Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned from Saccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. A gene homologous to scADAT1 has been identified in the region of Drosophila Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastoris and purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system (Keegan, 1999).
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 reveals 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 curtails 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 (Higuchi, 1993).
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. Transcript analysis of minigenes transiently expressed in PC-12 cells shows 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 (Herb, 1996).
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 promiscuously deaminate perfectly base-paired dsRNA, 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, the role of internal loops within ADAR substrates has been investigated. 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 (greater than or equal to six nucleotides) act as helix ends, whereas smaller loops (less than or equal to four nucleotides) do not. This work provides the first information about how secondary structure within ADAR substrates dictates selectivity, and suggests a rational approach for delineating minimal substrates for RNAs deaminated by ADARs in vivo (Lehmann, 1999).
The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA editing enzyme implicated in the site-selective deamination of adenosine to inosine in cellular pre-mRNAs. The pre-mRNA for the rat serotonin-2C receptor (5-HT2CR) possesses four editing sites (A, B, C, and D), which undergo A-to-I nucleotide conversions that alter the signaling function of the encoded G-protein-coupled receptor. Measurements of 5-HT2CR pre-mRNA editing in vitro revealed site-specific deamination catalyzed by ADAR1. Three splice site variants, ADAR1-a, -b, and -c, all efficiently edit the A site of 5-HT2CR pre-mRNA, but the D site does not serve as an efficient substrate for any of the ADAR1 variants. Mutational analysis of the three double-stranded (ds) RNA binding motifs present in ADAR1 reveal a different relative importance of the individual dsRNA binding motifs for deamination of the A site of 5-HT2CR and synthetic dsRNA substrates. Quantitative reverse transcription-polymerase chain reaction analyses demonstrate that the 5-HT2CR pre-mRNA is most highly expressed in the choroid plexus of rat brain. However, ADAR1 and the related deaminase ADAR2 shows significant expression in all regions of the brain examined, including cortex, hippocampus, olfactory bulb, and striatum, where the 5-HT2CR pre-mRNA is extensively edited (Liu, 1999a).
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. 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 has been examined. Measurement of GluR-B pre-mRNA editing in vitro reveals different site-specific deamination catalyzed by the three ADAR1 variants. The ADAR1-a, -b, and -c splice variants all efficiently edit the R/G site and the intron +60 hotspot but exhibit little editing activity at the Q/R site. ADAR1-b and -c show higher editing activity than ADAR1-a for the R/G site, whereas the intron +60 site is edited with comparable efficiency by all three ADAR1 splice variants. Mutational analysis reveals that the functional importance of each of the three RNA-binding motifs of ADAR1 varies 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 show 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 vary among the choroid plexus, cortex, hippocampus, olfactory bulb, and striatum, but in all regions of rat brain the editing of the flip isoform is greater than that of the flop isoform (Liu, 1999b).
The ADAR family of RNA-editing enzymes deaminates adenosines within RNA that are 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. Attempts have been made to determine if ADAR deamination specificity occurs because the enzymes bind to targeted regions with higher affinities. To explore this question, an examination was made of the binding of rat ADAR2 to a region of rat gluR-B pre-mRNA that contains the R/G editing site, and a wild-type molecule was compared with one containing mutations that decreased R/G site editing. Although binding affinity to the two sequences is 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 appears to be nonspecific (Ohman, 2000).
Structural analogs of a natural RNA editing substrate have been synthesized and editing reactions of these substrates by recombinant ADAR-2, an RNA-editing adenosine deaminase, were compared. 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 causes a large decrease in deamination rate, whereas 2[prime]-deoxyadenosine and 2[prime]-deoxy-2[prime]-fluoroadenosine are 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 (Yi-Brunozzi, 1999).
RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. The role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 was examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine that was be modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5' to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11-15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited (Herbert, 2001).
ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double-stranded. Site-selective A-->I editing has been detected at specific sites within a few structured pre-mRNAs of metazoans.The editing selectivity of ADAR enzymes was examined, and the naturally edited R/G site in the pre-mRNA of the glutamate receptor subunit B (GluR-B) was chosen. A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. These results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double-stranded stem. To further understand the mechanism of selective ADAR2 editing, the importance of internal loops in the RNA substrate was investigated. The immediate structure surrounding the editing site was found to be important. A purine opposite the editing site has a negative effect on both the selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affect editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events (Kallman, 2003).
