Functional analysis of the KCS-like element of the interferon-inducible RNA-specific adenosine deaminase ADAR1 promoter.
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.
Markle
Danielle
D
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106-9601, USA.
Das
Sonali
S
Ward
Simone Visosky
SV
Samuel
Charles E
CE
D000243:Adenosine Deaminase / Q000235:genetics*; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007372:Interferons / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000276:immunology / Q000378:metabolism; D019892:eIF-2 Kinase / Q000235:genetics
D000906:Antibodies; D009687:Nuclear Proteins; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D007372:Interferons; D015500:Chloramphenicol O-Acetyltransferase; D019892:eIF-2 Kinase; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase