end products of editing and not intermediates that serve as a repository for deleted U's.

Adenosine Triphosphatases↗
PubMed1999

The RNA-editing enzyme ADAR1 is localized to the nascent ribonucleoprotein matrix on Xenopus lampbrush chromosomes but specifically associates with an atypical loop.

Double-stranded RNA adenosine deaminase (ADAR1, dsRAD, DRADA) converts adenosines to inosines in double-stranded RNAs. Few candidate substrates for ADAR1 editing are known at this point and it is not known how substrate recognition is achieved. In some cases editing sites are defined by basepaired regions formed between intronic and exonic sequences, suggesting that the enzyme might function cotranscriptionally. We have isolated two variants of Xenopus laevis ADAR1 for which no editing substrates are currently known. We demonstrate that both variants of the enzyme are associated with transcriptionally active chromosome loops suggesting that the enzyme acts cotranscriptionally. The widespread distribution of the protein along the entire chromosome indicates that ADAR1 associates with the RNP matrix in a substrate-independent manner. Inhibition of splicing, another cotranscriptional process, does not affect the chromosomal localization of ADAR1. Furthermore, we can show that the enzyme is dramatically enriched on a special RNA-containing loop that seems transcriptionally silent. Detailed analysis of this loop suggests that it might represent a site of ADAR1 storage or a site where active RNA editing is taking place. Finally, mutational analysis of ADAR1 demonstrates that a putative Z-DNA binding domain present in ADAR1 is not required for chromosomal targeting of the protein.

Adenosine Deaminase↗
PubMed2006

Sequence elements critical for efficient RNA editing of a tobacco chloroplast transcript in vivo and in vitro.

In tobacco chloroplast transcripts 34 nt are efficiently edited to U. No common consensus region is present around all editing sites; however, sites can be grouped in clusters that share short common sequences. Transgene transcripts carrying either the wild-type -31/+22 or -31/+60 sequence near NTrpoB C473, an editing site within tobacco rpoB transcripts, or three different mutated sequences, were all highly edited in vivo. Endogenous transcripts of rpoB, psbL and rps14, all of which contain common sequences S1, S2 and S3 5' to NTrpoB C473, NTpsbL C2 and NTrps14 C80, were less edited in transgenic plants that over-express transcripts from NTrpoB C473 transgenes. Extent of reduction of endogenous editing differed between transgenic lines expressing mutated -31/+22 regions, depending on the abundance of the transgene transcripts. The -20/-5 sequence contains critical 5' sequence elements. Synthetic RNA templates with alterations within this 5' region were less efficiently edited in vitro than wild-type templates, by either tobacco or maize chloroplast extracts. The tobacco chloroplast extract supports both RNA editing and processing of 3' transcript termini. We conclude that within the -20/-5 region, sequences common to editing sites in the transcripts of rpoB, psbL and rps14 are critical for efficient NTrpoB C473 editing.

Base Sequence↗
PubMed1992

The psbL gene from bell pepper (Capsicum annuum): plastid RNA editing also occurs in non-photosynthetic chromoplasts.

We have determined the nucleotide sequence of the plastid psbL gene from bell pepper. This gene has an ACG as a first codon. Isolation of RNA from pepper leaves and ripe fruits and subsequent sequencing of the psbL cDNA revealed that this ACG codon is post-transcriptionally edited into an AUG initiation codon in both leaves and fruits. These data indicate that the RNA editing machinery which exists in chloroplasts is still functional in chromoplasts from ripe fruits.

Amino Acid Sequence↗
PubMed2001

Non-templated addition of nucleotides to the 3' end of nascent RNA during RNA editing in Physarum.