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, phylogenetic analysis of sequences required for editing at the R/G site was performed. Highly conserved sequences were found that are 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 is the bases in paired, helical regions whose identity is conserved, whereas bases in nonhelical regions vary, but maintain their nonhelical state. It is speculated that this pattern in part reflects constraints imposed by ADAR's unique specificity and support for these hypotheses was obtained with mutagenesis studies. Unexpectedly, some of the gluR introns are conserved beyond the sequences required for editing. The approximately 600-nt intron 13 of gluR-C is particularly remarkable, showing greater than 94% nucleotide identity between human and chicken, organisms estimated to have diverged 310 million years ago (Aruscavage, 2000).
The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNA-binding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm (Sansam, 2003).
The arginine residue at position 586 of the GluR-B subunit renders heteromeric AMPA-sensitive glutamate receptor channels impermeable to calcium. The codon for this arginine is introduced at the precursor messenger RNA (pre-mRNA) stage by site-selective adenosine editing of a glutamine codon. Heterozygous mice engineered by gene targeting to harbor an editing-incompetent GluR-B allele, synthesize unedited GluR-B subunits and, in principal neurons and interneurons, express AMPA receptors with increased calcium permeability. These mice develop seizures and die by 3 weeks of age, showing that GluR-B pre-mRNA editing is essential for brain function (Brusa, 1995).
Mouse mutants were generated with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines have increased AMPAR calcium permeabilities in pyramidal neurons, and one of these shows elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induces NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants have mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlates with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons (Feldmeyer, 1999).
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 an understanding of the biological roles of ADARs, a method was developed for systematically identifying ADAR substrates. In the first application of the method, five additional substrates in Caenorhabditis elegans were identified. 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 (Morse, 1999).
RNA transcripts encoding the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor (5-HT2CR) undergo adenosine-to-inosine RNA editing events at up to five specific sites. Compared with rat brain, human brain samples express higher levels of RNA transcripts encoding the amino acids valine-serine-valine (5-HT2C-VSV) and valine-glycine-valine (5-HT2C-VGV) at positions 156, 158, and 160, respectively. Agonist stimulation of the nonedited human receptor (5-HT2C-INI) and the edited 5-HT2C-VSV and 5-HT2C-VGV receptor variants stably expressed in NIH-3T3 fibroblasts demonstrate that serotonergic agonists are less potent at the edited receptors. Competition binding experiments reveal a guanine nucleotide-sensitive serotonin high affinity state only for the 5-HT2C-INI receptor; the loss of high affinity agonist binding to the edited receptor demonstrates that RNA editing generates unique 5-HT2CRs that couple less efficiently to G proteins. This reduced G protein coupling for the edited isoforms is primarily due to silencing of the constitutive activity of the nonedited 5-HT2CR. The distinctions in agonist potency and constitutive activity suggest that different edited 5-HT2CRs exhibit distinct responses to serotonergic ligands and further imply that RNA editing represents a novel mechanism for controlling physiological signaling at serotonergic synapses (Niswender, 1999).
RNA editing by site-selective deamination of adenosine to inosine alters codons and splicing in nuclear transcripts, and therefore protein function. ADAR2 is a candidate mammalian editing enzyme that is widely expressed in brain and other tissues, but its RNA substrates are unknown. ADAR2-mediated RNA editing has been studied by generating mice that are homozygous for a targeted functional null allele. Editing in ADAR2-/- mice is substantially reduced at most of 25 positions in diverse transcripts; the mutant mice become prone to seizures and die young. The impaired phenotype appears to result entirely from a single underedited position, as it reverts to normal when both alleles for the underedited transcript are substituted exonically, with alleles encoding the edited version. The critical position specifies an ion channel determinant, the Q/R site, in AMPAreceptor GluR-B pre-messenger RNA. It is concluded that this transcript is the physiologically most important substrate of ADAR2 (Higuchi, 2000).
