Functional analysis of the KCS-like element of the interferon-inducible RNA-specific adenosine deaminase ADAR1 promoter (original) (raw)
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Proceedings of the National Academy of Sciences, 1999
A BSTR ACT RNA-specific adenosine deaminase (ADAR1) catalyzes the deamination of adenosine to inosine in viral and cellular RNAs. Two size forms of the ADAR1 editing enzyme are known, an IFN-inducible Ϸ150-kDa protein and a constitutively expressed N-terminally truncated Ϸ110-kDa protein. We have now identified alternative exon 1 structures of human ADAR1 transcripts that initiate from unique promoters, one constitutively expressed and the other IFN inducible. Cloning and sequence analyses of 5-rapid amplification of cDNA ends (RACE) cDNAs from human placenta established a linkage between exon 2 of ADAR1 and two alternative exon 1 structures, designated herein as exon 1A and exon 1B. Analysis of RNA isolated from untreated and IFN-treated human amnion cells demonstrated that exon 1B-exon 2 transcripts were synthesized in the absence of IFN and were not significantly altered in amount by IFN treatment. By contrast, exon 1A-exon 2 transcripts were IFN inducible. Transient transfection analysis with reporter constructs led to the identification of two functional promoters, designated P C and P I . Exon 1B transcripts were initiated from the P C promoter whose activity in transient transfection reporter assays was not increased by IFN treatment. The 107-nt exon 1B mapped 14.5 kb upstream of exon 2. The 201-nt exon 1A that mapped 5.4 kb upstream of exon 2 was initiated from the interferon-inducible P I promoter. These results suggest that two promoters, one IFN inducible and the other not, initiate transcription of the ADAR1 gene, and that alternative splicing of unique exon 1 structures to a common exon 2 junction generates RNA transcripts with the deduced coding capacity for either the constitutively expressed Ϸ110-kDa ADAR1 protein (exon 1B) or the interferon-induced Ϸ150-kDa ADAR1 protein (exon 1A).
Journal of Biological Chemistry, 2005
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 5flanking 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 1Bcontaining 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 1Bcontaining transcript and encoded p110 protein product are abundantly expressed both in adult brain and during embryogenesis.
Journal of Biological Chemistry, 2003
Protein kinase regulated by RNA (PKR) plays important roles in many cellular processes including virus multiplication and cell growth, differentiation, and apoptosis. The promoter of the PKR gene possesses a novel 15-bp element designated KCS, positioned upstream of a consensus interferon (IFN)-stimulated response element, that is required for both basal and interferoninducible transcription. Protein binding to the KCS element is not dependent upon IFN treatment and correlates with transcriptional activity of the PKR promoter. The identity of KCS-binding proteins (KBP) that selectively bind at the KCS element is largely unknown, except for the transcription factor Sp1. We now have purified KBP from HeLa cell nuclear extracts by ionexchange and DNA-affinity chromatography steps and then identified four constituent proteins of the KBP complex by mass spectrometry and immunochemistry: KBP120 and KBP45 are the damaged DNA-binding protein subunits, p127 DDB1 and p48 DDB2, respectively; KBP100 is the transcription factor Sp1; and KBP35 is the heterogeneous nuclear ribonucleoprotein A1. The steady-state levels of these four KCS-binding proteins in human cells are not altered by IFN treatment. Components of the KBP complex bind selectively and constitutively to the KCS element in the absence of IFN treatment, both in vitro as measured by competition electrophoretic mobility shift assay (EMSA) and DNA pull-down assays and in vivo as measured by chromatin immunoprecipitation assays. Depletion of DDB2 by antisense strategy reduces KBP complex formation by EMSA. These results provide new insight into the biochemical identity and activity of proteins involved in PKR promoter function.
