Grass Carp (Ctenopharyngodon Idellus) Tarbp 2 (Trans-Activation-Responsive RNA-Binding Protein 2) Inhibits Apoptosis By Decreasing PKR Phosphorylation

TARBP2(Trans-Activation-Responsive RNA-Binding Protein 2)was originally identified as a dsRNA binding protein that binds to human immunodeficiency virus type 1(HIV-1)TAR RNA and promotes HIV-1replication.Later,it was found that PKR can bind directly to inhibit PKR activity.PKR plays a significant role in IFN antiviral responses and in mediating apoptosis.Its regulation is crucial for cellular antiviral and subsequent recovery.In mammalian cells,PACT(PKR activating protein)and TARBP2 play a key role in PKR activity regulation in a dsRNA independent manner.In previous studies we found that grass carp PACT can activate PKR,indicating that fish PKR also have dsRNA independent regulation.In this study,we dedicate to find out the function of grass carp TARBP2 on PKR regulation.First of all,we used Quantitative Real-time PCR(qRT-PCR)to detect the expression of grass carp(Ctenopharyngodon idella)TARBP2(CiTARBP2)in grass carp kidney(CIK)cells and grass carp tissues after poly I:C stimulation for different time.The expression of CiTARBP2 was significantly increased after poly I:C stimulation for 6h,suggesting that CiTARBP2 may play a role in poly I:C mediated immune response.Similarly,we also detected the expression levels of CiPKR and CiPACT in different grass carp tissues under poly I:C stimulation,and found that both of them were up-regulated to varying degrees.In order to investigate the relationship between PKR,TARBP2 and PACT in grass carp,we used subcellular localization to observe the positions of CiPKR,CiTARBP2 and CiPACT in CIK cells respectively.It was found that CiPKR and CiTARBP2 mainly distributed in cytoplasm,CiPACT existed in both the nucleus and cytoplasm.Then co-localization and immunoprecipitation experiments showed that TARBP2 could not only form heterodimers with PKR or PACT,but also form homologous dimer with itself,suggesting that TARBP2 plays an important role in PKR activity regulation.We also found that poly I:C promoted phosphorylation of CiTARBP2 and enhanced the interaction of CiTARBP2 and CiPKR,suggesting that activated CiTARBP2 binds to PKR to perform its function.Overexpression of CiTARBP2 decreased CiPKR phosphorylation and inhibited PKR-induced apoptosis,revealing its negative regulation mechanism on PKR.These results suggest that CiTARBP2 can directly or indirectly regulate PKR phosphorylation through protein-protein interaction to inhibit CiPKR activity and thereby inhibit apoptosis.