Characterization of the 5′-flanking region of the human RNA-specific adenosine deaminase ADAR1 gene and identification of an interferon-inducible ADAR1 promoter
Introduction
Interferons (IFN) are a family of regulatory cytokines that possess a wide range of biologic activities (Vilcek and Sen, 1996). These activities include the ability to mediate establishment of a potent antiviral state in uninfected animal cells; the regulation of cell proliferation and differentiation; and the modulation of the immune response. Among the recently identified IFN-inducible genes is an RNA editing enzyme, the double-stranded RNA adenosine deaminase (Patterson and Samuel, 1995, Wathelet et al., 1988) now known as ADAR1 (Bass et al., 1997).
RNA editing represents an important post-transcriptional process by which RNA transcripts are covalently modified in a manner that has the potential to alter either the coding capacity of the RNA or the function of the encoded product (Rueter and Emeson, 1998). The ADAR editing enzymes catalyze the C-6 deamination of adenosine in double-stranded (ds) structures present within viral RNAs and cellular pre-mRNAs as well as synthetic dsRNA substrates (Bass et al., 1997, Rueter and Emeson, 1998, Seeburg et al., 1998). Two classes of adenosine to inosine editing processes have been defined in naturally occurring candidate RNA substrates of ADAR. First, multiple clustered modifications characteristic of adenosine deamination are found in viral RNAs, exemplified by the extensive adenosine modifications present on the viral antisense RNA late in polyoma virus infection (Kumar and Carmichael, 1997) and by the biased hypermutations observed in negative-stranded RNA virus genomes during lytic and persistent infections, as in the case of the measles virus (Cattaneo, 1994). Second, highly site-specific adenosine to inosine modifications are found at one or a few sites in certain viral and cellular RNA transcripts, as exemplified by the editing of hepatitis delta virus (HDV) RNA (Polson et al., 1996) and the pre-mRNAs encoding two important neurotransmitter receptors, the serotonin-2C receptor (5-HT-2CR) (Burns et al., 1997) and the glutamate receptor (GluR) channel subunits (Rueter and Emeson, 1998, Seeburg et al., 1998). Editing of these viral and cellular RNAs results in codon changes and subsequently encoded HDV, GluR and 5-HT-2CR protein products with altered functional activities. These selective A-to-I deaminations found in HDV RNA and cellular GluR and 5-HT-2CR pre-mRNA transcripts are dependent upon double-stranded (ds) regions within the substrate RNAs (Bass et al., 1997, Rueter and Emeson, 1998).
Molecular cloning studies established that the ADAR enzymes constitute a multi-gene family of enzymes (Bass et al., 1997, Rueter and Emeson, 1998). We isolated the ADAR1 cDNA in a screen for interferon-inducible human sequences (Patterson and Samuel, 1995). ADAR1 is a 1226-amino-acid protein (Kim et al., 1994, O'Connell et al., 1995, Patterson and Samuel, 1995) that possesses both dsRNA-binding and Z-DNA-binding activities (Liu et al., 1998). The central region of the deduced ADAR1 protein sequence includes three copies of the highly conserved double-stranded RNA binding motif (Patterson and Samuel, 1995) first described in the interferon-inducible RNA-dependent protein kinase PKR (Samuel, 1993). The dsRBM motifs of ADAR1 are implicated in the recognition of dsRNA structures within the substrate RNAs. Three alternative-splice site variants of IFN-inducible ADAR1 are differentially expressed in a tissue-specific fashion and possess functionally distinct dsRNA binding domains (Liu et al., 1997). A repeated domain present in the N-terminal region of ADAR1 homologous to the N-terminal region of the vaccinia virus E3L protein (Patterson and Samuel, 1995) corresponds to two Z-DNA binding domains of ADAR1, designated Zα and Zβ (Herbert et al., 1997). Two immunologically related forms of the human ADAR1 deaminase are present in a variety of human cell lines: an interferon-inducible ∼150-kDa protein present in both the cytoplasm and nucleus and a constitutively expressed ∼110-kDa truncated protein present predominantly, if not exclusively, in the nucleus (Patterson and Samuel, 1995). The gene encoding the ADAR1 proteins maps to human chromosome 1q21.1–21.2 (Wathelet et al., 1988, Weier et al., 1995).
Little is known regarding the regulation of ADAR1 expression by IFN, other than that the gene is IFN-inducible (Patterson and Samuel, 1995, Patterson et al., 1995, Wathelet et al., 1988). Because of the possible role of ADAR1 during lytic and persistent viral infections in generating either mRNA transcripts with altered coding capacity or viral genomes possessing biased hypermutations (Cattaneo, 1994, Rueter and Emeson, 1998), and because of the recent identification of a ribonuclease specific for inosine-containing RNA (Scadden and Smith, 1997) suggestive of a potential role of ADAR1 in the antiviral actions of IFN in a manner conceptually similar to the 2′–5′-oligoadenylate synthetase-RNase L pathway (Samuel, 1991), it is important to define the molecular basis of ADAR1 transcriptional control by interferon. As a step towards this goal, we report herein the isolation of genomic clones that contain the functional IFN-responsive promoter region of the human ADAR1 gene. Sequence analysis revealed an ISRE element and, unexpectedly, a candidate KCS-like element previously identified only in the human and mouse PKR gene promoters (Kuhen and Samuel, 1997).
Section snippets
Promoter cloning
Genomic clones of ADAR1 were isolated by screening two types of libraries, a λ-phage library and a P1-phage library (Liu et al., 1997). A human genomic library in the λ phage vector EMBL-3 SP6/T7 prepared from human placenta DNA (Clontech, Palo Alto, CA) was screened by filter hybridization using random primed 32P-labeled cDNA fragments of the human ADAR cDNA as the probes (Sambrook et al., 1989). λ-phage DNA was prepared from twice-rescreened plaques, and genomic inserts were characterized by
Cloning of the 5′-untranslated region of the human interferon-inducible ADAR1 cDNA
The sequence of the complete 5′-UTR of the human ADAR1 mRNA is not known. The nucleotide sequence for the most 5′ cDNA clone previously determined for the human ADAR1 cDNA included a sequence of 164 nt flanking the ATG translation start site (Patterson and Samuel, 1995, Wang et al., 1995). To obtain the sequence of the 5′-UTR, 5′-RACE was performed as described in Section 2 using an ADAR1 minus primer corresponding to antisense nt +305 to +285 and then the nested ADAR1 minus primer +82 to +63,
Acknowledgements
This work was supported in part by Research Grant AI-12520 from the National Institute of Allergy and Infectious Diseases, US Public Health Service.
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