SMART MODE:

NORMAL GENOMIC

• Wong SK, Sato S, Lazinski DW
• Substrate recognition by ADAR1 and ADAR2.
• RNA. 2001; 7: 846-58
• Display abstract

• RNA editing catalyzed by ADAR1 and ADAR2 involves the site-specific conversion of adenosine to inosine within imperfectly duplexed RNA. ADAR1- and ADAR2-mediated editing occurs within transcripts of glutamate receptors (GluR) in the brain and in hepatitis delta virus (HDV) RNA in the liver. Although the Q/R site within the GluR-B premessage is edited more efficiently by ADAR2 than it is by ADAR1, the converse is true for the +60 site within this same transcript. ADAR1 and ADAR2 are homologs having two common functional regions, an N-terminal double-stranded RNA-binding domain and a C-terminal deaminase domain. It is neither understood why only certain adenosines within a substrate molecule serve as targets for ADARs, nor is it known which domain of an ADAR confers its specificity for particular editing sites. To assess the importance of several aspects of RNA sequence and structure on editing, we evaluated 20 different mutated substrates, derived from four editing sites, for their ability to be edited by either ADAR1 or ADAR2. We found that when these derivatives contained an A:C mismatch at the editing site, editing by both ADARs was enhanced compared to when A:A or A:G mismatches or A:U base pairs occurred at the same site. Hence substrate recognition and/or catalysis by ADARs could involve the base that opposes the edited adenosine. In addition, by using protein chimeras in which the deaminase domains were exchanged between ADAR1 and ADAR2, we found that this domain played a dominant role in defining the substrate specificity of the resulting enzyme.

• Maas S, Kim YG, Rich A
• Genomic clustering of tRNA-specific adenosine deaminase ADAT1 and two tRNA synthetases.
• Mamm Genome. 2001; 12: 387-93
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• Human tRNA-specific adenosine deaminase (hADAT1) specifically converts A37 in the anticodon loop of human tRNA(Ala) to inosine via a hydrolytic deamination mechanism. The enzyme is related to a family of RNA editing enzymes (ADARs) specific for pre-mRNA, and it has been cloned based on its sequence homology to the catalytic domain of ADARs. In the present study we have analyzed the 5'-flanking sequence of the murine ADAT1 gene, revealing that the first transcribed exon is located 1.1 kb downstream from the polyadenylation site of lysyl tRNA synthetase (KARS). The close proximity is conserved in the human genome with an intergenic distance of 5.5 kb. We determined the complete cDNA sequence as well as exon/intron organization of murine KARS. Significant sequence similarities between KARS and ADAT1 are apparent within their substrate interaction domains. Radiation hybrid panel analysis mapped human ADAT1 and human KARS to region q22.2--22.3 of Chromosome (Chr) 16 with alanyl tRNA synthetase (AARS) positioned centromeric to the KARS and ADAT1 genes. 16q22--24 has recently been recognized as a susceptibility candidate locus for several autoimmune inflammatory diseases. The clustering of three tRNA specific genes, of which two are specific for tRNA(Ala), may indicate their evolutionary relatedness or common factors involved in regulating their expression.

• Eckmann CR, Neunteufl A, Pfaffstetter L, Jantsch MF
• The Human But Not the Xenopus RNA-editing Enzyme ADAR1 Has an Atypical Nuclear Localization Signal and Displays the Characteristics of a Shuttling Protein.
• Mol Biol Cell. 2001; 12: 1911-24
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• The RNA-editing enzyme ADAR1 (adenosine deaminase that acts on RNA) is a bona fide nuclear enzyme that has been cloned from several vertebrate species. Putative nuclear localization signals (NLSs) have been identified in the aminoterminal regions of both human and Xenopus ADAR1. Here we show that neither of these predicted NLSs is biologically active. Instead, we could identify a short basic region located upstream of the RNA-binding domains of Xenopus ADAR1 to be necessary and sufficient for nuclear import. In contrast, the homologous region in human ADAR1 does not display NLS activity. Instead, we could map an NLS in human ADAR1 that overlaps with its third double-stranded RNA-binding domain. Interestingly, the NLS activity displayed by this double-stranded RNA-binding domain does not depend on RNA binding, therefore showing a dual function for this domain. Furthermore, nuclear accumulation of human (hs) ADAR1 is transcription dependent and can be stimulated by LMB, an inhibitor of Crm1-dependent nuclear export, indicating that hsADAR1 can move between the nucleus and cytoplasm. Regulated nuclear import and export of hsADAR1 can provide an excellent mechanism to control nuclear concentration of this editing enzyme thereby preventing hyperediting of structured nuclear RNAs.

• Gerber AP, Keller W
• RNA editing by base deamination: more enzymes, more targets, new mysteries.
• Trends Biochem Sci. 2001; 26: 376-84
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• 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.

• Charlab R, Valenzuela JG, Andersen J, Ribeiro JM
• The invertebrate growth factor/CECR1 subfamily of adenosine deaminase proteins.
• Gene. 2001; 267: 13-22
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• Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine. Its lack determines severe combined immunodeficiency in mammals, possibly due to accumulation of extracellular adenosine, which induces apoptosis in lymphocytes (Franco et al., 1998). Thus, presence of normal levels of ADA leads to normal growth and proliferation of lymphocytes. Several vertebrate and microbial ADA amino-acid sequences are known, with substantial similarity to each other. On the other hand, there are invertebrate growth factors as well as a candidate gene for the human cat eye syndrome (CECR1) (Riazi et al., 2000. Genomics 64, 277-285), which share substantial similarity to each other, and also to ADA. In this study, we report the expression and ADA enzymatic activity of a cDNA from the salivary glands of Lutzomyia longipalpis, a blood-sucking insect, with substantial similarity to insect growth factors and to human CECR1. We also demonstrate the existence of a subfamily of the adenosine deaminase family characterized by their unique amino-terminal region. Both Drosophila melanogaster and humans have both types of adenosine deaminases. Results indicate that these invertebrate proteins previously annotated as growth factors, as well as the human CECR1 gene product, may exert their actions through adenosine depletion. The different roles played by each type of adenosine deaminase in humans and Drosophila remains to be fully investigated.

• Raitskin O, Cho DS, Sperling J, Nishikura K, Sperling R
• RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery.
• Proc Natl Acad Sci U S A. 2001; 98: 6571-6
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• Multiple members of the ADAR (adenosine deaminases acting on RNA) gene family are involved in A-to-I RNA editing. It has been speculated that they may form a large multicomponent protein complex. Possible candidates for such complexes are large nuclear ribonucleoprotein (lnRNP) particles. The lnRNP particles consist mainly of four spliceosomal subunits that assemble together with the pre-mRNA to form a large particle and thus are viewed as the naturally assembled pre-mRNA processing machinery. Here we investigated the presence of ADARs in lnRNP particles by Western blot analysis using anti-ADAR antibodies and by indirect immunoprecipitation. Both ADAR1 and ADAR2 were found associated with the spliceosomal components Sm and SR proteins within the lnRNP particles. The two ADARs, associated with lnRNP particles, were enzymatically active in site-selective A-to-I RNA editing. We demonstrate the association of ADAR RNA editing enzymes with physiological supramolecular complexes, the lnRNP particles.

• Reenan RA
• The RNA world meets behavior: A-->I pre-mRNA editing in animals.
• Trends Genet. 2001; 17: 53-6
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• Speculations on the genetic component of animal behavior have been fueled primarily by single-gene mutations that affect specific behaviors in model organisms. Pre-mRNA editing by adenosine deaminases acting on RNA (ADARs) provides an additional mechanism for introducing protein diversity and has primarily been observed in signaling components of the nervous system. Two recent reports of mutant mice and Drosophila deficient in ADAR activities provide further evidence that pre-mRNA editing has an ancient and primary role in the evolution of nervous system function and behavior.

• Ansmant I, Motorin I
• [Identification of RNA modification enzymes using sequence homology]
• Mol Biol (Mosk). 2001; 35: 248-67
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• Modified nucleotides possessing diverse chemical structures are universally present in all cell RNAs. Their formation is a posttranscriptional process catalyzed by various RNA modification enzymes. Despite the large body of data concerning the localization of modified nucleotides in different RNAs, the enzymes involved in their biosynthesis remain largely unknown. In this review we discuss the recent achievements in application of the sequence homology approach to searching for and identifying RNA modification enzymes such as RNA-pseudouridine synthases, RNA-methyltransferases, and RNA-inosine synthases (adenosine deaminases).

• Chen CX, Cho DS, Wang Q, Lai F, Carter KC, Nishikura K
• A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains.
• RNA. 2000; 6: 755-67
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• Members of the double-stranded RNA- (dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C (5-HT(2C)R). We have isolated hADAR3, the third member of this class of human enzyme and investigated its editing site selectivity using in vitro RNA editing assay systems. As originally reported for rat ADAR3 or RED2, purified ADAR3 proteins could not edit GluR-B RNA at the "Q/R" site, the "R/G" site, and the intronic "hot spot" site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT(2C)R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses revealed that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibited in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing.

• Rutz B, Seraphin B
• A dual role for BBP/ScSF1 in nuclear pre-mRNA retention and splicing.
• EMBO J. 2000; 19: 1873-86
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• The MSL5 gene, which codes for the splicing factor BBP/ScSF1, is essential in Saccharomyces cerevisiae, yet previous analyses failed to reveal a defect in assembly of (pre)-spliceosomes or in vitro splicing associated with its depletion. We generated 11 temperature-sensitive (ts) mutants and one cold-sensitive (cs) mutant in the corresponding gene and analyzed their phenotypes. While all mutants were blocked in the formation of commitment complex 2 (CC2) at non-permissive and permissive temperature, the ts mutants showed no defect in spliceosome formation and splicing in vitro. The cs mutant was defective in (pre)-spliceosome formation, but residual splicing activity could be detected. In vivo splicing of reporters carrying introns weakened by mutations in the 5' splice site and/or in the branchpoint region was affected in all mutants. Pre-mRNA leakage to the cytoplasm was strongly increased (up to 40-fold) in the mutants. A combination of ts mutants with a disruption of upf1, a gene involved in nonsense-mediated decay, resulted in a specific synthetic growth phenotype, suggesting that the essential function of SF1 in yeast could be related to the retention of pre-mRNA in the nucleus.

• Higuchi M et al.
• Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2.
• Nature. 2000; 406: 78-81
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• 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.

