Human RNA-specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible

Abstract

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 approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-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 PC and PI. Exon 1B transcripts were initiated from the PC 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 PI 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 approximately 110-kDa ADAR1 protein (exon 1B) or the interferon-induced approximately 150-kDa ADAR1 protein (exon 1A).

Figure 1

Nucleotide sequence of the ADAR1 5′-RACE-derived cDNAs and comparison to corresponding genomic DNA sequences. Sequences of the two unique 5′-RACE-derived cDNAs are designated 5′-RACE-1A and 5′-RACE-1B. The previously described exon 1 (14) corresponds to the 5′-RACE-1A sequence, with the ATG translation initiation codon in bold font. The newly identified exon 1 corresponds to the 5′-RACE-1B sequence. The bold sequence shown by the lower lines corresponds to the 5′ portion of exon 2; the position of the exon 2 nested minus primer at nucleotides 48 to 67 is indicated by the underlined sequence. Nucleotide numbering begins with 1 at the 5′ end of each exon. Genomic DNA sequence was obtained from a subclone of the λ176 genomic clone for exon 1A and exon 2, and from a subclone of the P1–249 genomic clone for exon 1B (see Fig. 2B).

Figure 2

Genomic organization of the human ADAR1 gene. (A) Organization of the exons and introns within the 5′ region of the ADAR1 gene. The 5′-most seven exons are indicated to scale by filled boxes numbered 1B to 6; introns and the 5′ flanking regions are indicated by the solid lines. P_I corresponds to the IFN-inducible promoter upstream of exon 1A. The entire gene spans approximately 39 kb in length and contains 16 exons including exon 1B. Shown to scale are the P1-phage genomic clone P1–249, the overlapping λ-phage genomic clone λ176, and the 5′-portion of the overlapping P1 clones P1–652 and P1–959. The restriction map shows cleavage sites for BamHI (B) and XhoI (X) spanning the ≈60-kb region upstream of exon 6. For the 5.8-kb BamHI fragment that includes exon 1B, cleavage sites for AvaI, HindIII, and XbaI are included; no PstI sites are present in the fragment. (B) Southern gel-blot of P1- and λ-phage genomic clones, either uncut or digested with BamHI, HindIII, or PstI as indicated. The probe was ³²P-labeled exon 1B.

Figure 3

Quantitative RT-PCR analysis of ADAR1 mRNA expression in human amnion U cells. RNA isolated from monolayer cultures of U cells either left untreated or treated with IFN-α for 24 h was analyzed by RT-PCR as described in Experimental Procedures. (A) Schematic showing the locations of the alternative exons 1A and 1B and exon 2, with exons denoted by boxes and introns by solid lines; the exon-specific oligomer primers are denoted by arrows. (B) PCR products fractionated by gel electrophoresis after the indicated number of amplification cycles. cDNA templates were prepared by using RNA prepared from either IFN-α treated or untreated U cells. PCR reactions contained 0.25 μM of primer, by using the primer pairs E1B plus 12 and ³²P-labeled E2 minus 273, E1A plus 170 and ³²P-labeled E2 minus 273, or β-actin plus and minus primers as indicated.

Figure 4

Northern gel-blot analysis of ADAR1 mRNA expression in human amnion U cells. Northern gel-blot analysis of RNA from human U cells. Details are as described in Materials and Methods. The probe was ³²P-labeled exon 1A-exon 2 junction (16) or ³²P-labeled exon 1B. The positions of the two ribosomal RNAs are indicated on the sides of the blots. Lanes 1 and 3–5, RNA from untreated cells; lanes 2 and 6–8, RNA from cells treated with IFN-α for 24 h. Samples of 6 μg (lanes 1–3, 6), 12 μg (lanes 4, 7), and 24 μg (lanes 5, 8) of total RNA were analyzed.

Figure 5

Identification of a constitutively active ADAR1 gene promoter ≈9-kb upstream of the interferon-inducible promoter. Genomic DNA restriction fragments derived from the 5.8-kb BamHI fragment (Fig. 2) that includes exon 1B were inserted into the promoter-less pCAT-Basic plasmid. Promoter activities observed in human U cells transfected with the indicated CAT reporter plasmids are shown as percent of the conversion of [¹⁴C]chloramphenicol to the acetylated derivatives. Open bars refer to cells left untreated, and hatched bars refer to cells treated with IFN-α. pCAT-Control, the CAT reporter gene linked to the simian virus 40 promoter and enhancer; pCAT-Basic, the promoter-less plasmid vector without inserted human genomic DNA; pCAT-0.6Pi (S/X), the IFN-inducible P_I promoter present within the 591-bp Sac-Xho genomic DNA fragment inserted into the CAT-Basic plasmid (16).

Figure 6

Genomic DNA sequence of the promoter Pc region of the human ADAR1 gene. The sequence of the promoter region as well as exon 1B and the 5′-sequence of intron 1B are shown. The nucleotide numbers of the genomic DNA sequence upstream of the AvaI site present in exon 1B are relative. The sequence designated as exon 1B corresponds to nucleotides 1 to 107 of the cDNA as defined by 5′-RACE and shown in Fig. 1. Potential transcription factor binding sites as described in the text are shown.

Figure 7

Schematic summary of the organization of the human ADAR1 gene possessing alternative exon 1 structures of transcripts that initiate from the Pc and P_I promoters. (A) Organization of the promoter region of the ADAR1 gene. Pc, the constitutively active promoter that initiates transcription with exon 1B; P_I, the interferon-inducible promoter that initiates transcription with exon 1A. (B) Schematic representation of the ORFs of the ADAR1 transcripts. Transcripts with the alternative exon 1A possess a 1,226 amino acid-deduced ORF that begins at AUG1 (methionine 1) present in exon 1A and ends at an UAG in exon 15 (16). Transcripts with the alternative exon 1B possess a 931 amino acid-deduced ORF that begins at AUG296 (methionine 2) present in the unusually large (1,586 nt) exon 2 and terminate at the UAG in exon 15. R, dsRNA-binding domain.