Mammalian transcripts that are known to be edited by site-selective adenosine deamination are expressed largely in brain: most encode subunits of ionotropic glutamate receptors (GluRs) that mediate fast excitatory neurotransmission. The only position edited to nearly 100% is the Q/R site of GluR-B, for which the mRNA contains an arginine (R) codon (CIG) in place of the genomic glutamine (Q) codon (CAG). The physiological importance of this codon substitution wrought by RNA editing has been revealed by early onset epilepsy and premature death of mice heterozygous for an intron-11-modified GluR-BECS allele with Q/R site-uneditable transcripts (Higuchi, 2000).
Heterozygous ADAR2+/- mice are phenotypically normal, but ADAR2-/- mice die between P0 and P20 and become progressively seizure-prone after P12, akin to GluR-B+/delta ECS mice. Therefore, this investigation focussed on the effect of ADAR2 deficiency on Q/R site editing of GluR-B pre-mRNA, the substrate for a nuclear RNA-dependent adenosine deaminase activity. As determined from cloned polymerase chain reaction with reverse transcription (RT-PCR) products from brain RNA6, Q/R site editing in primary GluR-B transcripts is tenfold lower in ADAR2-/- than in wild-type mice (10% compared with 98%). This identifies ADAR2 as the principal RNA-editing enzyme at the Q/R site. The remaining low level of Q/R site editing in GluR-B pre-mRNA cannot be mediated by the residual, enzymatically inactive, truncated ADAR2 protein, but is mediated by another ADAR, perhaps ADAR1, for which gene expression appeared unchanged in ADAR2 -/- mice (Higuchi, 2000).
The low extent of Q/R site editing of GluR-B pre-mRNA led to nuclear accumulation of incompletely processed primary GluR-B transcripts and to a fivefold reduction in GluR-B mRNA, as assessed by RNase protection and quantitative RT-PCR. The increased level of intron 11-containing GluR-B transcripts and the decrease in GluR-B mRNA are easily visualized by in situ hybridization. Editing is thus a prerequisite for efficient splicing and processing of the pre-mRNA. The edited GluR-B transcripts are preferentially spliced, as revealed by a shift in Q/R site editing from 10% to 40% when comparing intron-11-containing transcripts with GluR-B mRNA. A defect in transcript processing caused by the interaction of the residual truncated ADAR2 protein with RNA can be excluded because GluR-B pre-mRNA accumulation is also observed in ADAR2+/+ mice expressing the Q/R site-uneditable GluR-BdeltaECS allele (Higuchi, 2000).
Strains are described containing homozygous deletions in each, or both, of the two C. elegans ADAR genes that have been characterized: adr-1 and adr-2. adr-1 is expressed in most, if not all, cells of the C. elegans nervous system and also in the developing vulva. Using chemotaxis assays, it has been shown that both ADARs are important for normal behavior. Biochemical, molecular and phenotypic analyses indicate that ADR-1 and ADR-2 have distinct roles in C. elegans, but sometimes act together (Tonkin, 2002).
The observation that animals with a deletion in adr-1 have vulva defects, as well as the strong expression of the adr-1::GFP construct in the developing vulva, implicates the ADR-1 protein in vulva morphogenesis. However, because the protruding-vulva defects are subtle, future studies will be needed to confirm this. ADARs could act in any of the signaling steps involved in vulval development. Since animals with a deletion in adr-2 have no detectable editing, it is perplexing that these animals do not have vulva defects. Possibly, adr-1 has functions beyond RNA editing or, alternatively, some adr-1-specific editing may exist in adr-2(gv42) animals but is beyond the limits of detection. The latter is consistent with the observation that the RNAi defects of the adr mutants are less severe in adr-2(gv42) animals compared with the double mutants. Although all mutant animals were back-crossed to wild-type animals eight times, since it has not been possible to rescue the Pvl defect, in theory the defect could derive from a mutation in a very closely linked gene. However, the strong vulva expression of the adr-1::GFP construct in multiple transgenic lines argues against this possibility. Finally, while there are no other genes with obvious sequence similarity to ADARs in the C. elegans genome, ADR-1 activity in the vulva could be mediated by interaction with an as yet unknown factor (Tonkin, 2002).
