Genome organization and transcriptional regulation of Adenosine Deaminase Acting on RNA gene 1 (ADAR1) in grass carp (Ctenopharyngodon idella).

ADAR1, involved in A-to-I RNA editing, belongs to adenosine deaminase acting on RNA (ADAR) family. A-to-I RNA editing is the most widespread editing phenomenon in higher eukaryotes. In the present study, we cloned and identified the full-length cDNA, complete genomic sequence and the promoter sequence of grass carp (Ctenopharyngodon idella) ADAR1 (CiADAR1) by homology cloning strategy and genome walking. CiADAR1 full-length cDNA is comprised of a 5'UTR (43  bp), a 3'UTR (229 bp) and a 4179 bp ORF encoding a polypeptide of 1392 amino acids. The deduced amino acid sequence of CiADAR1 contains two Z-DNA binding domains, three dsRNA binding motifs and a conserved catalytic domain. The complete genomic CiADAR1 has 16 exons and 15 introns. Phylogenetic tree analysis revealed that CiADAR1 shared high homology with Danio rerio ADAR1 (DrADAR1). RT-PCR showed that CiADAR1 were ubiquitously expressed and significantly up-regulated after stimulation with poly I:C. In spleen and liver, CiADAR1 mRNA reached the peak at 12 h and maintained the highest level during 12-24 h post-injection. CiADAR1 promoter was found to be 1102 bp in length and divided into two distinct regions, the proximal region containing three putative interferon regulatory factor binding elements (IRF-E) and the distal region containing only one IRF-E. To further study the transcriptional regulatory mechanism of CiADAR1, grass carp IRF1 (CiIRF1) and IRF3 (CiIRF3) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. Then, gel mobility shift assay was employed to analyze the affinity of CiADAR1 promoter sequence with CiIRF1 and CiIRF3 in vitro. The result revealed that CiIRF1 and CiIRF3 bound to CiADAR1 promoter with high affinity, indicating that IRF1 and IRF3 could be the potential transcriptional regulatory factor for CiADAR1. Co-transfection of pcDNA3.1-IRF1 (or pcDNA3.1-IRF3) with pGL3-CiADAR1 into C. idella kidney (CIK) cells showed that both IRF1 and IRF3 played a positive role in CiADAR1 transcription. In addition, the mutant assay revealed that the proximal region of CiADAR1 promoter is the main regulatory region in CiADAR1 transcription.

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