Cloning, Expressions, Subcellular Localizations And Immune Functions Of SARM1and Its Splice Variants In Grass Carp

Grass carp Ctenopharyngodon idella is one of the most important aquaculture species inChina. However, grass carp reovirus (GCRV), which causes hemorrhagic disease infreshwater fish culture, seriously threats the production of this economic species in China.Act as a lower vertebrate, grass carp mainly depends on innate immunity to prevent infectionof pathogens, thus this study was to explore the relationship between the immune-relatedgenes and GCRV-induced immune pathways, which laying a foundation for breeding anddisease control in grass carp.Toll-like receptors (TLRs) can sense pathogen-associated molecular patterns (PAMPs),and activate immune-related pathways. Sterile alpha and Toll/IL-1R motif containing1(SARM1) is the last discovered Toll-like receptors (TLRs) adaptor. Ulike to the other fouradaptors which have activating functions, SARM1was demonstrated to negatively regulateToll/IL-1R domain-containing adaptor inducing IFN-β (TRIF)-dependent TLRs signaling inhuman. Despite of the highly conserved structure in evolution, SARM1acts as diverse rolesin different species, and the role of SARM1in teleost is still poorly understood. This studymainly focuses on the following aspects:1. Identification and characterization of Ctenopharyngodon idella SARM1(CiSARM1).By homology-based cloning,3’RACE and5’RACE methods, we obtained thefull-length cDNA and DNA of CiSARM1, and identified its two novel splice variantsCiSARM1s1and CiSARM1s. Then we analyzed the three genes by bioinformatics.The genomic sequence of CiSARM1is9563bp in length, containing5′-flankingsequence of1050bp, seven exons and six introns. CiSARM1has the conserved proteinstructure which contains two N-terminal ARM domains, two central SAM motifs, and aC-terminal TIR domain; CiSARM1s1and CiSARM1s2are transcribed by intron retentionmechanism, and only retain N-terminal ARM motifs. 2. Subcellular localizations of CiSARM1, CiSARM1s1and CiSARM1s2in CIK cells.Constructed three gene-EGFP fusion expression vectors, transfected into CIK cells, andlabeled with MitoTracker Red CMXRos and DAPI. Images were collected by confocal laserscanning microscope.Confocal microscopy detected that CiSARM1specifically co-localizes withmitochondria, CiSARM1s1locates mostly in mitochondria and fuzzily in nucleus, whereasCiSARM1s2distributes in the whole cell.3. The temporal expression profiles of CiSARM1, CiSARM1s1and CiSARM1s2invitro and in vivo.For tissue distribution analysis of mRNA expression,15tissues were sampled fromhealthy grass carp. For the viral challenge experiment, Head kidney and spleen were collectedat different time points post injection. For virus or PAMPs stimulations, CIK cells werecollected at different time points after treatment. Real-time fluorescent quantitative RT-PCR(qRT-PCR) was used to quantify mRNA expressions of CiSARM1, CiSARM1s1andCiSARM1s2.qRT-PCR demonstrated that all the three genes ubiquitously expressed in15investigated tissues and could be up-regulated post grass carp reovirus (GCRV) challenge,suggesting they involve in antiviral defense. Additionally, CiSARM1and CiSARM1s1, butnot CiSARM1s2, were responsive to bacterial pathogen-associated molecular patterns,implying CiSARM1and CiSARM1s1could regulate antibacterial response.4. The temporal expression profiles of CiSARM1, CiSARM1s1, CiSARM1s2andimmune related genes in three overexpression cells.Constructed three overexpression vectors, transfected into CIK cells, and establishedstable cell lines. Then we analysed the temporal expressions of CiSARM1, CiSARM1s1,CiSARM1s2, CiTRIF, CiMyD88, CiIPS-1, CiTRAF6, CiTBK1, CiIRF3, CiIRF7, CiIFN-Iand CiMx1in transgenic cells.Results indicated that CiSARM1s1and CiSARM1s2were mutually repressed, but bothof them promoted CiSARM1, suggesting CiSARM1was in a dominant position; moreimportantly, from the mRNA expressions of the mediators in IFN-I pathways, overexpressionof CiSARM1may inhibit GCRV-triggered IFN-I response through not only TRIF-, but alsoMyD88-and IPS-1-dependent pathways; CiSARM1and CiSARM1s1may selectively targetMyD88-and IPS-1-dependent signalings to reduce GCRV-triggered IFN-I induction.5. Antiviral activity assays.96-well plate staining method was used to observe the cytopathic effect (CPE), the plateswere photographed under a light box. For further confirming the antiviral function, GCRV VP4expression was examined in six stable transgenic cells at72h post GCRV challenge byqRT-PCR.Antiviral activity assays indicated that all the three genes could promote GCRV-inducedcell death.6. Verified by RNAi experiments.Three pairs of oligonucleotides for CiSARM1or CiSARM1s were synthesized, annealedand inserted to pCMV-EGFP-CMV-SV40to generate shRNAs. Then transfected them intoCIK cells, and established stable cell lines. We examined the mRNA expressions of immunerelated genes and antiviral activity assays.RNAi experiments demonstrated that the three genes inhibited GCRV-triggered IFN-Iresponse and promoted GCRV-induced cell death again.Thus, CiSARM1possesses not only immune involvements in species of otherevolutionary status but also specific participation in fish, meantime alternative splicing mayprovide crucial mechanisms to assist CiSARM1in preventing excessive activation of the hostimmune response, these findings uncover the regulatory mechanisms of SARM1in teleostand serve the further functional and evolutionary researches on SARM1.