A splicing isoform of Ctenopharyngodon idella ADAR1 (CiADAR1-like): Genome organization, tissue specific expression and transcriptional regulation

• A splicing isoform of Ctenopharyngodon idella ADAR1 was cloned and identified.
• CiADAR1-like genome has 9 exons and 8 introns.
• CiADAR1-like promoter region contains 3 ISRE and 8 IRF-E.
• CiADAR1-like was significantly up-regulated after stimulation with poly I:C.
• Both IRF1 and IRF3 could activate the transcription of CiADAR1-like.

Catalyzing the deamination of adenosine to inosine in RNA, ADAR1 (adenosine deaminase that act on RNA 1) belongs to ADAR family. In our previous work, we have cloned the complete genomic sequence of ADAR1 from grass carp (Ctenopharyngodon idella), named CiADAR1. In the process, we found a splicing isoform of CiADAR1 (CiADAR1-like). CiADAR1 and CiADAR1-like are possessed by different promoters but share a common exon 2. The complete genomic CiADAR1-like has 9 exons and 8 introns. Its full-length cDNA is comprised of a 5′ UTR (417 bp), a 3′ UTR (118 bp) and a 3324 bp-long ORF encoding a polypeptide of 1107 amino acids. The deduced amino acid sequence of CiADAR1-like contains two Z-DNA binding domains, three dsRNA binding motifs and a truncate catalytic domain. CiADAR1-like shared higher homology with Danio rerio ADAR1 and lower homology with HsADAR1-like in phylogenetic tree. qRT-PCR showed that CiADAR1-like were ubiquitously expressed and significantly up-regulated after stimulation with Poly I:C. Its mRNA reached the peak at 12 h post-stimulation in all tested tissues. Western-blotting experiment proved CiADAR1-like was factually expressed in C. idella kidney (CIK) cells. To further study the transcriptional regulatory mechanism of CiADAR1-like, we cloned its promoter sequence. CiADAR1-like promoter is 1173 bp in length containing 3 ISRE and 8 IRF-E. Subsequently, 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. In vitro, CiIRF1 and CiIRF3 were able to bind to CiADAR1-like promoter with high affinity in gel mobility shift assays, revealing that IRF1 and IRF3 could be the potential transcriptional regulatory factors for CiADAR1-like. In vivo, Co-transfection of pcDNA3.1-IRF1 (or pcDNA3.1-IRF3) with pGL3-CiADAR1-like promoter into CIK cells showed that both IRF1 and IRF3 significantly increased the luciferase activity, suggesting that they play a positive role in CiADAR1-like transcription.