Fish Paralog Proteins RNASEK-a And-b Enhance Type Ⅰ Interferon Secretion And Promote Apoptosis

Type Ⅰ interferon is an essential component in the innate immune system.Once viruses infect host,pattern recognition receptors sense their pathogen-associated molecular patterns and activate downstream type Ⅰ interferon signaling pathways to protect host from viral diseases.Apoptosis is a form of programmed cell death and participates in antiviral response together with type Ⅰ interferon.RNASEK belongs to a conserved ribonuclease family,wildly expressing in nearly all tissues and developmental stages of all metazoans.It has been reported to modulate viral entries via interferon inducible gene LY6 E.In addition,RNASEK is related to multiple cancer progresses.In one study,RNASEK was significantly upregulated when paclitaxel induced cancer cell apoptosis.These evidences suggest that RNASEK is involved in the innate immune system and apoptosis.However,the regulation of RNASEK on type Ⅰ interferon secretion and apoptosis remains unclear.Grass carp(Ctenopharyngodon idella)is one of four traditional freshwater fish in China.Grass carp hemorrhage,as an epidemic induced by grass carp reovirus or small RNA virus,can kill 70%-80% of grass carp.Investigating the innate immune response of grass carp will provide direct theoretical basis for viral diseases.Meanwhile,fish innate immunity is similar to that of mammals and gradually attracts a lot of research.Therefore,in this study,we select grass carp as an experimental model to elucidate the regulation of RNASEK on type Ⅰ interferon and apoptosis.We first cloned two paralog genes RNASEK-a and-b in grass carp.The open reading frame of them is 966 bp and 1180 bp,respectively.The two coding regions have the same length of 306 bp composing of 3 exons with 73.27% of protein homology.RNASEK is conservative across species,suggesting it has important biological functions.To observe the distribution of RNASEK-a and-b in grass carp cells,subcellular localization was performed.RNASEK-a and-b are localized only in cytoplasm,and further fine localization showed that they localize in early and late endosomes and endoplasmic reticulum but rarely in mitochondria and lysosomes.We next stimulated grass carp kidney cells and tissues by using poly I:C(ds RNA)or grass carp reovirus.As a result of quantitative real-time PCR,both poly I:C and grass carp reovirus upregulated RNASEK-a and-b m RNA in vitro and in vivo,suggesting these two genes participate in the innate immune response.For validation of this hypothesis,we performed overexpression and knockdown of RNASEK-a and-b in grass carp kidney and ovary cells.Quantitative real-time PCR and western blot/immunofluorescence were used to test m RNA and protein expression of genes,respectively.Overexpression of RNASEK-a and-b individually promote type Ⅰinterferon expression,opposite to that observed in the knockdown experiments.Given previous study reported that IRF3/IRF7 phosphorylation can induce type Ⅰinterferon in grass carp,we tried to demonstrate whether RNASEK-a and-b enhance type Ⅰ interferon production via IRF3/IRF7.We overexpressed RNASEK-a and-b in grass carp kidney cells and observed that they upregulated the phosphorylation of IRF3 and IRF7.Hence,grass carp RNASEK-a and-b induce type Ⅰ interferon expression through activating IRF3 and IRF7.Apoptosis is of significance and closely related to the innate immune system.To investigate the regulation of RNASEK-a and-b on apoptosis,we conducted overexpression of RNASEK-a and-b in grass carp liver,kidney,and ovary cells.Several methods including Bax/Bcl-2 m RNA ratio,DNA Ladder,TUNEL stanning,and flow cytometry were used to evaluate the degree of cell apoptosis.Overexpression of RNASEK-a and-b individually increased Bax/Bcl-2 m RNA ratio,DNA fragmentations,TUNEL-positive cells,and Annexin V-positive cells.Thus,grass carp RNASEK-a and-b promote cell apoptosis.Our previous work proved e IF2α phosphorylation can induce apoptosis.To verify whether RNASEK-a and-b promote cell apoptosis via e IF2α,we overexpressed RNASEK-a and-b in grass carp kidney and liver cells.As results,grass carp RNASEK-a and-b enhanced the phosphorylation level of e IF2α.Therefore,grass carp RNASEK-a and-b promote apoptosis through activating e IF2α.Taken together,RNASEK is highly conservative across species and consist of two paralog proteins,RNASEK-a and-b,in fish.In grass carp,RNASEK-a and-b localize in cytoplasm(mainly in endosomes and endoplasmic reticulum)and widely express in nearly all tissues.Poly I:C(ds RNA)and grass carp reovirus can upregulate RNASEK-a and-b expression in grass carp cells and tissues.Interestingly,grass carp RNASEK-a and-b enhance IRF3/IRF7 phosphorylation-mediated type Ⅰ IFN production and promote e IF2α phosphorylation-induced cell apoptosis.Thus,our study reveals a previously unrecognized role of RNASEK as a new positive regulator of type I interferon and apoptosis,hence providing new insights or targets for treating related diseases.