Molecular Cloning And Functional Study Of PRMT6 From Black Carp

Protein arginine methylation(PRMT)is a prevalent posttranslational modification.Recent research has shown that protein arginine methyltransferases 6(PRMT6)has been identified as a suppressor of TBK1/IRF3 in human and mammals,it is important family members of PRMTs.To explore the role of PRMT6 in teleost fish,In this study,the CDS sequence of PRMT6 from black carp(Mylopharyngodon piceus)was cloned and its function in the antiviral innate immune response of black carp was initially explored,The specific research contents and results are as follows:1.In terms of evolution and structure,The CDS sequence of black carp PRMT6(bcPRMT6)gene contains 1068 nucleotides and encods 355 amino acids;the molecular weight of the bcPRMT6 protein was predicted by Ex PASy software to be 39.61 KDa and the isoelectric point was 5.61.In evolutionary history,BcPRMT6 has the highest homology with goldfish PRMT6,reaching 96.6%;followed by zebrafish PRMT6,with96.3% homology.According to the amino acid sequence alignment,it is found that it has high homology with mammalian PRMT6.Conservation in structural evolution is mainly reflected in the methylethyltransferase(MTA)domain.2.In terms of protein and RNA level expression,qPCR experiments showed that the transcription level of bcPRMT6 in black carp kidney cells(MPK)responded to different stimuli,indicating that bcPRMT6 is involved in the host's innate immune response caused by viruses or bacteria.Immunoblot experiments showed that bcPRMT6 can be expressed in EPC cells and the protein migration size is about 43 k Da.Like its mammalian counterpart,bc PRMT6 has been identified to distribute majorly in the nucleus through the immunofluorescent stainingassay.3.In terms of functional regulation,bc PRMT6 shows little interferon(IFN)promoter-inducing activity in the reporter assay and bcPRMT6 shows no antiviral activity against either grass carp reovirus(GCRV)or spring viremia of carp virus(SVCV)in plaque assay.When co-expressed with bcPRMT6,the IFN promoter-inducing abilities of black carp TBK1(bcTBK1)and IRF3/7(bcIRF3/7)are fiercely attenuated.Accordingly,bcTBK1-mediated antiviral activity in EPC cells is obviously dampened by bcPRMT6.The interaction between bcPRMT6 and bcIRF3/7 has been identified by coimmunoprecipitation assay;however,no direct association between bcPRMT6 and bcTBK1 has been detected.4.Taken together,our data elucidates for the first time in teleost fish that PRMT6 suppresses TBK1-IRF3/7 signaling during host antiviral innate immune activation.