• Ouaissi A, Vergnes B, Borges M, Guilvard E
• Identification and molecular characterization of two novel Trypanosoma cruzi genes encoding polypeptides sharing sequence motifs found in proteins involved in RNA editing reactions.
• Gene. 2000; 253: 271-80
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• We have previously identified a Trypanosoma cruzi cDNA encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we reported that Tc52 also plays a role in T. cruzi-associated immunosuppression observed during Chagas' disease. Moreover, Tc52 gene targeting deletion strategy allowed us to demonstrate that monoallelic disruption of Tc52 resulted in the alteration of the metacyclogenesis process and the production of less virulent parasites. Sequence analysis of a 7358 bp genomic fragment containing the Tc52 encoding gene revealed two additional open reading frames (ORF-A and C). The ORFs are likely to have protein coding function by a number of criteria, including reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunofluorescence analyses. The deduced amino-acid (aa) sequence of the ORF-A localized upstream of the Tc52 gene revealed that it contains within its N-terminus (aa 1 to 170) four RGG boxes known to act as RNA binding motifs in some proteins that interact with RNA, interspersed with a high density of glycine with regular spacing of tryptophan (WX(9-10)) in which X is often a glycine. Moreover, the C-terminal part of the ORF-C (aa 253-289) contains a motif that is strikingly similar (7-35% identity, 14-46% similarity over 28aa) to a short sequence (RNP1) comprising the consensus sequence RNA binding domain (CS-RBD) found in a number of proteins that interact with RNA. The aa sequence from the ORF-C localized downstream of the Tc52 gene showed significant homology to human adenosine deaminase acting on RNA (hADAT1) that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala) and to its homologue yeast protein (Tad1p) (22-25% identity and an additional 38-40% similarity over 177aa). Moreover, highly similar motifs of the deaminase domain are present in the T. cruzi ORF-C. Furthermore, the 5' flanking regions of the genes contained repeat TATA and CAAT nucleotide sequences which resemble the motifs found upstream of the transcription initiation sites in eukaryotic promoters. Therefore, the characterization of novel T. cruzi genes encoding proteins which show similarity to components of RNA processing reactions provides new tools to investigate the gene expression regulation in these parasitic organisms. Moreover, our recent findings on the Tc52 encoding gene underline the interest of genetic manipulation of T. cruzi, not only making it possible to use more closely an in vitro approach to find out how genes function, but also to obtain 'attenuated' strains that could be used in the development of vaccinal strategies.

• Slavov D, Crnogorac-Jurcevic T, Clark M, Gardiner K
• Comparative analysis of the DRADA A-to-I RNA editing gene from mammals, pufferfish and zebrafish.
• Gene. 2000; 250: 53-60
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• The DRADA gene in mammals encodes an A-to-I RNA editase, an adenosine deaminase that acts on pre-mRNAs to produce site specific inosines. DRADA has been shown to deaminate specific adenosine residues in a subset of glutamate and serotonin receptors, and this editing results in proteins of altered sequences and functional properties. DRADA thus plays a role in creating protein diversity. To study the evolutionary significance of this gene, we have characterized the genomic structure of DRADA from Fugu rubripes, and compared the protein sequences of DRADA from mammals, pufferfish and zebrafish. The DRADA gene from Fugu is three-fold compacted with respect to the human gene, and contains a novel intron within the large second coding exon. DRADA cDNAs were isolated from zebrafish and a second pufferfish, Tetraodon fluviatilis. Comparisons among fish, and between fish and mammals, of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA binding domains vary within a single protein in their levels of conservation. Conservation within the Z DNA binding domain has also been assessed. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.

• Kolesnikov AA, Shoenian G, Presber W
• [An edited segment of the kinetoplast gene MURF4 (ATPase 6) in leishmania has a similar structure]
• Mol Biol (Mosk). 2000; 34: 210-3

• Slavov D, Clark M, Gardiner K
• Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish.
• Gene. 2000; 250: 41-51
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• One type of RNA editing involves the deamination of adenosine (A) residues to inosines (I) at specific sites in specific pre-mRNAs. These inosines are subsequently read as guanosines by the ribosome, with potentially significant consequences for protein sequence. In mammals, two such A-to-I RNA editases are RED1, which edits some serotonin and glutamate receptors, and RED2, with unidentified substrates. To study the evolutionary conservation among these editases, we have isolated homologous genes from the Japanese pufferfish, Fugu rubripes. Fugu has two genes homologous to Red1 that are similar in size and organization and that show a fivefold compaction relative to the human gene; they differ, however, in their base compositional features. The Fugu gene for RED2 is unusually large, spanning more than 50kb; within the largest intron, there is evidence for a novel gene on the opposite strand. Because of these unusual features, the partial genomic structure was determined for the mouse RED2 gene. A partial cDNA for RED1 was also isolated from zebrafish. Comparisons between fish and between fish and mammals of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA-binding domains vary within a single protein in their levels of conservation. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.

• Aruscavage PJ, Bass BL
• A phylogenetic analysis reveals an unusual sequence conservation within introns involved in RNA editing.
• RNA. 2000; 6: 257-69
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• Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes that convert adenosines to inosines within cellular and viral RNAs. Certain glutamate receptor (gluR) pre-mRNAs are substrates for the enzymes in vivo. For example, at the R/G editing site of gluR-B, -C, and -D RNAs, ADARs change an arginine codon (AGA) to a glycine codon (IGA) so that two protein isoforms can be synthesized from a single encoded mRNA; the highly related gluR-A sequence is not edited at this site. To gain insight into what features of an RNA substrate are important for accurate and efficient editing by an ADAR, we performed a phylogenetic analysis of sequences required for editing at the R/G site. We observed highly conserved sequences that were shared by gluR-B, -C, and -D, but absent from gluR-A. Surprisingly, in contrast to results obtained in phylogenetic analyses of tRNA and rRNA, it was the bases in paired, helical regions whose identity was conserved, whereas bases in nonhelical regions varied, but maintained their nonhelical state. We speculate this pattern in part reflects constraints imposed by ADAR's unique specificity and gained support for our hypotheses with mutagenesis studies. Unexpectedly, we observed that some of the gluR introns were conserved beyond the sequences required for editing. The approximately 600-nt intron 13 of gluR-C was particularly remarkable, showing >94% nucleotide identity between human and chicken, organisms estimated to have diverged 310 million years ago.

• Maas S, Kim YG, Rich A
• Sequence, genomic organization and functional expression of the murine tRNA-specific adenosine deaminase ADAT1.
• Gene. 2000; 243: 59-66
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• We have recently identified the first mammalian tRNA-specific adenosine deaminase human ADAT1, a member of the ADAR family of RNA editing enzymes. This protein is responsible for the first step of the unique A(37) to m(1)I(37) modification in eukaryotic tRNA(Ala). Here, we present the genomic structure of murine ADAT1 and the functional expression of mADAT1 cDNA. In mouse, as well as in human, ADAT1 is expressed from a single copy gene. The coding region of the mADAT1 gene is spread over nine exons, covering approximately 30kb of genomic DNA and encodes a protein of 499 amino acids. Overall, mADAT1 shares 81% nucleotide homology and 87.5% protein homology with the human ortholog. The recombinant mouse protein is active specifically and with a high efficiency on human tRNA(Ala) in vitro. Its genomic organization is compared to the structures of the sequence-related, pre-mRNA specific adenosine deaminases ADAR1 and ADAR2.

• Bock R
• Sense from nonsense: how the genetic information of chloroplasts is altered by RNA editing.
• Biochimie. 2000; 82: 549-57
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• Plastid transcripts can be subject to an RNA processing mechanism changing the identity of individual nucleotides and thus altering the information content of the mRNA. This processing step was termed RNA editing and adds a novel mechanism to the multitude of RNA maturation events required before mRNAs can serve as faithful templates in plastid protein biosynthesis. RNA editing in chloroplasts proceeds by the conversion of individual cytidine residues to uridine and, in some bryophytes, also by the reverse event, uridine-to-cytidine transitions. The discovery of RNA editing in chloroplasts has provided researchers with a wealth of molecular and evolutionary puzzles, many of which are not yet solved. However, recent work employing chloroplast transformation technologies has shed some light on the molecular mechanisms by which RNA editing sites are recognized with extraordinarily high precision. Also, extensive phylogenetic studies have provided intriguing insights in the evolutionary dynamics with which editing sites may come and go. This review summarizes the state-of-the-art in the field of chloroplast RNA editing, discusses mechanistic and evolutionary aspects of editing and points out some of the important open questions surrounding this enigmatic RNA processing step.

• Kari L, Landweber LF
• Computing with DNA.
• Methods Mol Biol. 2000; 132: 413-30

• Liu Y, Lei M, Samuel CE
• Chimeric double-stranded RNA-specific adenosine deaminase ADAR1 proteins reveal functional selectivity of double-stranded RNA-binding domains from ADAR1 and protein kinase PKR.
• Proc Natl Acad Sci U S A. 2000; 97: 12541-6
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• The RNA-specific adenosine deaminase (ADAR1) and the RNA-dependent protein kinase (PKR) are both interferon-inducible double-stranded (ds) RNA-binding proteins. ADAR1, an RNA editing enzyme that converts adenosine to inosine, possesses three copies of a dsRNA-binding motif (dsRBM). PKR, a regulator of translation, has two copies of the highly conserved dsRBM motif. To assess the functional selectivity of the dsRBM motifs in ADAR1, we constructed and characterized chimeric proteins in which the dsRBMs of ADAR1 were substituted with those of PKR. Recombinant PKR-ADAR1 chimeras retained significant RNA adenosine deaminase activity measured with a synthetic dsRNA substrate when the spacer region between the RNA-binding and catalytic domains of the deaminase was exactly preserved. However, with natural substrates, substitution of the first two dsRBMs of ADAR1 with those from PKR dramatically reduced site-selective editing activity at the R/G and (+)60 sites of the glutamate receptor B subunit pre-RNA and completely abolished editing of the serotonin 2C receptor (5-HT(2C)R) pre-RNA at the A site. Chimeric deaminases possessing only the two dsRBMs from PKR were incapable of editing either glutamate receptor B subunit or 5-HT(2C)R natural sites but edited synthetic dsRNA. Finally, RNA antagonists of PKR significantly inhibited the activity of chimeric PKR-ADAR1 proteins relative to wild-type ADAR1, further demonstrating the functional selectivity of the dsRBM motifs.

• Reenan RA, Hanrahan CJ, Barry G
• The mle(napts) RNA helicase mutation in drosophila results in a splicing catastrophe of the para Na+ channel transcript in a region of RNA editing.
• Neuron. 2000; 25: 139-49
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• The mle(napts) mutation causes temperature-dependent blockade of action potentials resulting from decreased abundance of para-encoded Na+ channels. Although maleless (mle) encodes a double-stranded RNA (dsRNA) helicase, exactly how mle(napts) affects para expression remained uncertain. Here, we show that para transcripts undergo adenosine-to-inosine (A-to-I) RNA editing via a mechanism that apparently requires dsRNA secondary structure formation encompassing the edited exon and the downstream intron. In an mle(napts) background, >80% of para transcripts are aberrant, owing to internal deletions that include the edited exon. We propose that the Mle helicase is required to resolve the dsRNA structure and that failure to do so in an mle(napts) background causes exon skipping because the normal splice donor is occluded. These results explain how mlen(napts) affects Na+ channel expression and provide new insights into the mechanism of RNA editing.

• Charlab R, Rowton ED, Ribeiro JM
• The salivary adenosine deaminase from the sand fly Lutzomyia longipalpis.
• Exp Parasitol. 2000; 95: 45-53
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• In the process of sequencing a subtracted cDNA library from the salivary glands of the sand fly Lutzomyia longipalpis, we identified a cDNA with similarities to gene products of the adenosine deaminase family. Prompted by this cDNA finding, we detected adenosine deaminase activity at levels of 1 U/mg protein in salivary gland homogenates. The activity was significantly reduced following a blood meal indicating its apparent secretory fate. The native enzyme has a K(m) of approximately 10 microM, an isoelectric pH between 4.5 and 5.5, and an apparent molecular weight of 52 kDa by size exclusion chromatography. The possible role of this enzyme, which converts adenosine to inosine, in the feeding physiology of L. longipalpis is discussed.