C. elegans ADARs are required for normal chemotaxis. Once an odorant is detected by a sensory neuron, a particular behavioral response is elicited through specific connections to interneurons, other sensory neurons and motor neurons. The data collected so far are not sufficient to indicate where in the chemosensation pathway ADARs are acting. The adr-1::GFP construct is expressed in the sensory neurons and cilia, but also in the ventral nerve cord, motor neurons and interneurons; at present, it is possible that ADARs are acting in any or all of these cells. RNA editing could affect chemosensation by targeting RNAs that encode receptors or signaling molecules within the AWA or AWC neurons, or affect molecules in the downstream cells that mediate the response of these neurons. Two of the C. elegans ADAR substrates analyzed in this study, unc-64 syntaxin and laminin-gamma mRNAs, are important for proper function of the nervous system. Editing sites in the 3'-UTRs of both of these substrates are altered in the adr deletion mutants and, in theory, either of these substrates could be involved in the chemotaxis defects observed. However, since there are probably hundreds of ADAR substrates in the worm nervous system to choose from, future studies will be required to determine this (Tonkin, 2002).
One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR also edits non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Phenotypes of mice homozygous for ADAR1 null mutation have been investigated. Although live ADAR1-/- embryos with normal gross appearance can be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos are also prone to apoptosis induced by serum deprivation. These results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis (Wang, 2004).
The C. elegans T20H4.4 open reading frame predicted by Genefinder encodes a 367 amino acid protein that is 32%-35% identical to the C-terminal domain of adenosine deaminases that act on RNA T20H4.4 cDNAs encode a larger 495 amino acid protein that is extended at its N-terminus to include a single double-stranded RNA-binding motif, and T20H4.4 has been shown to occupy the second position in a six-gene operon. Ten different spliced-leader (SL) sequences were found attached to T20H4.4 mRNAs, including SL1, SL2 and eight SL2-like leaders that include two new variants. Characterization of cDNAs derived from all six genes confirms the essential features of C. elegans operons: intercistronic distances in the range of 104-257 nt between the upstream polyadenylation sites and the downstream trans-splice sites; SL2, or SL2-like leaders, attached to the downstream mRNAs. Polycistronic mRNA fragments reveal a 5'-untranslated region (5'-UTR) greater than 705 nt. The 5'-UTR is removed in mature mRNAs from the first gene (T20H4.5) and replaced primarily by SL1, and to a lesser extent by SL2. This study provides new information regarding operons and how they are processed (Hough, 1999).
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. Human genomic clones of the ADAR gene and cDNA clones encoding splice site variants of the ADAR protein have been isolated and characterized. Southern blot and sequence analyses reveal 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) is 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 differ in exons 6 and 7. ADAR-b is a 5'-splice site variant that possesses a 26-amino acid deletion within exon 7; ADAR-c is a 3'-splice site variant that possesses an additional 19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c proteins all possess comparable double-stranded RNA-specific adenosine deaminase activity. However, mutational analysis of the R-motifs reveals that the exon 6 and 7 deletions of ADAR-b and -c variants alter the functional importance of each of the three R-motifs (Liu, 1997).
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]. hADAR3, the third member of this class of human enzyme has been isolated and its editing site has been selectivity investigated using in vitro RNA editing assay systems. 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 reveal that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis has 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 inhibits in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing (Chen, 2000).
RNA-specific adenosine deaminase (ADAR1) catalyzes the deamination of adenosine to inosine in viral and cellular RNAs. Two size forms of the ADAR1 editing enzyme are known, an IFN-inducible approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-kDa protein. Alternative exon 1 structures of human ADAR1 transcripts have been identified that initiate from unique promoters, one constitutively expressed and the other IFN inducible. Cloning and sequence analyses of 5'-rapid amplification of cDNA ends (RACE) cDNAs from human placenta have established a linkage between exon 2 of ADAR1 and two alternative exon 1 structures, designated herein as exon 1A and exon 1B. Analysis of RNA isolated from untreated and IFN-treated human amnion cells demonstrates that exon 1B-exon 2 transcripts are synthesized in the absence of IFN and are not significantly altered in amount by IFN treatment. By contrast, exon 1A-exon 2 transcripts are IFN inducible. Transient transfection analysis with reporter constructs has identified two functional promoters, designated PC and PI. Exon 1B transcripts are initiated from the PC promoter, whose activity in transient transfection reporter assays is not increased by IFN treatment. The 107-nt exon 1B maps 14.5 kb upstream of exon 2. The 201-nt exon 1A that maps 5.4 kb upstream of exon 2 is initiated from the interferon-inducible PI promoter. These results suggest that two promoters, one IFN inducible and the other not, initiate transcription of the ADAR1 gene, and that alternative splicing of unique exon 1 structures to a common exon 2 junction generates RNA transcripts with the deduced coding capacity for either the constitutively expressed approximately 110-kDa ADAR1 protein (exon 1B) or the interferon-induced approximately 150-kDa ADAR1 protein (exon 1A) (George, 1999).
