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 singlestranded 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.

Different isoforms of serotonin subtype 2C receptor (5-HT 2C R) with altered G protein-coupling efficacy are generated by RNA editing, which converts genomically encoded adenosine residues into inosines. In combination, editing of five sites all located within the second intracellular loop region of 5-HT 2C R mRNA changes the gene-encoded Ile, Asn, and Ile at positions 156, 158, and 160, respectively. We analyzed the G protein-coupling functions of previously unreported editing isoform receptors. An ϳ13-fold reduction in the agonist potency for G protein-coupling stimulation as well as a significantly reduced basal level activity was observed with the thalamus-specific isoform carrying Ile 156 , Gly 158 , and Val 160 (5-HT 2C R-IGV). In contrast, the agonist was four-to fivefold less potent with 5-HT 2C R-MSV and -IDV, detected in the amygdala and choroid plexus, respectively, indicating a dominant role for the amino acid residue at position 158 in receptor functions. We also identified a splicing variant receptor with a truncated C terminus that displayed no ligand binding capacity or G protein-coupling activity. Examination of the alternatively spliced RNA encoding this truncated receptor suggests that editing of this variant RNA occurs after completion of splicing, resulting in complete editing at all five sites. Key Words: Serotonin 5-HT 2C receptor-RNA editing-RNA splicing-Doublestranded RNA adenosine deaminase -Adenosine deaminases that act on RNA-G protein coupling. J. Neurochem. 74, 1290Neurochem. 74, -1300Neurochem. 74, (2000.Members of the serotonin [5-hydroxytryptamine (5-HT)] receptor gene family are believed to play important roles in physiological and behavioral processes such as circadian rhythms and feeding behavior. Furthermore, 5-HT receptors may have a causative relevance to human mental abnormalities and pathology, including anxiety, psychotic depression, migraine, substance dependency, and schizophrenia (Baxter et al., 1995). In humans and rodents, three subtypes of the 5-HT 2 receptor (5-HT 2 R) gene family (5-HT 2A R, 5-HT 2B R, and 5-HT 2C R) have been identified (Baxter et al., 1995).RNA editing plays a critical role in the expression of certain gene products by changing the sequence context of mRNAs, which results in synthesis of proteins not encoded in the gene sequence (Smith et al., 1997). One type of RNA editing involves the conversion of adenosine residues into inosine in transcripts of genes such as glutamate receptor ion channels (Sommer et al., 1991;Lomeli et al., 1994) and 5-HT 2C Rs (Burns et al., 1997). The A-to-I RNA editing mechanism requires the doublestranded RNA (dsRNA) structure formed around the editing sites (Higuchi et al., 1993;Egebjerg et al., 1994;Lomeli et al., 1994;Burns et al., 1997) and adenosine deaminases that act on RNA (ADARs) (Kim et al., 1994;O'Connell et al., 1995;Dabiri et al., 1996; Melcher et al., 1996a,b;Bass et al., 1997;Gerber et al., 1997;Lai et al., 1997). Members of the ADAR gene family share some structural similarities, such as...

Double-stranded RNA induces the homology-dependent degradation of cognate mRNA in the cytoplasm via RNA interference (RNAi) but also is a target for adenosine-to-inosine (A-to-I) RNA editing by adenosine deaminases acting on RNA (ADARs). An interaction between the RNAi and the RNA editing pathways in Caenorhabditis elegans has been suggested recently, but the precise mode of interaction remains to be established. In addition, it is unclear whether this interaction is possible in mammalian cells with their somewhat different RNAi pathways. Here we show that ADAR1 and ADAR2, but not ADAR3, avidly bind short interfering RNA (siRNA) without RNA editing. In particular, the cytoplasmic full-length isoform of ADAR1 has the highest affinity among known ADARs, with a subnanomolar dissociation constant. Gene silencing by siRNA is significantly more effective in mouse fibroblasts homozygous for an ADAR1 null mutation than in wild-type cells. In addition, suppression of RNAi effects are detected in fibroblast cells overexpressing functional ADAR1 but not when overexpressing mutant ADAR1 lacking double-stranded RNA-binding domains. These results identify ADAR1 as a cellular factor that limits the efficacy of siRNA in mammalian cells.

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.adenosine deaminases acting on RNA ͉ double-stranded RNA adenosine deaminase ͉ nucleic acid-protein interactions ͉ large nuclear ribonucleoprotein