• Lehmann KA, Bass BL
• Double-stranded RNA adenosine deaminases ADAR1 and ADAR2 have overlapping specificities.
• Biochemistry. 2000; 39: 12875-84
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• Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to produce inosines within RNAs that are largely double-stranded (ds). Like most dsRNA binding proteins, the enzymes will bind to any dsRNA without apparent sequence specificity. However, once bound, ADARs deaminate certain adenosines more efficiently than others. Most of what is known about the intrinsic deamination specificity of ADARs derives from analyses of Xenopus ADAR1. In addition to ADAR1, mammalian cells have a second ADAR, named ADAR2; the deamination specificity of this enzyme has not been rigorously studied. Here we directly compare the specificity of human ADAR1 and ADAR2. We find that, like ADAR1, ADAR2 has a 5' neighbor preference (A approximately U > C = G), but, unlike ADAR1, also has a 3' neighbor preference (U = G > C = A). Simultaneous analysis of both neighbor preferences reveals that ADAR2 prefers certain trinucleotide sequences (UAU, AAG, UAG, AAU). In addition to characterizing ADAR2 preferences, we analyzed the fraction of adenosines deaminated in a given RNA at complete reaction, or the enzyme's selectivity. We find that ADAR1 and ADAR2 deaminate a given RNA with the same selectivity, and this appears to be dictated by features of the RNA substrate. Finally, we observed that Xenopus and human ADAR1 deaminate the same adenosines on all RNAs tested, emphasizing the similarity of ADAR1 in these two species. Our data add substantially to the understanding of ADAR2 specificity, and aid in efforts to predict which ADAR deaminates a given editing site adenosine in vivo.

• Mehta A, Kinter MT, Sherman NE, Driscoll DM
• Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA.
• Mol Cell Biol. 2000; 20: 1846-54
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• The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.

• Oppegard LM, Kabb AL, Connell GJ
• Activation of guide RNA-directed editing of a cytochrome b mRNA.
• J Biol Chem. 2000; 275: 33911-9
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• The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created only after the addition or deletion of specific uridines. Although in vitro systems have been valuable in characterizing the editing mechanism, only a limited number of mRNAs are accurately edited in vitro. We demonstrate here that in vitro editing of cytochrome b mRNA is inhibited by an A-U sequence present on both the 5'-untranslated sequence and on a cytochrome b guide RNA. Mutation of the sequence on the guide RNA stimulates directed editing and results in the loss of binding to at least one component within the editing extract. Mutation of the sequence on the mRNA increases the accuracy of the editing. Evidence is provided that suggests the A-U sequence interacts with the editing machinery both in vitro and in vivo.

• Barabino SM, Ohnacker M, Keller W
• Distinct roles of two Yth1p domains in 3'-end cleavage and polyadenylation of yeast pre-mRNAs.
• EMBO J. 2000; 19: 3778-87
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• Yth1p is the yeast homologue of the 30 kDa subunit of mammalian cleavage and polyadenylation specificity factor (CPSF). The protein is part of the cleavage and polyadenylation factor CPF, which includes cleavage factor II (CF II) and polyadenylation factor I (PF I), and is required for both steps in pre-mRNA 3'-end processing. Yth1p is an RNA-binding protein that was previously shown to be essential for polyadenylation. Here, we demonstrate that Yth1p is also required for the cleavage reaction and that two protein domains have distinct roles in 3'-end processing. The C-terminal part is required in polyadenylation to tether Fip1p and poly(A) polymerase to the rest of CPF. A single point mutation in the highly conserved second zinc finger impairs both cleavage and polyadenylation, and affects the ability of Yth1p to interact with the pre-mRNA and other CPF subunits. Finally, we find that Yth1p binds to CYC1 pre-mRNA in the vicinity of the cleavage site. Our results indicate that Yth1p is important for the integrity of CPF and participates in the recognition of the cleavage site.

• Ohman M, Kallman AM, Bass BL
• In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site.
• RNA. 2000; 6: 687-97
• Display abstract

• The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that is completely or largely double stranded. In mammals, most of the characterized substrates encode receptors involved in neurotransmission, and these substrates are thought to be targeted by the mammalian enzymes ADAR1 and ADAR2. Although some ADAR substrates are deaminated very promiscuously, mammalian glutamate receptor B (gluR-B) pre-mRNA is deaminated at a few specific adenosines. Like most double-stranded RNA (dsRNA) binding proteins, ADARs bind to many different sequences, but few studies have directly measured and compared binding affinities. We have attempted to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question we studied binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and compared a wild-type molecule with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences was almost identical, footprinting studies indicate ADAR2 binds to the wild-type RNA at a discrete region surrounding the editing site, whereas binding to the mutant appeared nonspecific.

• Mulligan RM, Williams MA, Shanahan MT
• RNA editing site recognition in higher plant mitochondria.
• J Hered. 1999; 90: 338-44
• Display abstract

• RNA editing is a process by which genomically encoded cytidines are converted to uridines in plant mitochondrial transcripts. This conversion usually changes the amino acid specified by a codon and converts an "aberrant" residue to the evolutionarily conserved amino acid. The selection of the edited cytidine is highly specific. The cis-acting sequences for editing site recognition have been examined in ribosomal protein S12 (rps12) transcripts and in transcripts for a second copy of an internal portion of the ribosomal protein S12 (rps12b). rps12b was created by recombination at 7 and 9 nucleotide sequences that included editing sites I and IV of rps12, thus affording an opportunity to study the editing of chimeric transcripts with rearrangement very near C to U editing sites. Rearrangements downstream of editing site IV did not affect the editing of that sequence, while rearrangement upstream of editing site I ablated editing at that cytidine residue. Secondary structure predictions indicated that RNA structure did not correlate with the editing of these substrates. These results taken together with other studies in the literature suggest that RNA editing site recognition is primarily dependent on the 5' flanking RNA sequence.

• Eckmann CR, Jantsch MF
• The RNA-editing enzyme ADAR1 is localized to the nascent ribonucleoprotein matrix on Xenopus lampbrush chromosomes but specifically associates with an atypical loop.
• J Cell Biol. 1999; 144: 603-15
• Display abstract

• 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.

• Matsumoto M, Briones AV, Nishigaki T, Hoshi M
• Sequence analysis of cDNAs encoding precursors of starfish asterosaps.
• Dev Genet. 1999; 25: 130-6
• Display abstract

• To understand how starfish sperm activating peptides (asterosaps) are synthesized in the ovary, we cloned cDNAs encoding asterosaps and elucidated their nucleotide sequences. The mRNA encoding asterosaps was synthesized only in the oocytes, but not in the follicle cells, and the length was 3.7 kb. The cDNA clones contained multiple isoforms of asterosaps. We assume that asterosap precursors are large prepolypeptide chains with an unusual "rosary-type" structure made of 10 successive similar stretches of 51-55 residues. Each stretch finishes with a "spacer" of 17-21 residues immediately followed by the sequence of one asterosap isoform. The N-terminal of this precursor has 19-21 successive glutamine-rich repeating units. Maturation of the precursor may require endopeptidases that cleave both C- and N-sites of lysine-arginine.

• Rueter SM, Dawson TR, Emeson RB
• Regulation of alternative splicing by RNA editing.
• Nature. 1999; 399: 75-80
• Display abstract

• The enzyme ADAR2 is a double-stranded RNA-specific adenosine deaminase which is involved in the editing of mammalian messenger RNAs by the site-specific conversion of adenosine to inosine. Here we identify several rat ADAR2 mRNAs produced as a result of two distinct alternative splicing events. One such splicing event uses a proximal 3' acceptor site, adding 47 nucleotides to the ADAR2 coding region, changing the predicted reading frame of the mature ADAR2 transcript. Nucleotide-sequence analysis of ADAR2 genomic DNA revealed the presence of adenosine-adenosine (AA) and adenosine-guanosine (AG) dinucleotides at these proximal and distal alternative 3' acceptor sites, respectively. Use of the proximal 3' acceptor depends upon the ability of ADAR2 to edit its own pre-mRNA, converting the intronic AA to an adenosine-inosine (AI) dinucleotide which effectively mimics the highly conserved AG sequence normally found at 3' splice junctions. Our observations indicate that RNA editing can serve as a mechanism for regulating alternative splicing and they suggest a novel strategy by which ADAR2 can modulate its own expression.

• Kim YG, Lowenhaupt K, Schwartz T, Rich A
• The interaction between Z-DNA and the Zab domain of double-stranded RNA adenosine deaminase characterized using fusion nucleases.
• J Biol Chem. 1999; 274: 19081-6
• Display abstract

• Zab is a structurally defined protein domain that binds specifically to DNA in the Z conformation. It consists of amino acids 133-368 from the N terminus of human double-stranded RNA adenosine deaminase, which is implicated in RNA editing. Zab contains two motifs with related sequence, Zalpha and Zbeta. Zalpha alone is capable of binding Z-DNA with high affinity, whereas Zbeta alone has little DNA binding activity. Instead, Zbeta modulates Zalpha binding, resulting in increased sequence specificity for alternating (dCdG)n as compared with (dCdA/dTdG)n. This relative specificity has previously been demonstrated with short oligonucleotides. Here we demonstrate that Zab can also bind tightly to (dCdG)n stabilized in the Z form in supercoiled plasmids. Binding was assayed by monitoring cleavage of the plasmids using fusion nucleases, in which Z-DNA-binding peptides from the N terminus of double-stranded RNA adenosine deaminase are linked to the nuclease domain of FokI. A fusion nuclease containing Zalpha shows less sequence specificity, as well as less conformation specificity, than one containing Zab. Further, a construct in which Zbeta has been replaced in Zab with Zalpha, cleaves Z-DNA regions in supercoiled plasmids more efficiently than the wild type but with little sequence specificity. We conclude that in the Zab domain, both Zalpha and Zbeta contact DNA. Zalpha contributes contacts that produce conformation specificity but not sequence specificity. In contrast, Zbeta contributes weakly to binding affinity but discriminates between sequences of Z-DNAs.

• Yi-Brunozzi HY, Easterwood LM, Kamilar GM, Beal PA
• Synthetic substrate analogs for the RNA-editing adenosine deaminase ADAR-2.
• Nucleic Acids Res. 1999; 27: 2912-7
• Display abstract

• We have synthesized structural analogs of a natural RNA editing substrate and compared editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase. Deamination rates were shown to be sensitive to structural changes at the 2[prime]-carbon of the edited adenosine. Methylation of the 2[prime]-OH caused a large decrease in deamination rate, whereas 2[prime]-deoxyadenosine and 2[prime]-deoxy-2[prime]-fluoroadenosine were deaminated at a rate similar to adenosine. In addition, a duplex containing as few as 19 bp of the stem structure adjacent to the R/G editing site of the GluR-B pre-mRNA supports deamination of the R/G adenosine by ADAR-2. This identification and initial characterization of synthetic RNA editing substrate analogs further defines structural elements in the RNA that are important for the deamination reaction and sets the stage for additional detailed structural, thermodynamic and kinetic studies of the ADAR-2 reaction.

• Gerber AP, Keller W
• An adenosine deaminase that generates inosine at the wobble position of tRNAs.
• Science. 1999; 286: 1146-9
• Display abstract

• Several transfer RNAs (tRNAs) contain inosine (I) at the first position of their anticodon (position 34); this modification is thought to enlarge the codon recognition capacity during protein synthesis. The tRNA-specific adenosine deaminase of Saccharomyces cerevisiae that forms I(34) in tRNAs is described. The heterodimeric enzyme consists of two sequence-related subunits (Tad2p/ADAT2 and Tad3p/ADAT3), both of which contain cytidine deaminase (CDA) motifs. Each subunit is encoded by an essential gene (TAD2 and TAD3), indicating that I(34) is an indispensable base modification in elongating tRNAs. These results provide an evolutionary link between the CDA superfamily and RNA-dependent adenosine deaminases (ADARs/ADATs).