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, it is readily detectable and quantifiable once it is purified away from the Watson-Crick nucleotides. 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 one IMP is present for every 17,000 ribonucleotides (Paul, 1998).
Adenosine deaminases that act on RNA (ADARs) constitute a family of RNA-editing enzymes that convert adenosine to inosine within double-stranded regions of RNA. A method to identify inosine-containing RNAs has been developed and has been used to identify ADAR substrates in Caenorhabditis elegans. All 10 of the C. elegans substrates identified by this method are edited in long stem-loop structures located in noncoding regions, and thus contrast with previously identified substrates of other organisms, in which ADARs target codons. To determine whether editing in noncoding regions is a conserved ADAR function, the method was applied to poly(A)+ RNA of human brain and 19 previously unknown ADAR substrates were identified. The substrates were strikingly similar to those observed in C. elegans, since editing was confined to 3' untranslated regions, introns, and a noncoding RNA. Also similar to what was found in C. elegans, 15 of the 19 substrates were edited in repetitive elements. The identities of the newly identified ADAR substrates suggest that RNA editing may influence many biologically important processes, and that for many metazoa, A-to-I conversion in coding regions may be the exception rather than the rule (Morse, 2002).
ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. ADAR2 can modify its own pre-mRNA, leading to the creation of a proximal 3'-splice junction containing a non-canonical adenosine-inosine (A-I) dinucleotide. Alternative splicing to this proximal acceptor shifts the reading frame of the mature mRNA transcript, resulting in the loss of functional ADAR2 expression. Both evolutionary sequence conservation and mutational analysis support the existence of an extended RNA duplex within the ADAR2 pre-mRNA formed by base-pairing interactions between regions approximately 1.3-kilobases apart in intron 4 and exon 5. Characterization of ADAR2 pre-mRNA transcripts isolated from adult rat brain identified 16 editing sites within this duplex region, and sites preferentially modified by ADAR1 and ADAR2 have been defined using both tissue culture and in vitro editing systems. Statistical analysis of nucleotide sequences surrounding edited and non-edited adenosine residues have identified a nucleotide sequence bias correlating with ADAR2 site preference and editing efficiency. Among a mixed population of ADAR substrates, ADAR2 preferentially favors its own transcript, yet mutation of a poor substrate to conform to the defined nucleotide bias increases the ability of that substrate to be modified by ADAR2. These data suggest that both sequence and structural elements are required to define adenosine moieties targeted for specific ADAR2-mediated deamination (Dawson, 2004).
The RNA-editing enzyme ADAR1 modifies adenosines by deamination and produces A-to-I mutations in mRNA. ADAR1 has been shown to function in host defense and in embryonic erythropoiesis during fetal liver development. The mechanisms for these phenotypic effects are not yet known. This study reports a novel function of ADAR1 in the regulation of gene expression by interacting with the nuclear factor 90 (NF90) proteins, known regulators that bind the antigen response recognition element (ARRE-2) and have been demonstrated to stimulate transcription and translation. ADAR1 upregulates NF90-mediated gene expression by interacting with the NF90 proteins, including NF110, NF90, and NF45. A knockdown of NF90 with small interfering RNA suppresses this function of ADAR1. Coimmunoprecipitation and double-stranded RNA (dsRNA) digestion demonstrate that ADAR1 is associated with NF110, NF90, and NF45 through the bridge of cellular dsRNA. Studies with ADAR1 deletions demonstrate that the dsRNA binding domain and a region covering the Z-DNA binding domain and the nuclear export signal comprise the complete function of ADAR1 in upregulating NF90-mediated gene expression. These data suggest that ADAR1 has the potential both to change information content through editing of mRNA and to regulate gene expression through interacting with the NF90 family proteins (Mie, 2005).