Virology, 1997
The RNA-dependent protein kinase (PKR) is inducible by interferon (IFN) and is implicated in the antiviral and antiproliferative actions of IFN. We have now isolated human genomic clones that contain the promoter region required for transcription of the Pkr gene. Transient transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5-flanking fragments of the Pkr gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed an element (5GGAAAACGAAACT3) involved in IFN inducibility that corresponds to the consensus sequence of the IFNstimulated response element (ISRE). Comparison of the promoter sequence of the human Pkr gene to that of the mouse homolog identified a novel element (5GGGAAGGCGGAGTCC3) immediately upstream of the ISRE element which so far is unique to the human and mouse Pkr gene promoters. We have designated this new motif as KCS, for kinase conserved sequence. Deletion and substitution mutants of the Pkr promoter region showed that the ISRE element was required for transcriptional induction by type I IFN, whereas the KCS motif increased promoter activity mediated by the ISRE. Additional potential regulatory cis-elements were identified in the human Pkr promoter that are commonly associated with growth control regulation and differentiation. Other than the ISRE and novel KCS elements, the overall organization of potential binding sites for transcription factors was not well conserved between the IFN-inducible promoters of the human and mouse Pkr genes. The strict conservation of sequence, distance, and position of KCS, relative to ISRE, together with mutagenesis results, suggest an important functional role for the newly recognized KCS motif.
Virology, 2002
The RNA-dependent protein kinase PKR plays important roles in the antiviral and antiproliferative actions of IFN. The IFN-inducible promoter of the human PKR gene contains a 15-bp DNA element designated KCS. The KCS element is located 4 bp upstream of the interferon-stimulated response element (ISRE) and is required for both basal and IFN-inducible transcription. We have examined the effect of insertion mutations between the KCS and the ISRE elements, as well as altered orientation of the KCS element relative to the ISRE element, to assess a possible functional interaction between them. Large insertions (Ն93 bp) between the KCS and ISRE elements significantly reduced both basal and IFN-inducible promoter activity. The function of the KCS element was dependent on the orientation of KCS relative to the ISRE element. Multimerization of the KCS element increased both basal and IFN-inducible transcription. Electrophoretic mobility shift analyses (EMSA) identified IFN-inducible protein complex formation that required both the KCS and the ISRE DNA element sequences. The novel IFN-inducible protein complexes contained the transcription factor STAT1, as shown by supershift analyses and by their presence in extracts prepared from STAT1 wild-type but not from STAT1 Ϫ/Ϫ null cells. These results, taken together, strongly suggest that the KCS and ISRE elements of the human PKR promoter represent a functional unit.
Molecular and Cellular Biology, 2005
Downstream elements are a newly appreciated class of core promoter elements of RNA polymerase IItranscribed genes. The downstream core element (DCE) was discovered in the human -globin promoter, and its sequence composition is distinct from that of the downstream promoter element (DPE). We show here that the DCE is a bona fide core promoter element present in a large number of promoters and with high incidence in promoters containing a TATA motif. Database analysis indicates that the DCE is found in diverse promoters, supporting its functional relevance in a variety of promoter contexts. The DCE consists of three subelements, and DCE function is recapitulated in a TFIID-dependent manner. Subelement 3 can function independently of the other two and shows a TFIID requirement as well. UV photo-cross-linking results demonstrate that TAF1/TAF II 250 interacts with the DCE subelement DNA in a sequence-dependent manner. These data show that downstream elements consist of at least two types, those of the DPE class and those of the DCE class; they function via different DNA sequences and interact with different transcription activation factors. Finally, these data argue that TFIID is, in fact, a core promoter recognition complex. . † Supplemental material for this article may be found at http://mcb .asm.org/. 9674 pH 7.9, 10 mM HEPES-KOH, pH 8.0, 4 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 100 g/ml bovine serum albumin, 10 mM dithiothreitol, and 250 M recombinant nucleoside triphosphates. Ad E3 in vitro transcription mixtures also contained baculovirus-expressed highly purified Sp1 and a partially purified mediator fraction in order to increase transcription signals .
