Functional analysis of the KCS-like element of the interferon-inducible RNA-specific adenosine deaminase ADAR1 promoter

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-ℓ) element. The KCS-ℓ 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-ℓ element of the ADAR1 PI promoter. Electrophoretic mobility shift assays (EMSAs) detected constitutively expressed nuclear proteins that bound selectively to the ADAR1 KCS-ℓ element. Competition EMSA and antibody supershift assays indicated that ADAR1 KCS-ℓ-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-ℓ element revealed that the ADAR1 KCS-ℓ element was not essential for either basal or IFN-inducible promoter activity. Substitution of the ADAR1 KCS-ℓ element with the PKR KCS element increased both basal and inducible ADAR1 PI promoter activity. These results suggest that the KCS-ℓ element of the ADAR1 PI promoter is not functionally equivalent to the KCS element of the PKR promoter.