ADAR2 is a nuclear enzyme essential for GluR2 pre-mRNA editing at Q/R site-607, which gates Ca2+ entry through AMPA receptor channels. Forebrain ischemia in adult rats selectively reduces expression of ADAR2 enzyme and, hence, disrupts RNA Q/R site editing of GluR2 subunit in vulnerable neurons. Recovery of GluR2 Q/R site editing by expression of exogenous ADAR2b gene or a constitutively active CREB, VP16-CREB, which induces expression of endogenous ADAR2, protects vulnerable neurons in the rat hippocampus from forebrain ischemic insult. Generation of a stable ADAR2 gene silencing by delivering small interfering RNA (siRNA) inhibits GluR2 Q/R site editing, leading to degeneration of ischemia-insensitive neurons. Direct introduction of the Q/R site edited GluR2 gene, GluR2(R607), rescues ADAR2 degeneration. Thus, ADAR2-dependent GluR2 Q/R site editing determines vulnerability of neurons in the rat hippocampus to forebrain ischemia (Peng, 2006).
Translation of mRNA is usually cytoplasmic. The RNA editing enzyme ADAR1, which catalyzes the deamination of adenosine to inosine in double-stranded RNA substrates, induces translation within the nucleus, possibly at the surface of the nucleolus. This activity does not depend on RNA editing. Two regions within ADAR1 are defined that act independently of each other to induce translation: the first includes the double-stranded RNA binding domains (DRBMs) of ADAR1 while the second maps to the C-terminal portion of the catalytic domain. Point mutations within each domain are identified that reduce nuclear translation; those in the DRBM region are also known to diminish RNA binding. This report adds to the growing functionality ascribed to the nucleus (Herbert, 2002).
The physiological significance of translation within the nucleus is not addressed in these studies. Nuclear translation would likely represent a very old function of eukaryotic cells. It has been suggested that a coupled transcription-translation mechanism in the nucleus may have developed from prokaryotic origins to become a quality assurance mechanism related to the nonsense-mediated decay of aberrant mRNA. ADAR1 might participate in this mechanism by inducing nuclear translation and using editing to mark flawed mRNAs as defective. Alternatively, the regulated translation of certain proteins in the nucleolus could be important for integrating various signaling pathways. For example, nucleolar pathways are already known to be obligatory for the maturation of numerous small RNAs that are essential for many aspects of RNA processing. Along these lines, a quality control checkpoint for ribosomes in the nucleolus would make sense. Such a pathway might involve the pre-assembly of ribosomes incorporating aminoacylated tRNAs and particular mRNAs. Production of protein from such messages could signal that functional ribosomes have been produced (Herbert, 2002 and references therein).
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. The effect of VAI RNA on ADAR function has been examined. 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 (Lei, 1998).
Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that deaminate adenosines to create inosines in double-stranded RNA (dsRNA). ADARs are not required for RNA interference (RNAi) in C. elegans and they do not antagonize the pathway to a detectable level when RNAi is initiated by injecting dsRNA. However, transgenes expressed in the somatic tissues of wild-type animals are silenced in strains with deletions in the two genes encoding ADARs, adr-1 and adr-2. Transgene-induced gene silencing in adr-1;adr-2 mutants depends on genes required for RNAi, suggesting that a dsRNA intermediate is involved. In wild-type animals, edited dsRNA corresponding to transgenes are detected, and it is proposed that editing of this dsRNA prevents somatic transgenes from initiating RNAi in wild-type animals (Knight, 2002).
Transgene-induced RNAi in the adr-1;adr-2 mutants appears remarkably similar to cosuppression. One mystery of cosuppression in C. elegans has been that it occurs almost exclusively in the germline. There has been only one previously reported instance of a transgene silencing an endogenous gene in the somatic tissues of a wild-type animal despite the thousands of transgenic strains that have been studied. The rarity of transgene-induced gene silencing in somatic tissues is in some instances attributable to ADAR activity. A distinct difference between cosuppression in the C. elegans germline and transgene-induced RNAi in the adr-1;adr-2 double mutants is their dependence on rde-1 (distantly related to Drosophila argonaute). Cosuppression is rde-1 independent, but transgene-induced RNAi in the somatic tissues of adr-1;adr-2 double mutants requires rde-1, indicating that although these two phenomenon appear related, subtle differences exist. rde-1 belongs to a large gene family in C. elegans, and therefore, it remains a possibility that one of the other gene family members may function in place of rde-1 in cosuppression (Knight, 2002).
