Identification And Characterization Of A Constitutively Expressed Ctenopharyngodon Idella ADAR1 Splicing Isoform (CiDAR1a)

As one member of ADAR family,ADAR1(adenosine deaminase acting on RNA1)can convert adenosine to inosine within dsRNA.There are many ADAR1 splicing isoforms in mammals,including an interferon(IFN)inducible ~150 kD protein(ADAR1-p150)and a constitutively expressed ~110 kD protein(ADAR1-p110).The structural diversity of ADAR1 splicing isoforms may reflect their multiple functions.ADAR1 splicing isoforms were also found in fish.In our previous study,we have cloned and indentified two different grass carp ADAR1 splicing isoforms,i.e.CiADAR1 and CiADAR1-like,both of them are IFN-inducible proteins.In this paper,we identified a novel CiADAR1 splicing isoform gene(named CiADAR1a).CiADAR1 a gene contains 15 exons and 14 introns.Its full-length cDNA is comprised of a 5? UTR(359 bp),a 3? UTR(229 bp)and a 2952 bp ORF encoding a polypeptide of 983 amino acids with one Z-DNA binding domain,three ds RNA binding motifs and a highly conserved hydrolytic deamination domain.CiADAR1 a was constitutively expressed in C.idella kidney(CIK)cells regardless of Poly I:C stimulation by Western blot assay.In normal condition,CiADAR1 a was found to be present mainly in the nucleus.After treatment with Poly I:C,it gradually shifted to cytoplasm,but the molecular machanism is unclear.To further investigate the mechanism of transcriptional regulation of CiADAR1 a,we cloned and identified its promoter sequence.The transcriptional start site of CiADAR1 a is mapped within the truncated exon 2.CiADAR1 a promoter is 1303 bp in length containing 4 IRF-Es.In the present study,we constructed pcDNA3.1 eukaryotic expression vectors with IRF1 and IRF3 and co-transfected them with pGL3-CiADAR1 a promoter into CIK cells.The results showed that neither the over-expression of IRF1 or IRF3 nor Poly I:C stimulation significantly impacted CiADAR1 a promoter activity in CIK cells.Together,according to the molecular and expression characteristics,subcellular localization and transcriptional regulatory mechanism,we deduced that CiADAR1 a shared a high degree of homology with mammalian ADAR1-p110.Subsequently,through sequence blast we deduced the nuclear export signal(NES)of grass carp ADAR1 gene.Then we constructed pEGFP-C1 eukaryotic expression vectors withCiADAR1a(ADAR1a ORF),CiADAR1N(ADAR1 N-terminal,1-505aa),CiADAR1N?NES(ADAR1 N-terminal with defective nuclear export signal)and transient transfected them into smcc cells.In fluorescence microscope images,we found that CIK cells transfected with pEGFP-C1-ADAR1 a or pEGFP-C1-ADAR1 N or pEGFP-C1-ADAR1N?NES vector,the GFP protein were present in nucleus,both nucleus and cytoplasm,nucleus,respectively.According to the consequence,we determined that there is a NES sequence localize in the N-terminal of CiADAR1.Above data has shown that CiADAR1 is a constitutively expressed isoform of CiADAR1.