Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp (Ctenopharyngodon idella) PKR.

Abstract

The protein kinase R (PKR) can inhibit protein translation and lead to apoptosis under the circumstances of virus invasion and multiple other stress conditions. PKR is a dsRNA binding protein with a dsRBD and a kinase domain (KD). dsRBD is mostly composed of two (in mammal PKR) or three (in some fish PKR) dsRNA binding motifs (dsRBMs). Multiple sequences alignment and Phylogenetic analysis indicate that the three dsRBMs of fish PKR share analogous structure but show to be divergence origination. In this study, we have identified and analyzed the three dsRBMs from grass carp (Ctenopharyngodon idellus) PKR (CiPKR), which was cloned previously in our laboratory. dsRBMs of CiPKR have two or three conserved regions involved in dsRNA binding. Among the three dsRBMs, dsRBM1 was peculiar to some fish PKRs, while dsRBM2 and dsRBM3 were closely related to the dsRBM1 and dsRBM2 of mammal PKRs respectively. Dimerization assay indicated that dsRBM1 and dsRBM2 formed not only homo-dimer but also homo-multimer; whereas dsRBM3 formed merely homo-dimer. Meanwhile, dsRBM1-2, dsRBM2-3 and dsRBM1-2-3 could homo-dimerize and homo-multimerize also. Poly I:C pull-down assay showed that the binding of dsRBM to Poly I:C needed two or three dsRBMs to cooperate invitro, meaning one dsRBM from CiPKR could not bind to dsRNA efficiently. To further investigate the effect of dsRBM on the function of CiPKR, we constructed pcDNA3.1/CiPKR-wt and a series of CiPKR mutants recombined plasmids including pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1. The recombined plasmids respectively were co-transfected with plasmid PGL3 promoter into CIK cells. In comparison with the control group, the luciferase translation inhibitions were 78.7%, 15%, 0, 0.5%, 61.8%, 67.3% respectively. The results indicated that the protein translation inhibition caused by CiPKR mutants with only one dsRBM were very weak, while those with two or three dsRBMs inhibited the protein translation powerfully. Cell viability were 34.2%, 98.2%, 112%, 108%, 50.3%, 47.5% respectively after transfected with pcDNA3.1/CiPKR-wt, pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1 in order into CIK cells for 48h. The results from cell counting also indicated that transfection of CiPKR-wt and the mutants CiPKR-ΔdsRBM3, CiPKR-ΔdsRBM1 could inhibit the protein translation and facilitated the decrease of CIK cells number. In conclusion, our observations suggested that two dsRBMs ranking in tandem at N terminal were essential for the function of CiPKR, and the presence of the extra dsRBM1 enhanced its function.