Characterization Of Four Genes In RIG-Ⅰ-like Receptor Signaling Pathway And MiRNA Transcriptome In Yellow Catfish(Pelteobagrus Fulvidraco)

Yellow catfish(Pelteobagrus fulvidraco)is an economically important freshwater fish in China,has been suffering an increasing damage from infectious diseases.Thus,a better understanding of the function and mechanism of the immune-related genes is necessary to make a disease control.Innate immunity is the first line of defense against pathogenic infections.Pattern recognition receptors(PRR)recognize a variety of pathogens and initiate specialized cytokine signaling to eliminate invading pathogens.Micro RNA(mi RNA)has emerged as essential post-transcriptional regulators of gene expression in a wide range of physiological processes including innate immune response.In this study,four key genes in the RIG-Ⅰ-like receptors(RLRs)signaling pathway of yellow catfish were identified,namely RIG-Ⅰ,LGP2,STING,and MAVS.Their tissue distributions and expression patterns following pathogen stimulation were determined and the subcellular localization was detected.Subsequently,their modulation of fish IFN promoters and NF-κB,as well as their antiviral effects were investigated.Additionally,mi RNA transcriptomes in response to Edwardsiella ictaluri and poly I:C were analyzed.The mi RNAs related to innate immune genes were characterized.1.Identification and functional studies of four genes in RLR pathway of yellow catfish1.1.Sequence characterization of RLR pathway genesRIG-Ⅰ,LGP2,STING and MAVS were cloned from yellow catfish.Sequence analysis showed that both RIG-Ⅰ and LGP2 harbour conserved Dex D/H helicase domain(DEXDc),RNA helicase domain(HELICc)and RD domain.However,RIG-Ⅰ contains two tandem repeat CARD domains in the N-terminus,while LGP2 does not.MAVS possesses a N-terminal CARD and a C-terminal transmembrane(TM)domain.STING comprises a CTD domain and a TM domain in the N-and C-terminus,respectively.1.2.Expression patterns of RLR pathway genesThe tissue-specific abundance of these genes in healthy yellow catfish was measured by q PCR.These four genes were widely distributed in the detected tissues including liver,spleen,head kidney,trunk kidney,heart,intestine,gill,and skin.RIG-Ⅰ and LGP2 were highly expressed in head kidney.STING and MAVS expressed at a high level in spleen and gill,respectively.Next,in-vivo modulations of these genes were measured in spleen and head kidney following stimulation with E.ictaluri,poly I:C,and SVCV(Spring viraemia of carp virus).Following poly I:C challenge,the expression levels of RIG-Ⅰ,LGP2,MAVS and STING in head kidney peaked at 72 h,12h,3h and 3h,respectively.In spleen treated with poly I:C,their expression reached maximum at 24 h.After E.ictaluri stimulation,RIG-Ⅰ displayed the highest expression at 6h,while LGP2,MAVS and STING showed the highest levels at 72 h in head kidney.In spleen induced by E.ictaluri,the expression of these genes reached the peak at 72 h.Upon SVCV infection,RIG-Ⅰ,LGP2,MAVS and STING exhibited the highest levels at 24 h,3h,24 h,and 72 h in head kidney.In spleen stimulated with SVCV,RIG-Ⅰ and STING peaked at 6h,while LGP2 and MAVS showed the highest expression at 12 h.1.3.Subcellular localization of RLR pathway genesTo validate their localization,GFP-tagged RIG-Ⅰ,LGP2,MAVS and STING were expressed in EPC cells.Laser scanning microscopy revealed that GFP fluorescence could only be detected in the cytoplasms,suggesting their intracellular localization.1.4.Effects of RLR pathway genes on the promoter activities of IFN and NF-κBTo identify the role these four genes in activating the signaling pathway,EPC cells were co-transfected with vectors expressing these genes and fish IFN promoter or NF-κB-driven luciferase reporter construct.Overexpression of RIG-Ⅰ enhanced the promoter activities of fish IFN1,IFN3,and NF-κB by 2.19-fold,11.4-fold and 50.0-fold,respectively.Similarly,the transfection of MAVS increased the promoter activities of IFN1,IFN3,and NF-κB by2.7-fold,5.9-fold and 25.7-fold.STING overexpression stimulated the promoter activities of IFN1,IFN3,and NF-κ B by 3.1-fold,56.8-fold and 14.6-fold.Furthermore,the dominant negative mutant of TBK1,IRF3,and IRF7 can inhibit the activation of IFN promoters by RIG-Ⅰ and STING.The result indicated that RIG-Ⅰ and STING may stimulate IFN via TBK1-IRF3/7.Finally,LGP2 negatively regulated RIG-Ⅰ-mediated IFN activation in the cells co-transfected with RIG-Ⅰ and LGP2.1.5.Antiviral effect of RLR pathway genesTo further determinate the role of these four genes in host immune response against viral infection,EPC cells were transfected with these genes and then infected by SVCV.At 48 h post infection,overexpression of RIG-Ⅰ,MAVS and STING reduced the viral titer 5,632-fold,17,782-fold,and 5,623-fold,respectively.2.Characterization of mi RNA transcriptome in response to E.ictaluri or poly I:C in yellow catfishMi RNA transcriptomes in spleen of yellow catfish challenged with E.ictaluri and poly I:C were characterized by high-through sequencing.In total,83 differently expressed mi RNAs and 1,932,192 novel mi RNA were found in the mi RNA transcriptome derived from E.ictaluri-infected yellow fish.106 differentially expressed mi RNAs and 3,209,927 novel mi RNA were identified in the mi RNA transcriptome in response to poly I:C.Moreover,mi RNA target predictions revealed the presence of 16 putative mi RNAs related to innate immune response,including RLR and TLR signaling pathways,complement and apoptosis.