Transcriptional Regulation Of ADAR1,ADAR1-like In Grass Carp(Ctenopharyngodon Idella)

ADAR1(adenosine deaminase acting on RNA 1), an IFN-induced protein, plays antiviral activity via its A-to-I RNA editing activity. In addition to the effects of A-to-I editing on RNA structure and function, as a dsRNA-binding protein, ADAR1 can participate in many biological processes including innate immune response by interacting with other dsRNA-binding proteins. In mammals, ADAR1 has many isoforms transcribed from different promoters and the well-known isoforms of ADAR1 are known as p150 and p110. p150 is the full length protein while p110 lacks Z-DNA binding domains(Z?). In our previous work, we have cloned and identified two isoforms of ADAR1 from grass carp(Ctenopharyngodon idella), named CiADAR1 and CiADAR1-like, respectively. The expressions of CiADAR1 and CiADAR1-like are significantly up-regulated under stimulation with poly I:C, indicating that they are involved in the antiviral immune response in cells. Further study shows that the 5'UTR of CiADAR1 and CiADAR1-like are different, suggesting that they are transcribed from different promoters.To further study the genome organizations of two isoforms of grass carp ADAR1, we identified the transcription start site(TSS) of CiADAR1 and CiADAR1-like by using 5'-Full RACE Kit with TAP. CiADAR1-like and CiADAR1 were generated from a single gene by alternative promoter usage and differential splicing, leading to the distinction in C-terminal deaminase domain.To study the transcriptional regulatory mechanisms of two isoforms of grass carp ADAR1, we cloned their promoter sequences according to the genomic sequence of grass carp ADAR1. CiADAR1 promoter was found to be 1080 bp in length and divided into two distinct regions, the proximal region(Pp) containing three putative interferon regulatory factor binding elements(IRF-E) and the distal region(Pd) containing only one IRF-E. CiADAR1-like promoter was found to be 1173 bp in length, containing eight IRF-E and three IFN-stimulated response elements(ISRE).In vitro, the results of gel mobility shift assays showed that the migration rates of CiADAR1 and CiADAR1-like promoter fragments were clearly blocked by the purified CiIRF1 and Ci IRF3 protein, indicating that Ci IRF1 and Ci IRF3 could be the potential transcriptional regulatory factor for CiADAR1 and CiADAR1-like.To further study the effects of Ci IRF1 and Ci IRF3 on promoter activity of CiADAR1 and CiADAR1-like in vivo, the recombinant plasmid of pGL3-ADAR1 pro and pGL3-ADAR1-likepro was constructed and transiently co-transfected into Ctenopharyngodon idella kidney(CIK) cells with pcDNA3.1-IRF1 or pcDNA3.1-IRF3, respectively. The results of luciferase assays showed that Ci IRF1 and Ci IRF3 significantly up-regulated the activity of luciferase, demonstrating that CiIRF1 and Ci IRF3 could activate the transcription of CiADAR1 and CiADAR1-like.For further study the roles of different promoter segments in CiADAR1 transcription, three different regions of CiADAR1 promoter luciferase reporter vector pGL3-ADAR1 Pp, pGL3-ADAR1 Pd and pGL3-ADAR1 Pf was constructed and transiently co-transfected into CIK cells with pcDNA3.1-IRF1, respectively. The results of luciferase assays in CIK cells indicated that the proximal region is the main regulatory region in CiADAR1 transcription.