• Maas S, Gerber AP, Rich A
• Identification and characterization of a human tRNA-specific adenosine deaminase related to the ADAR family of pre-mRNA editing enzymes.
• Proc Natl Acad Sci U S A. 1999; 96: 8895-900
• Display abstract

• The mammalian adenosine deaminases acting on RNA (ADARs) constitute a family of sequence-related proteins involved in pre-mRNA editing of nuclear transcripts through site-specific adenosine modification. We report here the identification and characterization of a human ADAR protein, hADAT1, that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala). It represents the functional homologue of the recently identified yeast protein Tad1p [Gerber, A., Grosjean, H., Melcher, T. & Keller, W. (1998) EMBO J. 17, 4780-4789]. The hADAT1 cDNA predicts a protein of 502 aa whose sequence displays strongest overall homology to a Drosophila melanogaster ORF (50% similarity, 32% identity), and the catalytic domain is closely related to the other ADAR proteins. In vitro, the recombinantly expressed and purified hADAT1 protein efficiently and specifically deaminates A(37) in the anticodon loop of tRNA(Ala) from higher eukaryotes and with lower efficiency from lower eukaryotes. It does not modify adenosines residing in double-stranded RNA or in pre-mRNAs that serve as substrates for ADAR1 or ADAR2. The anticodon stem-loop of tRNA(Ala) alone is not a functional substrate for hADAT1. The enzyme is expressed ubiquitously in human tissues and is represented by a single gene. The identification and cloning of hADAT1 should help to elucidate the physiological significance of this unique modification in tRNA(Ala), which is conserved from yeast to man.

• Lehmann KA, Bass BL
• The importance of internal loops within RNA substrates of ADAR1.
• J Mol Biol. 1999; 291: 1-13
• Display abstract

• Adenosine deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that convert adenosines to inosines within double-stranded RNA (dsRNA). Although ADARs deaminate perfectly base-paired dsRNA promiscuously, deamination is limited to a few, selected adenosines within dsRNA containing mismatches, bulges and internal loops. As a first step in understanding how RNA structural features promote selectivity, we investigated the role of internal loops within ADAR substrates. We observed that a dsRNA helix is deaminated at the same sites whether it exists as a free molecule or is flanked by internal loops. Thus, internal loops delineate helix ends for ADAR1. Since ADAR1 deaminates short RNAs at fewer adenosines than long RNAs, loops decrease the number of deaminations within an RNA by dividing a long RNA into shorter substrates. For a series of symmetric internal loops related in sequence, larger loops (>/=six nucleotides) acted as helix ends, whereas smaller loops (

• Liu Y, Samuel CE
• Editing of glutamate receptor subunit B pre-mRNA by splice-site variants of interferon-inducible double-stranded RNA-specific adenosine deaminase ADAR1.
• J Biol Chem. 1999; 274: 5070-7
• Display abstract

• The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA-editing enzyme that catalyzes the deamination of adenosine in double-stranded RNA structures. Three alternative splice-site variants of ADAR1 (ADAR1-a, -b, and -c) occur that possess functionally distinct double-stranded RNA-binding motifs as measured with synthetic double-stranded RNA substrates. The pre-mRNA transcript encoding the B subunit of glutamate receptor (GluR-B) has two functionally important editing sites (Q/R and R/G sites) that undergo selective A-to-I conversions. We have examined the ability of the three ADAR1 splice-site variants to catalyze the editing of GluR-B pre-mRNA at the Q/R and R/G sites as well as an intron hotspot (+60) of unknown function. Measurement of GluR-B pre-mRNA editing in vitro revealed different site-specific deamination catalyzed by the three ADAR1 variants. The ADAR1-a, -b, and -c splice variants all efficiently edited the R/G site and the intron +60 hotspot but exhibited little editing activity at the Q/R site. ADAR1-b and -c showed higher editing activity than ADAR1-a for the R/G site, whereas the intron +60 site was edited with comparable efficiency by all three ADAR1 splice variants. Mutational analysis revealed that the functional importance of each of the three RNA-binding motifs of ADAR1 varied with the specific target editing site in GluR-B RNA. Quantitative reverse transcription-polymerase chain reaction analyses of GluR-B RNA from dissected regions of rat brain showed significant expression and editing at the R/G site in all brain regions examined except the choroid plexus. The relative levels of the alternatively spliced flip and flop isoforms of GluR-B RNA varied among the choroid plexus, cortex, hippocampus, olfactory bulb, and striatum, but in all regions of rat brain the editing of the flip isoform was greater than that of the flop isoform.

• Kobielak K, Kobielak A, Trzeciak WH
• Cloning of the lymphoid enhancer binding factor-1 (Lef-1) cDNA from rat kidney: homology to the mouse sequence.
• Acta Biochim Pol. 1999; 46: 885-8
• Display abstract

• We have cloned and sequenced rat cDNA that encodes the Lef-1 protein. The cDNA, containing 1194 nt exhibits 94% similarity to the mouse Lef-1 cDNA. The deduced amino-acids sequence of rat Lef-1 protein, consisting of 397 amino acids, exhibited 98% homology with the known sequence of mouse Lef-1 protein.

• Vanfleteren JR, Vierstraete AR
• Insertional RNA editing in metazoan mitochondria: the cytochrome b gene in the nematode Teratocephalus lirellus.
• RNA. 1999; 5: 622-4

• Hanrahan CJ, Palladino MJ, Bonneau LJ, Reenan RA
• RNA editing of a Drosophila sodium channel gene.
• Ann N Y Acad Sci. 1999; 868: 51-66
• Display abstract

• Extensive analysis of cDNAs from the para locus in D. melanogaster reveals posttranscriptional modifications indicative of adenosine-to-inosine RNA editing. Most of these edits occur in highly conserved regions of the Na+ channel, and they occur in distant relatives of D. melanogaster as well. Sequence comparison between species has identified putative cis-acting elements important for each RNA editing site. Double-stranded RNA secondary structures with striking similarity to known RNA editing sites were generated based on these data. In addition, the RNA editing sites appear to be developmentally regulated. We have cloned a potential RNA editase, DRED, with a high degree of homology to the mammalian RED1,2 genes. The DRED locus itself is highly regulated by transcription from alternative promoters and alternative splicings.

• Scott J, Navaratnam N, Carter C
• Molecular modelling of the biosynthesis of the RNA-editing enzyme APOBEC-1, responsible for generating the alternative forms of apolipoprotein B.
• Exp Physiol. 1999; 84: 791-800
• Display abstract

• We discovered in 1987 that the shorter form of apolipoprotein B (B48) synthesized in the intestine is due to the action, previously unrecognized in mammalian cells, of an mRNA-editing process, and more recently we demonstrated that this was due to a specific enzyme (APOBEC-1) with cytidine deaminase activity. We show here, by sequence alignment, molecular modelling and mutagenesis, that APOBEC-1 is a cytidine deaminase, responsible for editing apoB mRNA, and that is related in crystal structure to the cytidine deaminase of Escherichia coli (ECCDA). The two enzymes are both homodimers with composite active sites formed with loops from each monomer. In the sequence of APOBEC-1, three gaps compared with ECCDA match the size and contour of the minimal RNA substrate. We propose a model in which the asymmetric binding of one active site to the substrate cytidine which is positioned by the downstream binding of the product uridine and that this helps to target the other active site for deamination.

• Wahle E, Ruegsegger U
• 3'-End processing of pre-mRNA in eukaryotes.
• FEMS Microbiol Rev. 1999; 23: 277-95
• Display abstract

• 3'-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail. In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA. Six factors have been identified that carry out the two reactions. With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned. Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown. Several examples of regulated polyadenylation are being analyzed at the molecular level. In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified. The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown. 3'-Processing is known to be coupled to transcription. This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase II.

• Brennicke A, Marchfelder A, Binder S
• RNA editing.
• FEMS Microbiol Rev. 1999; 23: 297-316
• Display abstract

• The term RNA editing describes those molecular processes in which the information content is altered in an RNA molecule. To date such changes have been observed in tRNA. rRNA and mRNA molecules of eukaryotes, but not prokaryotes. The demonstration of RNA editing in prokaryotes may only be a matter of time, considering the range of species in which the various RNA editing processes have been found. RNA editing occurs in the nucleus, as well as in mitochondria and plastids, which are thought to have evolved from prokaryotic-like endosymbionts. Most of the RNA editing processes, however, appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing mechanisms includes nucleoside modifications such as C to U and A to I deaminations, as well as non-templated nucleotide additions and insertions. RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence.

• Kudla J, Bock R
• RNA editing in an untranslated region of the Ginkgo chloroplast genome.
• Gene. 1999; 234: 81-6
• Display abstract

• mRNAs in plant cell organelles can be subject to RNA editing, an RNA processing step altering the identity of single nucleotide residues. In higher plant chloroplasts, editing proceeds by C-to-U conversions at highly specific sites. All known plastid RNA editing sites are located in protein-coding regions and, typically, change the coding properties of the mRNA. To gain more insight into the evolution of editing, we have determined the molecular structure and RNA editing pattern of the psbE operon of the primitive seed plant Ginkgo biloba. We report here the identification of altogether four sites of C-to-U editing, two of which are unique to Ginkgo and have not been found in other species. Surprisingly, one of the sites is located in an intercistronic spacer, thus being the first chloroplast editing site detected outside a protein-coding region. This indicates that the plastid editing machinery can operate also in untranslated regions and without having apparent functional consequences.

• Morse DP, Bass BL
• Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA.
• Proc Natl Acad Sci U S A. 1999; 96: 6048-53
• Display abstract

• Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in Caenorhabditis elegans. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.

• Seeburg PH, Higuchi M, Sprengel R
• RNA editing of brain glutamate receptor channels: mechanism and physiology.
• Brain Res Brain Res Rev. 1998; 26: 217-29
• Display abstract

• Glutamate-gated cation selective channels mediate fast excitatory neurotransmission in the mammalian brain. Functionally critical channel positions contain amino acid residues not predicted from the exonic sequence for the channel subunits. The codons for these residues are created in the respective primary gene transcripts by the site selective deamination of adenosine to inosine. This type of RNA editing requires a short double-stranded RNA structure formed by the exonic sequence around the adenosine targeted for deamination with a complementary sequence in the downstream intron and hence, it precedes splicing. Candidate enzymes for nuclear transcript editing currently comprise three molecularly cloned mammalian RNA-dependent adenosine deaminases. Two of these are expressed in most body tissues, perhaps indicating that adenosine deamination in transcripts is more global than has been recognized. Indeed, numerous mRNAs in different tissues may contain inosine residues and encode proteins with amino acid substitutions and different properties relative to the exonically encoded forms. If so, RNA editing by adenosine deamination may significantly enlarge the functional repertoire of the mammalian genome.

• Paul MS, Bass BL
• Inosine exists in mRNA at tissue-specific levels and is most abundant in brain mRNA.
• EMBO J. 1998; 17: 1120-7
• Display abstract

• The general view that mRNA does not contain inosine has been challenged by the discovery of adenosine deaminases that act on RNA (ADARs). Although inosine monophosphate (IMP) cannot be detected in crude preparations of nucleotides derived from poly(A)+ RNA, here we show it is readily detectable and quantifiable once it is purified away from the Watson-Crick nucleotides. We report that IMP is present in mRNA at tissue-specific levels that correlate with the levels of ADAR mRNA expression. The amount of IMP present in poly(A)+ RNA isolated from various mammalian tissues suggests adenosine deamination may play an important role in regulating gene expression, particularly in brain, where we estimate one IMP is present for every 17 000 ribonucleotides.