RNAs in Physarum: mitochondria contain extra nucleotides that are not encoded by the mitochondrial genome, at least in the traditional sense. While it is known that insertion of non-encoded nucleotides is linked to RNA synthesis, the exact nature of this relationship remains unclear. Here we demonstrate that the efficiency of editing is sensitive not only to the concentration of the nucleotide that is inserted, but also to the concentration of the nucleotide templated just downstream of an editing site. These data strongly support a co-transcriptional mechanism of Physarum: RNA editing in which non-encoded nucleotides are added to the 3' end of nascent RNAs. These results also suggest that transcription elongation and nucleotide insertion are competing processes and that recognition of editing sites most likely involves transient pausing by the Physarum: mitochondrial RNA polymerase. In addition, the pattern of nucleotide concentration effects, the context of editing sites and the accuracy of the mitochondrial RNA polymerase argue that the mechanism of Physarum: editing is distinct from that of other co-transcriptional editing systems.

Adenosine Triphosphatases↗
PubMed2006

RNA editing and alternative splicing: the importance of co-transcriptional coordination.

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (pol II) is essential for several co-transcriptional pre-messenger RNA processing events, including capping, 3'-end processing and splicing. We investigated the role of the CTD of RNA pol II in the coordination of A to I editing and splicing of the ADAR2 (ADAR: adenosine deaminases that act on RNA) pre-mRNA. The auto-editing of Adar2 intron 4 by the ADAR2 adenosine deaminase is tightly coupled to splicing, as the modification of the dinucleotide AA to AI creates a new 3' splice site. Unlike other introns, the CTD is not required for efficient splicing of intron 4 at either the normal 3' splice site or the alternative site created by editing. However, the CTD is required for efficient co-transcriptional auto-editing of ADAR2 intron 4. Our results implicate the CTD in site-selective RNA editing by ADAR2 and in coordination of editing with alternative splicing.

Adenosine Deaminase↗
PubMed2003

Modulation of RNA editing by functional nucleolar sequestration of ADAR2.

The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. Here we demonstrate that ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNA-binding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm.

Adenosine Deaminase↗
PubMed2003

Structure and sequence determinants required for the RNA editing of ADAR2 substrates.

ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. We have demonstrated previously that ADAR2 can modify its own pre-mRNA, leading to the creation of a proximal 3'-splice junction containing a non-canonical adenosine-inosine (A-I) dinucleotide. Alternative splicing to this proximal acceptor shifts the reading frame of the mature mRNA transcript, resulting in the loss of functional ADAR2 expression. Both evolutionary sequence conservation and mutational analysis support the existence of an extended RNA duplex within the ADAR2 pre-mRNA formed by base-pairing interactions between regions approximately 1.3-kilobases apart in intron 4 and exon 5. Characterization of ADAR2 pre-mRNA transcripts isolated from adult rat brain identified 16 editing sites within this duplex region, and sites preferentially modified by ADAR1 and ADAR2 have been defined using both tissue culture and in vitro editing systems. Statistical analysis of nucleotide sequences surrounding edited and non-edited adenosine residues have identified a nucleotide sequence bias correlating with ADAR2 site preference and editing efficiency. Among a mixed population of ADAR substrates, ADAR2 preferentially favors its own transcript, yet mutation of a poor substrate to conform to the defined nucleotide bias increases the ability of that substrate to be modified by ADAR2. These data suggest that both sequence and structural elements are required to define adenosine moieties targeted for specific ADAR2-mediated deamination.

Adenosine Deaminase↗
PubMed1997

Developmental changes of RNA editing of glutamate receptor subunits GluR5 and GluR6: in vivo versus in vitro.