It is somewhat surprising that cosuppression functions in the C. elegans germline since there is evidence that adr-2 is expressed in this tissue. Perhaps the level of ADAR activity in the germline is insufficient to prevent cosuppression, or cosuppression may not be mediated by dsRNA. If cosuppression involves a dsRNA intermediate, it may be protected from editing by localization to the cytoplasm, where ADARs are rarely found, or rapidly cleaved to siRNAs that may be too short to serve as ADAR substrates. In plants the correlation between the effectiveness of cosuppression and the absence of ADARs is strong. Plants are highly susceptible to cosuppression and also lack genes encoding ADARs (Knight, 2002).
Perhaps the simplest interpretation of these data is that the activities of ADARs and Dicer are physically separated. ADARs are primarily localized to the nucleus, but Dicer is found in the cytoplasm of embryonal carcinoma and HeLa cells. If the two activities are separated, dsRNA delivered by injection may trigger RNAi before ever being exposed to ADARs in the nucleus. When dsRNA is produced in the nucleus at low concentrations from transgenes, however, ADARs may first edit the dsRNA before the duplex is exposed to Dicer. In other organisms, highly edited RNAs are retained in the nucleus. If a similar retention mechanism exists in C. elegans, it could further prevent edited dsRNAs from entering the cytoplasm and being cleaved by Dicer. At high concentrations of dsRNA, however, ADARs are subject to substrate inhibition, and this may explain why RNAi that is triggered in wild-type animals by inducing synthesis of dsRNA is not inhibited by ADARs (Knight, 2002).
Alternatively, it is possible that both ADARs and Dicer are present in the nucleus. In C. elegans the intracellular localization of Dicer remains unknown. dcr-1 is required for RNAi in the C. elegans germline, and reducing dcr-1 levels by RNAi results in a decreased sensitivity to RNAi in the somatic tissues. C. elegans dcr-1 homozygous strains, however, only have defects in somatic RNAi when the triggering dsRNA is provided from a transgene. Although the reason dcr-1 homozygotes only have defects in somatic RNAi triggered from transgenes remains unclear, it is intriguing that deletions in both dcr-1 and the two genes encoding ADARs only effect somatic RNAi initiated in the nucleus. If ADARs and Dicer are nuclear, then at low concentrations of dsRNA, ADARs may simply have a higher affinity for dsRNA than Dicer (Knight, 2002).
In C. elegans, several ADAR substrates have been identified, but it is unclear exactly how editing of these substrates leads to the phenotypes of animals lacking ADARs. Many ADAR substrates in C. elegans and humans are edited in noncoding regions that are predicted to form long double-stranded regions, suggesting that, in addition to changing the coding potential of mRNAs, editing may also play other roles. It is intriguing to speculate that editing of naturally occurring dsRNAs may prevent these dsRNAs from triggering gene silencing as observes with dsRNAs originating from transgenes (Knight, 2002).
The fate of double-stranded RNA (dsRNA) in the cell depends on both its length and location. The expression of dsRNA in the nucleus leads to several distinct consequences: (1) the promiscuous deamination of adenosines to inosines by dsRNA-specific adenosine deaminase (ADAR) can lead to the nuclear retention of edited transcripts; (2) dsRNAs might induce heterochromatic gene silencing through an RNAi-related mechanism. Is RNA editing also connected to heterochromatin? Members of the conserved Vigilin class of proteins have a high affinity for inosine-containing RNAs. In agreement with other work, these proteins were found to localize to heterochromatin and mutation or depletion of the Drosophila Vigilin, DDP1, leads to altered nuclear morphology and defects in heterochromatin and chromosome segregation. Furthermore, nuclear Vigilin is found in complexes containing not only the editing enzyme ADAR1 but also RNA helicase A and Ku86/70. In the presence of RNA, the Vigilin complex recruits the DNA-PKcs enzyme, which appears to phosphorylate a discrete set of targets, some or all of which are known to participate in chromatin silencing. These results are consistent with a mechanistic link between components of the DNA-repair machinery and RNA-mediated gene silencing (Wang, 2005).
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