• Lei M, Liu Y, Samuel CE
• Adenovirus VAI RNA antagonizes the RNA-editing activity of the ADAR adenosine deaminase.
• Virology. 1998; 245: 188-96
• Display abstract

• The virus-associated VAI RNA of adenovirus is a small highly structured RNA that is required for the efficient translation of cellular and viral mRNAs at late times after infection. VAI RNA antagonizes the activation of the interferon-inducible RNA-dependent protein kinase, PKR, an important regulator of translation. The RNA-specific adenosine deaminase, ADAR, is an interferon-inducible RNA-editing enzyme that catalyzes the site-selective C-6 deamination of adenosine to inosine. ADAR possesses three copies of the highly conserved RNA-binding motif (dsRBM) that are similar to the two copies found in PKR, the enzyme in which the prototype dsRBM motif was discovered. We have examined the effect of VAI RNA on ADAR function. VAI RNA impairs the activity of ADAR deaminase. This inhibition can be observed in extracts prepared from interferon-treated human cells and from monkey COS cells in which wild-type recombinant ADAR was expressed. Analysis of wild-type and mutant forms of VA RNA suggests that the central domain is important in the antagonism of ADAR activity. These results suggest that VAI RNA may modulate viral and cellular gene expression by modulating RNA editing as well as mRNA translation.

• Chang BH, Chan L
• Evolutionary analysis of RNA editing enzymes.
• Methods. 1998; 15: 41-50
• Display abstract

• This article focuses on the evolution of apolipoprotein B (apoB) mRNA editing. We review the tools commonly used in homology search and phylogenetic analysis and demonstrate their use in the analysis of RNA-editing enzymes. The ultimate goal is to apply these tools to answer two questions: How did apoB mRNA editing come about? How might it be related to other base substitution editing in the course of evolution.

• Anant S, Yu H, Davidson NO
• Evolutionary origins of the mammalian apolipoproteinB RNA editing enzyme, apobec-1: structural homology inferred from analysis of a cloned chicken small intestinal cytidine deaminase.
• Biol Chem. 1998; 379: 1075-81
• Display abstract

• Mammalian apolipoproteinB (apoB) RNA editing is a site-specific deamination reaction that mediates the C to U conversion responsible for apoB48 production in the mammalian small intestine. This process is not detected in chicken apoB RNA. Mammalian apoB RNA editing is mediated by a multicomponent enzyme complex that includes a single catalytic subunit, apobec-1. In order to examine the evolution of apobec-1, we have cloned and characterized an orthologous cytidine deaminase cDNA isolated from chicken small intestine. Northern blot analysis revealed expression restricted to the small intestine, colon and lung but not the liver or other tissues. The cDNA encodes a single 31 kDa protein with features reminiscent of other cytidine deaminases and with approximately 39% overall homology to rat apobec-1. The recombinant protein is a cytidine deaminase with activity on a monomeric substrate that was found to be zinc-dependent. However, no RNA editing activity was detectable towards cytidine nucleotides presented in the context of an optimally configured mammalian apoB RNA template. These studies provide information concerning the evolution of the apoB RNA editing machinery and indicate that a chicken small intestinal cytidine deaminase with homology to apobec-1 demonstrates no activity on an RNA substrate.

• Kostyrko A, Trzeciak WH
• [Mechanisms of nuclear mRNA editing]
• Postepy Biochem. 1998; 44: 94-101

• Brooks R, Eckmann CR, Jantsch MF
• The double-stranded RNA-binding domains of Xenopus laevis ADAR1 exhibit different RNA-binding behaviors.
• FEBS Lett. 1998; 434: 121-6
• Display abstract

• We have cloned cDNAs encoding two versions of Xenopus double-stranded RNA adenosine deaminase (ADAR1). Like ADAR1 proteins from other species Xenopus ADAR1 contains three double-stranded RNA-binding domains (dsRBDs) which are most likely required for substrate binding and recognition of this RNA-editing enzyme. Analysis of mammalian ADAR1 identified the third dsRBD in this enzyme as most important for RNA binding. Here we analyzed the three dsRBDs of Xenopus ADAR1 for their in vitro RNA-binding behavior using two different assays. Northwestern assays identified the second dsRBD in the Xenopus protein as most important for RNA binding while in-solution assays demonstrated the importance of the third dsRBD for RNA binding. The differences between these two assays are discussed and we suggest that both the second and third dsRBD of Xenopus ADAR1 are important for RNA binding in vivo. We show further that all three dsRBDs can contribute to a cooperative binding effect.

• Berger I et al.
• Spectroscopic characterization of a DNA-binding domain, Z alpha, from the editing enzyme, dsRNA adenosine deaminase: evidence for left-handed Z-DNA in the Z alpha-DNA complex.
• Biochemistry. 1998; 37: 13313-21
• Display abstract

• Double-stranded RNA adenosine deaminase (ADAR1) is an ubiquitous enzyme in metazoa that edits pre-mRNA changing adenosine to inosine in regions of double-stranded RNA. Zalpha, an N-terminal domain of human ADAR1 encompassing 76 amino acid residues, shows apparent specificity for the left-handed Z-DNA conformation adopted by alternating (dGdC) polymers modified by bromination or methylation, as well as for (dGdC)13 inserts present in supercoiled plasmids. Here, a combination of circular dichroism, fluorescence, and gel-retardation studies is utilized to characterize recombinant Zalpha peptide and to examine its interaction with DNA. Results from laser-Raman spectroscopy experiments provide direct evidence for the existence of Z-DNA in peptide-DNA complexes.

• Maas S, Melcher T, Seeburg PH
• Mammalian RNA-dependent deaminases and edited mRNAs.
• Curr Opin Cell Biol. 1997; 9: 343-9
• Display abstract

• The past year has witnessed major progress in the field of mammalian nuclear RNA editing. Two new sequence-related RNA-dependent adenosine deaminases, distantly related to the previously characterized double-stranded RNA adenosine deaminase DRADA/dsRAD, have been molecularly characterized. One of these deaminases edits in vitro with precision for the molecular determinant that controls the Ca2+ permeability of fast synaptic glutamate-gated cation channels. This deaminase, like DRADA, is expressed in many tissues and the search is now on for more substrates of these RNA-editing enzymes. Moreover, the physiological role of the apolipoprotein B RNA editing enzyme APOBEC-1 has been investigated in genetically manipulated mice.

• Bass BL
• RNA editing and hypermutation by adenosine deamination.
• Trends Biochem Sci. 1997; 22: 157-62
• Display abstract

• Double-stranded RNA adenosine deaminase (dsRAD) was discovered ten years ago. In the intervening decade, research on dsRAD has progressed not only predictably, such as with the purification of the enzyme and identification of cDNAs, but also in some quite surprising ways. This review covers both areas of progress, but will concentrate on the surprises, which include the discovery that dsRAD is a member of a larger family of deaminases and the identification of RNAs that appear to be targets for these deaminases in vivo.

• Mittaz L, Scott HS, Rossier C, Seeburg PH, Higuchi M, Antonarakis SE
• Cloning of a human RNA editing deaminase (ADARB1) of glutamate receptors that maps to chromosome 21q22.3.
• Genomics. 1997; 41: 210-7
• Display abstract

• RED1 is a double-stranded RNA-specific editase characterized in the rat and is implicated in the editing of glutamate receptor subunit pre-mRNAs, particularly in the brain. Starting from human ESTs homologous to the rat RED1 sequence, we have characterized two forms of human RED1 cDNAs, one form coding for a putative peptide of 701 amino acids (similar to the shorter of two rat mRNAs) and a long form coding for a putative protein of 741 amino acids, the extra 120 bp of which are homologous to an AluJ sequence. Both forms were observed at approximately equal levels in cDNA clones and in seven different human tissues tested by RT-PCR. The human and rat short isoforms have 95 and 85% sequence identity at the amino acid and nucleotide levels, respectively. The human sequence (designated ADARB1 by the HGMW Nomenclature Committee) contains two double-stranded RNA-binding domains and a deaminase domain implicated in its editing action. Northern blot analysis detected two transcripts of 8.8 and 4.2 kb strongly expressed in brain and in many human adult and fetal tissues. ADARB1 maps to human chromosome 21q22.3, a region to which several genetic disorders map, including one form of bipolar affective disorder. Recently it was shown that heterozygous mice harboring an editing-incompetent glutamate receptor B allele have early onset fatal epilepsy. Since glutamate receptor channels are essential elements in synaptic function and plasticity and mediate pathology in many neurological disorders, and since RED1 is central in glutamate receptor channel control, ADARB1 is a candidate gene for diseases with neurological symptoms, such as bipolar affective disorder and epilepsy.

• Yang JH, Sklar P, Axel R, Maniatis T
• Purification and characterization of a human RNA adenosine deaminase for glutamate receptor B pre-mRNA editing.
• Proc Natl Acad Sci U S A. 1997; 94: 4354-9
• Display abstract

• The glutamate receptor subunit B (GluR-B) pre-mRNA is edited at two adenosine residues, resulting in amino acid changes that alter the electrophysiologic properties of the glutamate receptor. Previous studies showed that these amino acid changes are due to adenosine to inosine conversions in two codons resulting from adenosine deamination. Here, we describe the purification and characterization of an activity from human HeLa cells that efficiently and accurately edits GluR-B pre-mRNA at both of these sites. The purified activity contains a human homolog of the recently reported rat RED1 (rRED1) protein, a member of the family of double-stranded RNA-dependent deaminase proteins. Recombinant human RED1 (hRED1), but not recombinant dsRAD, another member of the family, efficiently edits both the Q/R and R/G sites of GluR-B RNA. We conclude that the GluR-B editing activity present in HeLa cell extracts and the recombinant hRED1 protein are indistinguishable.

• Gaur RK, McLaughlin LW, Green MR
• Functional group substitutions of the branchpoint adenosine in a nuclear pre-mRNA and a group II intron.
• RNA. 1997; 3: 861-9
• Display abstract

• Splicing of nuclear mRNA precursors (pre-mRNAs) takes place in the spliceosome, a large and complex ribonucleoprotein. Nuclear pre-mRNA splicing and group II intron self-splicing occur by a chemically identical pathway involving recognition of a specific branchpoint adenosine and nucleophilic activation of its 2'-hydroxyl group. The chemical similarity between these two splicing reactions, as well as other considerations, have suggested that the catalytic core of the spliceosome and group II introns may be related. Here we test this hypothesis by analyzing splicing and RNA branch formation of a pre-mRNA and a group II intron in which the branchpoint adenosine was substituted with purine base analogues. We find that replacement of the branchpoint adenosine with either of two modified adenosine analogues or guanosine leads to remarkably similar patterns of splicing and RNA branch formation in the two systems.

• Herbert A, Alfken J, Kim YG, Mian IS, Nishikura K, Rich A
• A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase.
• Proc Natl Acad Sci U S A. 1997; 94: 8421-6
• Display abstract

• Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zalpha) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5' to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zalpha has allowed identification of motifs common to this class of nucleic acid binding domain.

