Functional analysis of the KCS-like element of the interferon-inducible RNA-specific adenosine deaminase ADAR1 promoter Danielle Markle, Sonali Das, Simone Visosky Ward, Charles E. Samuel * Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106-9601, USA Received 2 October 2002; received in revised form 11 November 2002; accepted 4 December 2002 Received by A.J. van Wijnen Abstract The ADAR1 gene encodes an RNA-specific adenosine deaminase that alters the functional activity of both viral and cellular RNAs by posttranscriptional adenosine-to-inosine RNA editing. The interferon (IFN) responsive PI promoter of the ADAR1 gene possesses an IFN- stimulated response element (ISRE) responsible for IFN-inducibility, as well as an adjacent upstream sequence, designated kinase conserved sequence-like (KCS-‘) element. The KCS-‘ element is similar to the 15-bp KCS element so far unique to the human and mouse RNA- dependent PKR kinase gene promoters. The KCS element of the PKR kinase (PKR) promoter is essential for both basal and IFN-inducible PKR promoter activity. We have now examined the functional properties of the KCS-‘ element of the ADAR1 PI promoter. Electrophoretic mobility shift assays (EMSAs) detected constitutively expressed nuclear proteins that bound selectively to the ADAR1 KCS-‘ element. Competition EMSA and antibody supershift assays indicated that ADAR1 KCS-‘-binding proteins shared some properties with PKR KCS- binding proteins. However, transient transfection analyses performed with ADAR1 PI promoter constructs possessing deletion and substitution mutant forms of the KCS-‘ element revealed that the ADAR1 KCS-‘ element was not essential for either basal or IFN-inducible promoter activity. Substitution of the ADAR1 KCS-‘ element with the PKR KCS element increased both basal and inducible ADAR1 PI promoter activity. These results suggest that the KCS-‘ element of the ADAR1 PI promoter is not functionally equivalent to the KCS element of the PKR promoter. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Interferon; ADAR deaminase; RNA editing; Transcriptional control 1. Introduction Interferons (IFNs), originally discovered as antiviral agents, affect a broad range of biologic processes in addition to inhibiting virus multiplication, such as cell proliferation and differentiation, apoptosis, and the immune response (Pestka et al., 1987; Samuel, 2001). The actions of IFNs are mediated by the products of the genes that they regulate. IFN treatment activates the transcription of a large number of cellular genes (Chang and Laimins, 2000; de Veer et al., 2001; Knipe et al., 2001). IFN-mediated signaling and transcriptional activation occurs principally by the JAK- STAT signaling pathway (Stark et al., 1998; Samuel, 2001; Levy and Darnell, 2002). The 13-bp IFN-stimulated response element (ISRE), found in promoters of genes regulated by the type I (a and b) IFNs, is responsible for their IFN-inducibility. ISGF-3, a trimeric protein complex consisting of Stat1, Stat2 and IRF-9, is activated by IFN-a/b and binds to the ISRE element to drive transcription (Stark et al., 1998; Kisseleva et al., 2002). Among the IFN-inducible proteins are two enzymes that bind double-stranded RNA: the RNA-specific adenosine deaminase (ADAR1) and the RNA-dependent protein kinase (PKR). ADAR1 is an RNA editing enzyme that catalyzes the C-6 deamination of adenosine (A) to produce inosine (I) in double-stranded structures found in both cellular and viral RNAs. This A-to-I editing thereby alters the functional activity of the RNA, because I is read as G 0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1119(02)01200-3 Gene 304 (2003) 143–149 www.elsevier.com/locate/gene * Corresponding author. Tel.: þ 1-805-893-3097; fax: þ1-805-893-5780. E-mail address: samuel@lifesci.ucsb.edu (C.E. Samuel). Abbreviations: bp, basepair; IFN, interferon; PKR, the RNA-dependent eIF-2a protein kinase inducible by interferon; PKR, gene encoding the PKR kinase; ADAR1, double-stranded RNA adenosine deaminase; ADAR1, gene encoding the double-stranded RNA adenosine deaminase; ISRE, interferon-stimulated response element; KCS, kinase conserved sequence of the PKR promoter; KCS-‘, KCS-like element of the human ADAR1 promoter.