In the present series of experiments we compared the up-regulation of GluR5 and GluR6 mRNA editing during the transition from the embryonic to the adult state with changes in the extent of editing during neuronal development in vitro. RNA was isolated from rats, from the cerebral cortex, hippocampus and cerebellum of embryonic brains (E19) and adult brains (2 months old), as well as from neurons prepared from the cortex, hippocampus and cerebellum of embryonic brains (E19) and held in tissue culture for 2, 8 or 16 days. Quantification of mRNA editing was achieved by using standards prepared from plasmids with cDNA inserts derived from the edited and unedited state of both GluR5 and GluR6 mRNA. In addition, GluR5 mRNA levels were determined in brain tissue and neuronal cells in culture by quantitative PCR. Developmental changes in the extent of GluR5 and GluR6 mRNA editing were different in vivo compared to in vitro. For GluR5 mRNA editing these differences were most pronounced in cerebellar neurons compared to cerebellar tissue: the extent of GluR5 mRNA editing found in vivo at E19 was significantly down-regulated in cerebellar neurons during the first 8 days in culture, and after 16 days in vitro the extent of editing was still about 50% of that found in the adult state in vivo. For GluR6 mRNA editing these differences were most pronounced in hippocampal neurons compared to the hippocampus in vivo: the extent of GluR6 mRNA editing found in vivo at E19 was significantly down-regulated in vitro during the whole culturing period, most pronounced after 8 days in vivo (to below 40% of that found at E19 and to below 30% of that found in adult hippocampus). GluR5 mRNA levels increased markedly from E19 to the adult brain. However, we could not find any specific pattern of changes in mRNA levels which might account for the development changes in the profile of GluR5 mRNA editing. Comparing developmental changes in the extent of mRNA editing of glutamate receptor subunits may help to elucidate the molecular and regulatory mechanisms of this important editing reaction. Strict control and clear indication of the age of primary neuronal cell cultures used should be required in accounts of electrophysiological or neurotoxicological studies as this would increase comparative usefulness of such experiments, since calcium fluxes through glutamate receptor ion channels are likely to influence the system significantly.

Analysis of Variance↗
PubMed1992

[RNA editing].

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Animals↗
PubMed2003

RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts.

We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression.

Amino Acid Sequence↗
PubMed2001

RNA editing by base deamination: more enzymes, more targets, new mysteries.

The posttranscriptional modification of messenger RNA precursors (pre-mRNAs) by base deamination can profoundly alter the physiological function of the encoded proteins. The recent identification of tRNA-specific adenosine deaminases (ADATs) has led to the suggestion that these enzymes, as well as the cytidine and adenosine deaminases acting on pre-mRNAs (CDARs and ADARs), belong to a superfamily of RNA-dependent deaminases. This superfamily might have evolved from an ancient cytidine deaminase. This article reviews the reactions catalysed by these enzymes and discusses their evolutionary relationships.

Adenosine Deaminase↗
PubMed1995

A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited.

RNA editing occurs in two higher-plant organelles, chloroplasts and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components.

Amino Acid Sequence↗
PubMed2005

Nucleotide specificity of the RNA editing reaction in pea chloroplasts.

A sensitive in vitro editing assay for the pea chloroplast petB editing site has been developed and utilized to study the mechanism of C-to-U editing in chloroplast extracts. The in vitro editing assay was characterized by several criteria including: linearity with extract amount; linearity over time; dependence on assay components; and specificity of editing site conversion. The increase in the extent C-to-U conversion of the petB editing site was nearly linear with the amount chloroplast protein extract added, although the reaction appeared to decline in rate after approximately 30 min. The assay was tested for the importance of various assay components, and the omission of protease inhibitor and ATP was shown to dramatically reduce the extent of the editing reaction. Sequence analysis of cDNA clones obtained after an in vitro editing reaction demonstrated that 12 of 17 (71%) clones were edited, and that no other nucleotide changes in these cDNAs were detected. Thus, the fidelity and specificity of the in vitro editing system appears to be excellent, and this system should be suitable to study both mechanism of the editing reaction and editing site selection. The in vitro editing reaction was strongly stimulated by the addition of ATP, and all four NTPs and dNTPs stimulated the editing reaction except for rGTP, which had no effect. Thus, the nucleotide specificity of the editing reaction is broad, and is similar in this respect to the mitochondrial editing system. Most enzyme or processes specifically utilize ATP or GTP for phosphorylation and the ability to substitute other NTPs and dNTPs is unusual. RNA helicases have a similar broad nucleotide specificity and this may reflect the involvement of an RNA helicase in plant organelle editing.

Chloroplasts↗