• Hough RF, Bass BL
• Analysis of Xenopus dsRNA adenosine deaminase cDNAs reveals similarities to DNA methyltransferases.
• RNA. 1997; 3: 356-70
• Display abstract

• We isolated two similar, but distinct, cDNA classes that encode Xenopus double-stranded RNA (dsRNA) adenosine deaminase. The longest, full-length open reading frame (ORF) predicts a 1,270-amino acid protein of 138,754 Da that is similar in size and about 50% identical to proteins encoded by mammalian cDNAs, yet larger than the 120-kDa protein purified from Xenopus eggs. Alignments of the Xenopus and mammalian ORFs show N-terminal heterogeneity, three conserved dsRNA binding motifs (dsRBMs), and strongly conserved carboxyl termini. Consistent with the observation of two cDNA classes, northern analyses of Xenopus oocyte poly A+ RNA show at least three mRNA species. Multiple nuclear polyadenylation hexamers and putative cytoplasmic polyadenylation elements were found in the 3' UTRs of cDNAs corresponding to the largest mRNA. In vitro translation experiments show that the cDNAs encode active deaminases and that the entire N-terminus and first dsRBM are dispensable for deaminase activity. Importantly, an analysis of the C-termini of five known dsRNA adenosine deaminases, and two putative deaminases, reveals motifs that are strikingly similar to the conserved motifs of the DNA-(adenine-N6alpha)-aminomethyltransferases and the DNA-(cytosine-5)-methyltransferases.

• Smith HC, Gott JM, Hanson MR
• A guide to RNA editing.
• RNA. 1997; 3: 1105-23

• Belcher SM, Howe JR
• Characterization of RNA editing of the glutamate-receptor subunits GluR5 and GluR6 in granule cells during cerebellar development.
• Brain Res Mol Brain Res. 1997; 52: 130-8
• Display abstract

• The non-NMDA class of ionotropic glutamate receptors are subject to RNA editing resulting in single amino acid changes within individual subunits that make up these oligomeric receptors. These amino acid changes result in significant alterations of important channel properties. Both edited and unedited versions of the kainate-receptor subunits GluR5 and GluR6 are present in brain, but whether this reflects the expression of both versions in individual types of neurons or differences in editing between different cell types is unclear. To characterize editing in a single identified type of central neuron, we have determined the extent to which GluR5 and GluR6 mRNAs are edited in acutely isolated cerebellar granule cells. RT-PCR analysis revealed that editing at each site in GluR5 and GluR6 increased during early postnatal development. The Q/R site was predominantly unedited in GluR5, whereas GluR6 was mostly edited. The Q/R and Y/C sites of GluR6 were edited to similar extents, whereas a smaller percentage of transcripts were edited at the I/V site. The expression of two double-stranded RNA adenosine deaminases implicated in GluR editing (DRADA and RED1) increased in granule cells between postnatal days 1 and 15. Finally, cerebellar granule cells express a previously unreported variant of RED1 which appears to arise from developmentally regulated alternative splicing.

• Lai F, Chen CX, Carter KC, Nishikura K
• Editing of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2 double-stranded RNA adenosine deaminases.
• Mol Cell Biol. 1997; 17: 2413-24
• Display abstract

• Double-stranded (ds) RNA-specific adenosine deaminase converts adenosine residues into inosines in dsRNA and edits transcripts of certain cellular and viral genes such as glutamate receptor (GluR) subunits and hepatitis delta antigen. The first member of this type of deaminase, DRADA1, has been recently cloned based on the amino acid sequence information derived from biochemically purified proteins. Our search for DRADA1-like genes through expressed sequence tag databases led to the cloning of the second member of this class of enzyme, DRADA2, which has a high degree of sequence homology to DRADA1 yet exhibits a distinctive RNA editing site selectivity. There are four differentially spliced isoforms of human DRADA2. These different isoforms of recombinant DRADA2 proteins, including one which is a human homolog of the recently reported rat RED1, were analyzed in vitro for their GluR B subunit (GluR-B) RNA editing site selectivity. As originally reported for rat RED1, the DRADA2a and -2b isoforms edit GluR-B RNA efficiently at the so-called Q/R site, whereas DRADA1 barely edits this site. In contrast, the R/G site of GluR-B RNA was edited efficiently by the DRADA2a and -2b isoforms as well as DRADA1. Isoforms DRADA2c and -2d, which have a distinctive truncated shorter C-terminal structure, displayed weak adenosine-to-inosine conversion activity but no editing activity tested at three known sites of GluR-B RNA. The possible role of these DRADA2c and -2d isoforms in the regulatory mechanism of RNA editing is discussed.

• Scott J
• RNA editing. Message change for a fat controller.
• Nature. 1997; 387: 242-3

• Morse DP, Bass BL
• Detection of inosine in messenger RNA by inosine-specific cleavage.
• Biochemistry. 1997; 36: 8429-34
• Display abstract

• Double-stranded RNA adenosine deaminases catalyze the conversion of adenosine to inosine within double-stranded RNA. A few candidate biological substrates for these enzymes have been discovered by noticing discrepancies between genomic and cDNA sequences. Toward the goal of finding a systematic approach to identify new deaminase substrates, we developed a method to cleave RNA specifically after inosine and an amplification strategy to identify the cleavage sites. We tested our method on a candidate substrate, the messenger RNA for glutamate receptor subunit B (GluR-B). We detected cleavage of the endogenous GluR-B message from rat brain at two known RNA editing sites, thus providing the first direct evidence for the presence of inosine at these sites. The described method will facilitate the mapping of inosines within RNA and, most importantly, will provide a way to identify new deaminase substrates.

• Scadden AD, Smith CW
• A ribonuclease specific for inosine-containing RNA: a potential role in antiviral defence?
• EMBO J. 1997; 16: 2140-9
• Display abstract

• RNA transcripts in which all guanosine residues are replaced by inosine are degraded at a highly accelerated rate when incubated in extracts from HeLa cells, sheep uterus or pig brain. We report here the partial purification and characterization of a novel ribonuclease, referred to as I-RNase, that is responsible for the degradation of inosine-containing RNA (I-RNA). I-RNase is Mg2+ dependent and specifically degrades single-stranded I-RNA. Comparison of the Km of the enzyme for I-RNA with the Ki for inhibition by normal RNA suggests a approximately 300-fold preferential binding to I-RNA, which can account for the specificity of degradation. The site of cleavage by I-RNase is non-specific; I-RNase acts as a 3'-->5' exonuclease generating 5'-NMPs as products. The presence of alternative unconventional nucleotides in RNA does not result in degradation unless inosine residues are also present. We show that I-RNase is able to degrade RNAs that previously have been modified by the RED-1 double-stranded RNA adenosine deaminase (dsRAD). dsRADs destabilize dsRNA by converting adenosine to inosine, and some of these enzymes are interferon inducible. We therefore speculate that I-RNase in concert with dsRAD may form part of a novel cellular antiviral defence mechanism that acts to degrade dsRNA.

• Mittaz L, Antonarakis SE, Higuchi M, Scott HS
• Localization of a novel human RNA-editing deaminase (hRED2 or ADARB2) to chromosome 10p15.
• Hum Genet. 1997; 100: 398-400
• Display abstract

• RNA-editing deaminase 2 (RED2; ADARB2) is a newly identified potential double-stranded RNA adenosine deaminase. It is the third member of this family, which includes DRADA and RED1. Genes of this family are candidates for involvement in neurological diseases such as epilepsy, because of their expression patterns and described functions. All three described genes are well expressed in brain, and DRADA and RED1 have been shown to play a role in the editing of mRNAs coding for glutamate receptor subunits in vitro, thereby changing the properties of these channels, which are the main excitatory neurotransmitter receptors in brain. Here we report the mapping of the human RED2 (hRED2; ADARB2) gene. Using the sequence of rat RED2, we identified a homologous human expressed sequence tag, and subsequently designed primers in the 3' untranslated region of the hRED2 transcript to perform polymerase chain reaction amplification on two somatic cell hybrid mapping panels. This allowed us to localize hRED2 on chromosome 10p15; until now, no genetic diseases have been mapped in this region or in the syntenic mouse chromosomal region that may involve RED2.

• Liu Y, George CX, Patterson JB, Samuel CE
• Functionally distinct double-stranded RNA-binding domains associated with alternative splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase.
• J Biol Chem. 1997; 272: 4419-28
• Display abstract

• The double-stranded RNA-specific adenosine deaminase (ADAR) is an interferon-inducible RNA-editing enzyme implicated in the site-selective deamination of adenosine to inosine in viral RNAs and cellular pre-mRNAs. We have isolated and characterized human genomic clones of the ADAR gene and cDNA clones encoding splice site variants of the ADAR protein. Southern blot and sequence analyses revealed that the gene spans about 30 kilobase pairs and consists of 15 exons. The codon phasing of the splice site junctions of exons 3, 5, and 7 that encode the three copies of the highly conserved RNA-binding R-motif (RI, RII, and RIII) was exactly conserved and identical to those R-motif exons of the interferon-inducible RNA-dependent protein kinase. Alternative splice site variants of the 1226-amino acid ADAR-a protein, designated b and c, were identified that differed in exons 6 and 7. ADAR-b was a 5'-splice site variant that possessed a 26-amino acid deletion within exon 7; ADAR-c was a 3'-splice site variant that possessed an additional 19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c proteins all possessed comparable double-stranded RNA-specific adenosine deaminase activity. However, mutational analysis of the R-motifs revealed that the exon 6 and 7 deletions of ADAR-b and -c variants altered the functional importance of each of the three R-motifs.

• Melcher T, Maas S, Herb A, Sprengel R, Seeburg PH, Higuchi M
• A mammalian RNA editing enzyme.
• Nature. 1996; 379: 460-4
• Display abstract

• Editing of RNA by site-selective adenosine deamination alters codons in brain-expressed pre-messenger RNAs for glutamate receptor (GluR) subunits including a codon for a channel determinant (Q/R site) in GluR-B, which controls the Ca2+ permeability of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Editing of GluR pre-mRNAs requires a double-stranded RNA (dsRNA) structure formed by exonic and intronic sequences and is catalysed by an unknown dsRNA adenosine deaminase. Here we report the cloning of complementary DNA for RED1, a dsRNA adenosine deaminase expressed in brain and peripheral tissues that efficiently edits the Q/R site in GluR-B pre-mRNA in vitro. This site is poorly edited by DRADA, which is distantly sequence-related to RED1. Both deaminases edit the R/G site in GluR-B pre-mRNA, indicating that members of an emerging gene family catalyse adenosine deamination in nuclear transcripts with distinct but overlapping substrate specificities.

• Grosjean H et al.
• Enzymatic conversion of adenosine to inosine and to N1-methylinosine in transfer RNAs: a review.
• Biochimie. 1996; 78: 488-501
• Display abstract

• Inosine (6-deaminated adenosine) is a characteristic modified nucleoside that is found at the first anticodon position (position 34) of several tRNAs of eukaryotic and eubacterial origins, while N1-methylinosine is found exclusively at position 37 (3' adjacent to the anticodon) of eukaryotic tRNA(Ala) and at position 57 (in the middle of the psi loop) of several tRNAs from halophilic and thermophilic archaebacteria. Inosine has also been recently found in double-stranded RNA, mRNA and viral RNAs. As for all other modified nucleosides in RNAs, formation of inosine and inosine derivative in these RNA is catalysed by specific enzymes acting after transcription of the RNA genes. Using recombinant tRNAs and T7-runoff transcripts of several tRNA genes as substrates, we have studied the mechanism and specificity of tRNA-inosine-forming enzymes. The results show that inosine-34 and inosine-37 in tRNAs are both synthesised by a hydrolytic deamination-type reaction, catalysed by distinct tRNA:adenosine deaminases. Recognition of tRNA substrates by the deaminases does not strictly depend on a particular "identity' nucleotide. However, the efficiency of adenosine to inosine conversion depends on the nucleotides composition of the anticodon loop and the proximal stem as well as on 3D-architecture of the tRNA. In eukaryotic tRNA(Ala), N1-methylinosine-37 is formed from inosine-37 by a specific SAM-dependent methylase, while in the case of N1-methylinosine-57 in archaeal tRNAs, methylation of adenosine-57 into N1-methyladenosine-57 occurs before the deamination process. The T psi-branch of fragmented tRNA is the minimalist substrate for the N1-methylinosine-57 forming enzymes. Inosine-34 and N1-methylinosine-37 in human tRNA(Ala) are targets for specific autoantibodies which are present in the serum of patients with inflammatory muscle disease of the PL-12 polymyositis type. Here we discuss the mechanism, specificity and general properties of the recently discovered RNA:adenosine deaminases/editases acting on double-stranded RNA, intron-containing mRNA and viral RNA in relation to those of the deaminases acting on tRNAs.

