The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1E861A, where E861A denotes Glu861→Ala861). Adar1E861A/E861A embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)–sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3′ untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1E861A/E861A were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

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.

Adenosine deaminases acting on RNA (ADARs) are involved in editing of adenosine residues to inosine in double-stranded RNA (dsRNA). Although this editing recodes and alters functions of several mammalian genes, its most common targets are noncoding repeat sequences, indicating the involvement of this editing system in currently unknown functions other than recoding of protein sequences. Here we show that specific adenosine residues of certain microRNA (miRNA) precursors are edited by ADAR1 and ADAR2. Editing of pri-miR-142, the precursor of miRNA-142, expressed in hematopoietic tissues, resulted in suppression of its processing by Drosha. The edited pri-miR-142 was degraded by Tudor-SN, a component of RISC and also a ribonuclease specific to inosinecontaining dsRNAs. Consequently, mature miRNA-142 expression levels increased substantially in ADAR1 null or ADAR2 null mice. Our results demonstrate a new function of RNA editing in the control of miRNA biogenesis.ADARs bind dsRNAs and deaminate adenosine residues to inosine. The resulting A→I conversions replace A-U Watson-Crick pairs with I•U wobble pairs in the dsRNA1 , 2. Although I•U and isosteric G•U wobble base pairs, like Watson-Crick pairs, participate in forming helical regions in RNA folding, they seem to have unique conformational and biological features3. Three members of the ADAR gene family (ADAR1-3) have been identified in vertebrates4 , 5. In addition, two isoforms of ADAR1, an interferon-inducible, cytoplasmic 150-kDa protein (p150) and a constitutive, nuclear 110-kDa protein (p110) are synthesized by translation initiation at alternative methionine codons6. Members of the ADAR gene family contain multiple dsRNA-binding domains and a separate deaminase domain7 -10. Although apparently functional domain features are conserved in ADAR3, its enzymatic activity has not yet been demonstrated9.An inosine residue converted from adenosine in RNA is detected as an A→G change of the complementary DNA sequence, and the translation machinery reads inosine as guanosine, leading to alterations of codons. Relatively few editing sites located within the coding sequences of target genes have been identified by comparing individual cDNA sequences to their corresponding genomic sequences 4,5 37 . In the present study, we set out to investigate the interaction between the A→I RNA-editing and miRNA-biogenesis pathways. Both ADAR1 and ADAR2 edit specific adenosine residues of precursors of certain miRNAs including mouse miR-142. We demonstrate here that A→I editing alters the dsRNA structure of pri-miR-142, inhibits miR-142 processing by Drosha and, consequently, decreases mature miR-142-5p and miR-142-3p RNA levels. Thus, we reveal, for the first time, a new function of A→I RNA editing in the regulation of processing and expression of miRNAs. RESULTS Editing of miRNA precursors by ADAR1p110 and ADAR2We chose eight miRNA precursors: pri-miR-142, pri-miR-181a, pri-miR-181b1, primiR-181b2, pri-miR-223 (expressed mainly in hematopoietic cells), pri-122α (liver), pri...