Analysis Of Transcriptional Regulation Of PRKRA And TARBP2 Mediated By IRF2 In Grass Carp(Ctenopharyngodon Idella)

PRKRA(interferon-inducible double-stranded RNA-dependent protein kinase activator A)is a cellular protein which can activate PKR in dsRNA-independent manner.TARBP2(transactivation response RNA binding protein 2)is a protein which inhibits PKR activity.IRF2 is a multifunctional transcription factor and plays a key role in the regulation of apoptosis and cell cycle.The existing results confirm that in addition to regulate the transcription of IFN and ISG genes,IRF2 also plays a role in the regulation of other cytokine genes.PRKRA and TARBP2 whcih plays a critical characteristic in the innate immune response by regulating PKR activity through a variety of pathways in mammals.Therefore,their transcriptional regulation has become a hot topic in related fields.To further explore of grass crap IRF2 regulating the transcription of PRKRA and TARBP2,In the present study,grass carp(Ctenopharyngodon idella)PRKRA full-length cDNA(named CiPRKRA,KT891991)and grass carp(Ctenopharyngodon idella)TARBP2 full-length cDNA(named CiTARBP2,KT891992)were cloned and identified.The full length of CiPRKRA cDNA is 1268 bp along with 36 bp of 5'UTR,350 bp of 3'UTR and the largest open reading frame(882bp)encoding a poly-peptide of 293 amino acids with a typical dsRNA binding motifs(dsRBM).The full length of CiTARBP2 cDNA is 2117 bp along with 215 bp of 5'UTR,861 bp of 3'UTR and the longest ORF(1041bp)encoding a poly-peptide of 346 amino acids with three typical dsRNA binding motifs(dsRBM).Phylogenetic tree analysis revealed that grass carp PRKRA and TARBP2 shared higher similarity with other fish species than to mammals.It distributed in the same branch,and the most nearest evolutionary relationship with Danio rerio.RT-PCR showed that all the expressions of CiPRKRA,CiTARBP2 and CiIRF2 have the same trend,the three expression levels were increased significantly in C.idella kidney(CIK)cells stimulated with poly I: C.After 12 h stimulation,the highest expression levels of the three are achieved,after that,the expression levels gradually were decreased.After 72 hours of stimulation,the expressions returned to the level of constitutively expression.These results indicate that PRKRA,TARBP2,and IRF2 all participate in antiviral responses in grass carp,and the three show a certain degree of synergy.In order to further study the IRF2 transcriptional regulation of PRKRA and TARBP2,the partial promoter sequence of CiPRKRA(1463 bp)containing one ISRE and the partial promoter sequence of CiTARBP2(1016 bp)containing three ISRE were cloned by Tail-PCR.Subsequently,CiIRF2 and Ci IRF2-nDBD(DNA-binding domain,DBD)were expressed and purified.In vitro,Ci IRF2 bound to CiPRKRA and CiTARBP2 promoters with high affinity by gel mobility shift assays,however,CiRF2-nDBD had no obvious blocking effect on CiPRKRA and CiTARBP2 promoter fragments,revealing that IRF2 might be a potential transcriptional regulatory factor for CiPRKRA and CiTARBP2.Dual-luciferase reporter assays were applied to further investigate the transcriptional regulations of CiPRKRA and CiTARBP2 in vivo.Recombinant plasmids of pGL3-PRKRAPro and pGL3-TARBP2 Pro were constructed and transiently cotransfected into CIK cells with pcDNA3.1-Ci IRF2,respectively.The results showed that CiIRF2 significantly decreased the luciferase activity of pGL3-PRKRAPro and increased the luciferase activity of pGL3-TARBP2 Pro,suggesting that it plays an egative role in CiPRKRA transcription and a positive role in CiTARBP2 transcription.