• Herbert A
• RNA editing, introns and evolution.
• Trends Genet. 1996; 12: 6-9

• Herb A, Higuchi M, Sprengel R, Seeburg PH
• Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences.
• Proc Natl Acad Sci U S A. 1996; 93: 1875-80
• Display abstract

• RNA editing by adenosine deamination in brain-expressed pre-mRNAs for glutamate receptor (GluR) subunits alters gene-specified codons for functionally critical positions, such as the channel's Q/R site. We show by transcript analysis of minigenes transiently expressed in PC-12 cells that, in contrast to GluR-B pre-mRNA, where the two editing sites (Q/R and R/G) require base pairing with nearby intronic editing site complementary sequences (ECSs), editing in GluR5 and GluR6 pre-mRNAs recruits an ECS located as far as 1900 nucleotides distal to the Q/R site. The exon-intron duplex structure of the GluR5 and GluR6 pre-mRNAs appears to be a substrate of double-stranded RNA-specific adenosine deaminase. This enzyme when coexpressed in HEK 293 cells preferentially targets the adenosine of the Q/R site and of an unpaired position in the ECS which is highly edited in brain.

• Dabiri GA, Lai F, Drakas RA, Nishikura K
• Editing of the GLuR-B ion channel RNA in vitro by recombinant double-stranded RNA adenosine deaminase.
• EMBO J. 1996; 15: 34-45
• Display abstract

• Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme responsible for the editing of RNA transcripts encoding glutamate-gated ion channel subunits (GLuR) in brain. In one case, the editing alters the gene-encoded glutamine (Q) to an arginine (R) located within the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunit GLuR-B. The result of editing at this site, called the 'Q/R' site, is a profound alteration of the Ca2+ permeability of the GLuR channel. Using recombinantly expressed DRADA proteins, we now demonstrate in vitro that DRADA is indeed involved in editing of the GLuR-B RNA. In addition to the formation of an RNA duplex structure involving exon and intron sequences, Q/R site-selective editing by DRADA also requires a cofactor protein(s) commonly present even in non-neuronal cells. The accuracy and efficiency of this RNA editing system appear to be determined by the quantitative balance between DRADA, cofactor and substrate GLuR-B RNA.

• Liu Y, Samuel CE
• Mechanism of interferon action: functionally distinct RNA-binding and catalytic domains in the interferon-inducible, double-stranded RNA-specific adenosine deaminase.
• J Virol. 1996; 70: 1961-8
• Display abstract

• The 1,226-amino-acid sequence of the interferon-inducible double-stranded RNA-specific adenosine deaminase (dsRAD) contains three copies (RI, RII, and RIII) of the highly conserved subdomain R motif commonly found in double-stranded RNA-binding proteins. We have examined the effects of equivalent site-directed mutations in each of the three R-motif copies of dsRAD on RNA-binding activity and adenosine deaminase enzyme activity. Mutations of the R motifs were analyzed alone as single mutants and in combination with each other. The results suggest that the RIII copy is the most important of the three R motifs for dsRAD activity and that the RII copy is the least important. The RIII mutant lacked detectable enzymatic activity and displayed greatly diminished RNA-binding activity. Site-directed mutations within the highly conserved CHAE sequence of the postulated C-terminal deaminase catalytic domain destroyed enzymatic activity but did not affect RNA-binding activity. These results indicate that the three copies of the RNA-binding R subdomain are likely functionally distinct from each other and also from the catalytic domain of dsRAD.

• Kelly MA, Vestling MM, Murphy CM, Hua S, Sumpter T, Fenselau C
• Primary structure of bovine adenosine deaminase.
• J Pharm Biomed Anal. 1996; 14: 1513-9
• Display abstract

• Derivatized bovine adenosine deaminase is used in enzyme replacement therapy and as an adjunct to gene therapy against severe combined immunodeficiency syndrome. Although a gene sequence is known for human adenosine deaminase, the structure of the bovine enzyme has not been characterized. Structure studies using mass spectrometry are reported here that evaluate sequence, processing, post-translational modifications and the extent of homology between the human protein and its therapeutic surrogate.

• Benne R
• RNA editing: how a message is changed.
• Curr Opin Genet Dev. 1996; 6: 221-31
• Display abstract

• Considerable progress has been made in unraveling the mechanistic features of RNA editing processes in a number of genetic systems. Recent highlights include the identification of the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme as a zinc-dependent cytidine deaminase that binds to RNA, the demonstration that adenosines in brain glutamate receptor pre-mRNAs are converted into inosines and that double-stranded RNA A deaminase (dsRAD), the candidate enzyme, is another zinc-dependent RNA nucleotide deaminase, and a mounting body of evidence for a cleavage-ligation mechanism for U insertion/deletion editing in kinetoplastid protozoa.

• Melcher T, Maas S, Herb A, Sprengel R, Higuchi M, Seeburg PH
• RED2, a brain-specific member of the RNA-specific adenosine deaminase family.
• J Biol Chem. 1996; 271: 31795-8
• Display abstract

• The mammalian RNA-specific adenosine deaminases DRADA/dsRAD (alias ADAR) and RED1 (alias ADARB1) have been implicated in the site-selective editing of brain-expressed pre-mRNAs for glutamate receptor subunits and of antigenomic RNA of hepatitis delta virus. These enzymes are expressed in many if not all tissues, predicting an as yet unappreciated significance for adenosine deamination-mediated recoding of gene transcripts in the mammalian organism. We now report the molecular cloning of cDNA for RED2 (alias ADARB2), a third member of the RNA-specific adenosine deaminase family in the rodent. RED2 is closely sequence-related to RED1 but appears to be expressed only in the brain, where expression is widespread reaching highest levels in olfactory bulb and thalamus. RED2 further differs from RED1 in having a 54-residue amino-terminal extension which includes an arginine-rich motif. Different from DRADA and RED1, recombinantly expressed RED2 did not deaminate adenosines in extended synthetic dsRNA or in GluR-B pre-mRNA. However, a chimera of RED1 and RED2 edited the GluR-B Q/R and R/G sites with moderate efficiency. Our data suggest that RED2 may edit brain-specific transcripts with distinct structural features.

• Yang JH, Sklar P, Axel R, Maniatis T
• Editing of glutamate receptor subunit B pre-mRNA in vitro by site-specific deamination of adenosine.
• Nature. 1995; 374: 77-81
• Display abstract

• Editing of the glutamate receptor subunit B (GluR-B) pre-mRNA at a single adenosine residue results in an amino-acid change that profoundly alters the electrophysiological properties of the receptor. Here we show that the GluR-B pre-mRNA is efficiently and accurately edited in vitro, and that base-pair interactions between the editing site and a sequence in the downstream intron are required for substrate recognition. In addition, we directly demonstrate that editing results from the conversion of adenosine to inosine by enzymatic deamination. The biochemical properties of this GluR-B editing activity are similar to those of a double-stranded-RNA-dependent adenosine deaminase, but RNA competition and column fractionation experiments indicate that the GluR-B editing and deaminase activities are distinct. Thus, the GluR-B editing enzyme may contain the adenosine deaminase, or a similar activity, and an RNA recognition subunit that specifically targets the enzyme to the editing site.

• Rueter SM, Burns CM, Coode SA, Mookherjee P, Emeson RB
• Glutamate receptor RNA editing in vitro by enzymatic conversion of adenosine to inosine.
• Science. 1995; 267: 1491-4
• Display abstract

• RNA encoding the B subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptor (GluR-B) undergoes a posttranscriptional modification in which a genomically encoded adenosine is represented as a guanosine in the GluR-B complementary DNA. In vitro editing of GluR-B RNA transcripts with HeLa cell nuclear extracts was found to result from an activity that converts adenosine to inosine in regions of double-stranded RNA by enzymatic base modification. This activity is consistent with that of a double-stranded RNA-specific adenosine deaminase previously described in Xenopus oocytes and widely distributed in mammalian tissues.

• Wang Y, Zeng Y, Murray JM, Nishikura K
• Genomic organization and chromosomal location of the human dsRNA adenosine deaminase gene: the enzyme for glutamate-activated ion channel RNA editing.
• J Mol Biol. 1995; 254: 184-95
• Display abstract

• The structure of the human gene encoding the double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was characterized. This nuclear localized enzyme is involved in the RNA editing required for the expression of certain subtypes of glutamate-gated ion channel subunits. The DRADA gene span 30 kb pairs and harbors 15 exons. The transcription of the DRADA gene driven by the putative promoter region, which contains no typical TATA or CCAAT box-like sequences, is initiated at multiple sites, 164 to 216 nucleotides upstream of the translation initiation codon. The three dsRNA binding motifs (DRBM), 70 amino acid residues long, are each encoded by two exons plus an intervening sequence that interrupts the motif at the identical amino acid position. This finding is consistent with the notion that the dsRNA binding domains may be composed of two separate functional subdomains. Fluorescent in situ hybridization localized the DRADA gene on the long arm chromosome 1, region q21. The gene structure and sequence information reported in this study will facilitate the investigation of involvement of DRADA in hereditary diseases that may be the result of malfunction of glutamate-gated ion channels.

• Patterson JB, Samuel CE
• Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase.
• Mol Cell Biol. 1995; 15: 5376-88
• Display abstract

• A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of approximately 6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.

• Horikami SM, Moyer SA
• Double-stranded RNA adenosine deaminase activity during measles virus infection.
• Virus Res. 1995; 36: 87-96
• Display abstract

• It has been postulated that the cellular double-stranded (ds) RNA adenosine deaminase enzyme is responsible for biased hypermutation during persistent SSPE measles infections in humans. As a test of this hypothesis we studied the effect of negative-strand RNA virus infection on enzyme activity. The adenosine deaminase activity was found in nuclear extracts of both uninfected CV-1 and A549 cells and in cytoplasmic extracts of A549, but not CV-1, cells. During measles or Sendai virus infection of either CV-1 or A549 cells the adenosine deaminase activity in the nucleus remained fairly constant up to 24 h post infection, and there was no apparent re-partitioning of the enzyme between the nucleus and the cytoplasm. Transcription complexes of Sendai virus in vitro or measles virus in vivo did not serve as substrates for the enzyme. These data suggest that even though some portion of the adenosine deaminase enzyme may be present in the cytoplasm of at least some cells during virus infection, modification of the viral RNAs by this enzyme, if it occurs at all, must be at a very low level not directly detectable by biochemical analysis.