Virology, 2008
The p150 form of the RNA-specific adenosine deaminase ADAR1 is interferon-inducible and catalyzes A-to-I editing of viral and cellular RNAs. We have characterized mouse genomic clones containing the promoter regions required for Adar1 gene transcription and analyzed interferon induction of the p150 protein using mutant mouse cell lines. Transient transfection analyses using reporter constructs led to the identification of three promoters, one interferon-inducible (P A ) and two constitutively active (P B and P C ). The TATA-less P A promoter, characterized by the presence of a consensus ISRE element and a PKR kinase KCS-like element, directed interferon-inducible reporter expression in rodent and human cells. Interferon induction of p150 was impaired in mouse cells deficient in IFNAR receptor, JAK1 kinase or STAT2 but not STAT1. Whereas Adar1 gene organization involving multiple promoters and alternative exon 1 structures was highly preserved, sequences of the promoters and exon 1 structures were not well conserved between human and mouse.
Journal of Molecular Biology, 1989
The Ada protein of Escherichia, coli catalyzes transfer of methyl groups from methylated DNA to its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, ada. Using an in vitro reconstituted system, we found that both the sigma factor and the methylated Ada protein are required for transcription of the ada gene. To elucidate molecular mechanisms involved in the regulation of the ada transcription, we investigated interactions of the non-methylated and methylated forms of Ada protein and the RKA polymerase holo enzyme (the core enzyme and sigma factor) with a DNA fragment, carrying the ada promoter region. Footprinting analyses revealed that t,he methylated Ada protein binds t,o a region from positions-63 to-31, which includes the ada regulatory sequence AAAGCGCA. No firm binding was observed with the nonmethylated Ada protein, although some DNase I-hypersensitive sites were produced in the promo& by both types of Ada protein. RNA polymerase did bind to the promoter once the methylat,ed Ada protein had bound to the upstream sequence. To correlate these phenomena with the process in ,uivo, we used the DKAs derived from promoter-defective mutants. ?l'o binding of Ada protein nor of RNA polymerase occurred with a mutant DNA having a C to G substitution at position-47 within the ada regulatory sequence. In the case of a-35 box mutant with a T to A change at position-34, the methylated Ada protein did bind to the ada regulatory sequence. yet there was no RNA polymerase binding. Thus, the binding of the methylated Ada protein to the upstream region apparently facilitates binding of the RNA polymerase to the proper region of the promot.er. The Ada protein possesses two known methyl acceptor sites, Cys69 and Cys321. The role of methvlation of each cysteinr residue was investigated using mutant forms of the Ada protein."The Ada protein with the cysteine residue at posit*ion 69 replaced by alanine was incapable of binding to the ada promoter even when the cysteine residue at position 321 of the protein was methylated. When the Ada protein wrth alanine at posit,ion 321 was methylated. it acquired the potential to bind to the ada promoter. These results arc compatible with the notion that methylation of the cysteine residue at position 69 causes a conformational change of the Ada protein, thereby faeilit,ating binding of the protein to the upstream regulatory sequence.
Journal of Molecular Biology, 2009
ADAR1 (adenosine deaminase acting on RNA) catalyses the deamination of adenosine to inosine on RNA substrates with double-stranded character. Here, we show that co-expression of ADAR1 in mammalian cells markedly increases plasmid-based gene expression in transfected cells. The enhanced expression was independent of the nature of the promoter (viral, cellular) used to drive gene expression, was independent of the protein reporter (luciferase, RRP) tested, and was independent of the human cell line examined (293T, HeLa). Exogenous protein levels were increased 20 to ~50-fold when ADAR1 was co-expressed, whereas RNA transcript levels changed less than 2-fold. The activation of PKR protein kinase and the phosphorylation of translation initiation factor eIF-2α seen following plasmid DNA transfection were both greatly reduced in ADAR1-transfected cells. Stable knockdown of the PKR kinase increased reporter gene expression in the absence, but not the presence of ADAR1 co-expression. Both size forms of ADAR1, the p150 inducible form and the p110-like constitutive form, enhanced plasmid-based gene expression. Taken together, these results indicate that the ADAR1 deaminase increases exogenous gene expression at the translational level by decreasing PKR-dependent eIF-2α phosphorylation.