Expression of Interferon-inducible RNA Adenosine Deaminase ADAR1 during Pathogen Infection and Mouse Embryo Development Involves Tissue-selective Promoter Utilization and Alternative Splicing

Abstract

ADAR1 (adenosine deaminase acting on RNA) is widely expressed in adult mammals and has a critical role during embryogenesis. Two size forms of ADAR1 are known that possess adenosine-to-inosine editing activity: an interferon (IFN)-inducible ϳ150-kDa protein and a constitutively expressed N-terminally truncated ϳ110-kDa protein. We defined the structure of the 5 -flanking region of the mouse Adar1 gene, and we show here that mouse Adar1 transcripts possess alternative exon 1 structures (1A, 1B, and 1C) that initiate from unique promoters and are spliced to a common exon 2 junction. Exon 1A-containing transcripts encoding p150 were expressed in all tissues examined from adult mice (brain, cecum, heart, kidney, liver, lung, spleen, and Peyer's patches) and were elevated most significantly in liver but remained lowest in brain following oral infection with Salmonella. Exon 1B-containing RNA was most abundant in brain and was not increased in any tissue examined following infection. Exon 1C-containing RNA was very scarce. Exon 1A, but not exon 1B or 1C, expression was increased in fibroblast L cells treated with IFN, and a consensus ISRE element was present in the promoter driving exon 1A expression. Exon 1B, but not 1A, was detectable in embryonic day 10.5 embryos and was abundantly expressed in embryonic day 15 embryos. Furthermore, the ADAR1 p110 protein isoform was detected in embryonic tissue, whereas both p110 and the inducible p150 proteins were found in IFN-treated L cells. Finally, the presence of alternative exon 7a correlated with exon 1B-containing RNA, and alternative exon 7b correlated with exon 1A-containing RNA. These results establish that multiple promoters drive the expression of the Adar1 gene in adult mice, that the IFN inducible promoter and exon 1A-containing RNA are primarily responsible for the increased ADAR1 observed in Salmonella-infected mice, and that the constitutive exon 1B-containing transcript and encoded p110 protein product are abundantly expressed both in adult brain and during embryogenesis.Adenosine (A) 1 to inosine (I) RNA editing is a post-transcriptional process by which an RNA sequence is modified by C-6 deamination of one or more As, leading to the formation of transcripts whose sequence differs from that found in the genome (1-4). Such A-to-I editing represents a form of genetic recoding, because inosine is recognized as guanosine by decoding ribosomes and by transcribing polymerases. Enzymes that catalyze A-to-I editing are known as ADARs (adenosine deaminases that act on RNA) (5). ADARs deaminate RNA substrates with double-stranded structure. In fact, the enzymatic activity of ADARs was discovered as an RNA duplex unwinding activity (6, 7).A-to-I editing is of broad biologic significance. Among the best characterized cellular and viral RNAs that undergo A-to-I editing with high selectivity at one or a few positions are transcripts encoding glutamate receptor (GluR) channels, the serotonin-2C receptor, and hepatitis delta virus RNA (8, 9). RNA editing of the neurotransmit...

Identification of the Zebrafish IFN Receptor: Implications for the Origin of the Vertebrate IFN System

“…Alternative promoter usage and splicing of virus-induced IFNs after viral infection thus appears as a general mechanism in teleosts. It is reminiscent of the situation recently described for the Adar1, Irgd, and Irga6 genes in mouse where two different promoters, one constitutive and one IFN inducible, lie ahead of alternative first exons (31,32).…”

Host Response to Polyomavirus Infection Is Modulated by RNA Adenosine Deaminase ADAR1 but Not by ADAR2

“…RT-PCR analyses of the adar1 Ϫ/Ϫ and adar2 Ϫ/Ϫ MEF cells, and transformants expressing human ADAR1, were carried out essentially as described previously (7,8). Total RNA was isolated by the TRIzol method by following the manufacturer's recommendations.…”

“…Proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes which were blocked using phosphate-buffered saline containing 5% (wt/vol) nonfat milk, and subsequently probed with primary antibody as indicated. Rabbit polyclonal antibody was used to detect PyV capsid protein VP1; guinea pig polyclonal antibody was used to detect mouse ADAR1 (8), and mouse monoclonal antibodies were used to detect PyV large T antigen and Alternatively, secondary anti-IgG IRDye-conjugated antibodies (Li-Cor) were used, and immunoreactive bands were visualized and quantitated using an Odyssey infrared imaging system (Li-Cor).…”

“…The mouse adar1 gene maps to a single locus on chromosome 3 band F2 (52) and is inducible by interferon (IFN) (8,9,36,43). Two size forms of ADAR1 protein are expressed by a mechanism involving alternative adar1 promoter usage and alternative exon 1 splicing: a short (p110) nuclear form that is constitutively made and a long (p150) form that is interferon inducible and present in both the cytoplasm and nucleus (7,8,36,37).…”

“…Two size forms of ADAR1 protein are expressed by a mechanism involving alternative adar1 promoter usage and alternative exon 1 splicing: a short (p110) nuclear form that is constitutively made and a long (p150) form that is interferon inducible and present in both the cytoplasm and nucleus (7,8,36,37). The mouse adar2 gene maps to chromosome 10 C1, is constitutively expressed, and is not known to be regulated by IFN or infection (10,16,29).…”