• Hajjar AM, Linial ML
• Modification of retroviral RNA by double-stranded RNA adenosine deaminase.
• J Virol. 1995; 69: 5878-82
• Display abstract

• In this report, we describe a recombinant provirus generated during in vitro passage that contains a short region of adenosine-to-guanosine hypermutation. The hypermutated region is restricted to complementary sequences present in the recombinant provirus. We propose that a duplex was formed in the recombinant RNA prior to reverse transcription. This duplex was a substrate for double-stranded RNA adenosine deaminase, an activity found in all cells examined that deaminates A in double-stranded RNA, converting it to inosine, which is further converted to a guanosine by reverse transcription. It appears that cis viral sequences facilitated the A-->G transitions.

• Hurst SR, Hough RF, Aruscavage PJ, Bass BL
• Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.
• RNA. 1995; 1: 1051-60
• Display abstract

• Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.

• Bass BL
• RNA editing. An I for editing.
• Curr Biol. 1995; 5: 598-600
• Display abstract

• In vitro editing in mammalian nuclear extracts reveals adenosine-to-inosine conversions in glutamate receptor messenger RNAs.

• Scott J
• A place in the world for RNA editing.
• Cell. 1995; 81: 833-6

• Lai F, Drakas R, Nishikura K
• Mutagenic analysis of double-stranded RNA adenosine deaminase, a candidate enzyme for RNA editing of glutamate-gated ion channel transcripts.
• J Biol Chem. 1995; 270: 17098-105
• Display abstract

• Mutagenic analysis of the substrate binding and catalytic domains of double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was carried out. This nuclear enzyme is likely to be involved in the RNA editing of glutamate-gated ion channels that are essential for fast excitatory neurotransmission in mammalian brain. The deletion of the first or the third of the three dsRNA binding motifs within the substrate binding domain dramatically decreases enzyme activity, whereas the second motif seems to be dispensable. The results indicate that the three motifs are not functionally equivalent in the catalytic action of DRADA. Mutation of the putative zinc-coordinating residues, His910, Cys966, and Cys1036, abolished the DRADA activity. Similarly, the Glu912 residue, predicted to be involved in the proton transfer functions of the enzyme, was found to be indispensable. Our results reinforce the previous proposal that the hydrolytic deamination mechanism of DRADA may be more similar to that of the cytidine deaminases than of adenosine deaminases.

• Melcher T, Maas S, Higuchi M, Keller W, Seeburg PH
• Editing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA in vitro reveals site-selective adenosine to inosine conversion.
• J Biol Chem. 1995; 270: 8566-70
• Display abstract

• In neurons of the mammalian brain primary transcripts of genes encoding subunits of glutamate receptor channels can undergo RNA editing, leading to altered properties of the transmitter-activated channel. Editing of these transcripts is a nuclear process that targets specific adenosines and requires a double-stranded RNA structure configured from complementary exonic and intronic sequences. We show here that the two independent editing sites in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA are edited with positional accuracy by nuclear extract from HeLa cells. Nucleotide analysis by thin layer chromatography of the edited RNA sequences revealed selective adenosine to inosine conversion, most likely reflecting the participation of double-stranded RNA adenosine deaminase. Our results predict the presence of inosine-containing codons in other mammalian mRNAs.

• Woolf TM, Chase JM, Stinchcomb DT
• Toward the therapeutic editing of mutated RNA sequences.
• Proc Natl Acad Sci U S A. 1995; 92: 8298-302
• Display abstract

• 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.

• Herbert A, Lowenhaupt K, Spitzner J, Rich A
• Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.
• Proc Natl Acad Sci U S A. 1995; 92: 7550-4
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• A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.

• Chen Z, Hirano A, Wong TC
• Isolation and characterization of intranuclear ribonucleoprotein complexes associated with double-stranded RNA adenosine deaminase from brain cells: implications for RNA-editing and hypermutation of viral RNA in the CNS.
• J Neurovirol. 1995; 1: 295-306
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• Double-stranded RNA adenosine deaminase (DsRAD), which converts adenosine in duplex RNA to inosine, has been implicated in editing of cellular mRNA and hypermutation of viral RNA in the central nervous system (CNS). We used subcellular fractionation to show that DsRAD in bovine brain tissues is associated with high-molecular-weight ribonucleoprotein (RNP) complexes in the nuclei. DsRAD-associated RNP complexes have apparent molecular mass of up to 500 kDa and buoyant density of 1.35 to 1.42 g cc-1 in CsCl solution. In human glioma cells, DsRAD is also found exclusively in intranuclear RNP complexes that co-sediment with the largest RNA species. These DsRAD-associated RNP complexes are dissociated by RNase A or high salt. The RNA component is not essential for DsRAD activity, and the protein component can be separated by dsRNA-affinity column, gel filtration column, and glycerol gradient into enzymatically active protein species with apparent molecular mass ranging from 120 kDa to 70 kDa in polyacrylamide gel. The bovine brain DsRAD has no apparent requirement for low-molecular-weight cofactors or metal ions. These results provide insight into the native state of DsRAD in brain cells and have interesting implications for its putative roles in RNA-editing and hypermutation of viral RNA in the CNS.

• Kim U, Garner TL, Sanford T, Speicher D, Murray JM, Nishikura K
• Purification and characterization of double-stranded RNA adenosine deaminase from bovine nuclear extracts.
• J Biol Chem. 1994; 269: 13480-9
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• The double-stranded RNA (dsRNA) adenosine deaminase (DRADA) deaminates adenosine residues to inosines and creates I-U mismatched base pairs in dsRNAs. Its involvement in RNA editing of glutamate-gated ion channel gene transcripts in mammalian brains has been proposed as one of the biological functions for this recently identified cellular enzyme. We purified a mixture of three forms, 93, 88, and 83 kDa, of bovine DRADA proteins, all likely to be active enzymes. We determined that DRADA has a native molecular mass of approximately 100 kDa, suggesting that the enzyme exists as a monomer. The purified enzyme was not inhibited by 2'-deoxycoformycin, a transition state analog inhibitor of adenosine deaminase and AMP deaminase, suggesting that the catalytic mechanism of DRADA might be different from that of other deaminases. DRADA binds specifically to dsRNA with a dissociation constant of 0.23 nM for a synthetic dsRNA, and the Michaelis constant is 0.85 nM. These values indicate that DRADA has a much higher affinity for its substrate than other deaminases such as adenosine deaminase and AMP deaminase. DRADA may need this extremely high affinity to catalyze efficiently the modification of relatively rare substrate RNAs in the cell nucleus.

• Polson AG, Bass BL
• Preferential selection of adenosines for modification by double-stranded RNA adenosine deaminase.
• EMBO J. 1994; 13: 5701-11
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• Double-stranded RNA adenosine deaminase (dsRAD), previously called the double-stranded RNA (dsRNA) unwinding/modifying activity, modifies adenosines to inosines within dsRNA. We used ribonuclease U2 and a mutant of ribonuclease T1 to map the sites of modification in several RNA duplexes. We found that dsRAD had a 5' neighbor preference (A = U > C > G) but no apparent 3' neighbor preference. Further, the proximity of the strand termini affected whether an adenosine was modified. Most importantly, dsRAD exhibited selectivity, modifying a minimal number of adenosines in short dsRNAs. Our results suggest that the specific editing of glutamate receptor subunit B mRNA could be performed in vivo by dsRAD without the aid of specificity factors, and support the hypothesis that dsRAD is responsible for hypermutations in certain RNA viruses.

• Atasoy U, Norby-Slycord CJ, Markert ML
• A missense mutation in exon 4 of the human adenosine deaminase gene causes severe combined immunodeficiency.
• Hum Mol Genet. 1993; 2: 1307-8

• Higuchi M, Single FN, Kohler M, Sommer B, Sprengel R, Seeburg PH
• RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency.
• Cell. 1993; 75: 1361-70
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• A functionally critical position (Q/R site) of the AMPA receptor subunit GluR-B is controlled by RNA editing that operates in the nucleus, since in brain and clonal cell lines of neural origin, unspliced GluR-B transcripts occur edited in the Q/R site CAG codon and, additionally, in intronic adenosines. Transfection of GluR-B gene constructs into PC12 cells revealed that the proximal part of the intron downstream of the unedited exonic site is required for Q/R site editing. This intron portion contains an imperfect inverted repeat preceding a 10 nt sequence with exact complementarity to the exon centered on the unedited codon. Single nucleotide substitutions in this short intronic sequence or its exonic complement curtailed Q/R site editing, which was recovered by restoring complementarity in the respective partner strand. Base conversion in the channel-coding region of GluR-B directed by base paired sequences may be executed by a ubiquitous nuclear adenosine deaminase specific for double-stranded RNA.

• Kim U, Nishikura K
• Double-stranded RNA adenosine deaminase as a potential mammalian RNA editing factor.
• Semin Cell Biol. 1993; 4: 285-93
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• Double-stranded RNA (dsRNA) adenosine deaminase, or DRADA, is a cellular enzyme that modifies adenosine residues to inosines in dsRNA by hydrolytic deamination, replacing A-U with mismatched I-U base pairs. Since it alters the base composition in its substrate RNA, one possible role played by DRADA is to participate in RNA editing. In this article, a brief review is given of characteristics of DRADA. Its possible involvement in RNA editing is also discussed in detail, including specific cases in which DRADA has been implicated as an RNA editing factor.

• Adrian GS, Wiginton DA, Hutton JJ
• Structure of adenosine deaminase mRNAs from normal and adenosine deaminase-deficient human cell lines.
• Mol Cell Biol. 1984; 4: 1712-7
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• The structure of human adenosine deaminase mRNA from normal and mutant lymphoblasts was examined by sequence analysis of a cDNA for normal mRNA and electrophoretic analyses of DNA fragments generated by S1 endonuclease cleavage of mRNA-cDNA hybrids. The 1,533-base sequence of the cloned cDNA represents the complete mRNA sequence with the possible exception of some of the 5' untranslated region. S1 nuclease analyses of hybrids between cloned cDNA and normal adenosine deaminase mRNA confirmed that a 76-base sequence in a previously examined adenosine deaminase cDNA is an intron. S1 nuclease analyses of mRNAs from seven mutant cell lines demonstrated that four of the mutants, those in the GM-2471, GM-2756, GM-4258, and GM-2606 cells, contain small defects, such as single-base changes, that are not detectable by the S1 nuclease technique. Three of the mRNAs, those in GM-3043, GM-2294, and GM-2825A cells, do contain defects detectable with S1 nuclease. These defects differ from each other and have been mapped to specific regions of the mRNA. Some or all of these defective mRNAs are postulated to result from anomalous RNA processing.

• Orkin SH et al.
• Molecular cloning of human adenosine deaminase gene sequences.
• J Biol Chem. 1983; 258: 12753-6
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• Using a mixture of synthetic 17-mer oligonucleotides encoding the 64 possible sequences for a peptide of adenosine deaminase as probe, we have isolated a clone for adenosine deaminase mRNA sequences from a collection of T-cell cDNA recombinants. This cDNA clone, phADA-1, contains an insert of 0.8 kilobase. In addition to the peptide chosen for synthesis of the oligonucleotide probe, the complete DNA sequence predicts 16 other experimentally determined peptides. Mapping of total cellular human DNAs with several restriction enzymes revealed relatively simple patterns of hybridization with phADA-1 as probe, including a polymorphism for PvuII cleavage. In agreement with previous studies, the adenosine deaminase gene was localized by blot hybridization to chromosome 20 in a hybrid cell mapping panel. Using the cDNA as probe, an 18-kilobase EcoRI fragment of human cellular DNA was also cloned in bacteriophage Charon 4A. These adenosine deaminase clones will prove valuable in the full characterization of the cellular gene, molecular analysis of inherited enzyme deficiency associated with immunodeficiency, and regional mapping of human chromosome 20.

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