Grass carp (10–20 g, Nanchang Shenlong Fisheries Development Co., Ltd., Jiangxi, China) were acclimatized in a specific pathogen-free animal tank for at least 2 weeks at 28°C prior to the experiments. For each experiment, three different fishes were killed to obtain the mixed sample.

Three stable cell lines, C. idella kidney (CIK), ovary (CO), and liver (CL, L8824) cells, were used in this study. CIK and CO cells were a gift from Professor Pin Nie (Institute of Hydrobiology, Chinese Academy of Sciences). CL cells were provided by Professor Jinnian Li (Anhui Agricultural University, China). Keeping in view CO cells were sensitive to the alteration of pH but CIK and CL cells were not, they were grown in different conditions. CIK (25) and CL (26) cells were cultured in Medium 199 (Corning, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 0.6% penicillin–streptomycin liquid (Beijing Solarbio Science & Technology Co., Ltd., China) at 28°C. CO (25) cells were grown in the same condition as that of CIK cells except for the addition of 5% CO₂. These cell lines were used for functional and confocal assays.

Genomic DNA and total RNA were extracted from grass carp tissues (50 mg for each tissue) by using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (Takara Bio, China) and RNA simple Total RNA Kit (Tiangen Biotech, China), respectively. The RNA sample was synthesized into cDNA by use of the PrimeScript RT reagent Kit with gDNA Eraser Perfect Real Time (Takara Bio, China). Homologous primers RNASEK-a-ORF-F/R and -b-ORF-F/R were designed according to the CDS of Carassius auratus RNASEK-a (XM_026267089) and RNASEK-b (XM_026258901). The above cDNA templates were used to perform polymerase chain reaction (PCR) with specific primers, the programs of which were 95°C for 3 min followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and then a final elongation at 72°C for 10 min. The PCR products were observed through agarose gel electrophoresis. Target DNA fragments were recycled from agarose gels, then cloned into pEASY-T1 vectors (Takara Bio, China), and finally confirmed by DNA sequencing (Tsingke, Beijing, China). Genomic DNA was subjected to the same cloning method as that of total RNA in the absence of reverse transcription. Thus, the CDS region and genome of both RNASEK-a and -b were obtained. The genomic structure of RNASEK from various taxa was compared. Sequence similarity analysis was conducted by Nucleotide BLAST of NCBI (http://www.ncbi.nlm.nih.gov/blast). Protein domains were predicted by the SMART program (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1). According to the amino acid sequences of the RNASEK family, multiple sequence alignment was performed via BioEdit software, and the phylogenetic tree was constructed by using the neighbor-joining algorithm from MEGA6.0 program. The primers used are listed in Table S1 . GenBank accession numbers of selected RNASEK orthologs from various species are listed in Table S2 .

PcDNA3.1, p3×FLAG-Myc-CMV™-24, pEGFP-C1, pDsRed2-C1, pGPU6/Neo, and PLKO-U6-PGK-mCherry (RFP)-2A-NEO (G418) plasmids were purchased from Invitrogen (USA), Transgen (Beijing, China), Promega (USA), BioVector (Chongqing, China), GenePharma (Suzhou, China), and Tsingke (Beijing, China), respectively. The CDS regions of grass carp RNASEK-a and -b were separately inserted into pcNDA3.1, pEGFP-C1, and p3×FLAG-Myc-CMV™-24 vectors to construct recombinant plasmids. Additionally, grass carp TBK1 CDS (MH545564.1) was used to obtain pcDNA3.1-TBK1 recombinant. The CDS regions of grass carp Rab5 (MF598473.1) and Rab7 (MF598474.1) were individually constructed into pDsRed2-C1 vectors (finished by Sangon Biotech, China). Subsequently, short hairpin RNA (shRNA) was designed. The shNC and shRNASEK-b-22 were cloned into pGPU6/Neo plasmids (named GP-shNC and GP-shRNASEK-b-22) as well as another shNC, shRNASEK-b-120, and shRNASEK-b-226 were inserted into PLKO-U6-PGK-mCherry (RFP)-2A-NEO (G418) vectors. The constructed recombinants pcDNA3.1-RNASEK-a/b, pEGFP-RNASEK-a/b, and pcDNA3.1-TBK1 were used for overexpression experiments. The recombinant plasmids pEGFP-RNASEK-a/b, p3×FLAG-RNASEK-a/b, and pDsRed2-Rab5/7 were utilized for subcellular localization assays. The shRNA recombinants GP-shNC, GP-shRNASEK-b-22, shNC, shRNASEK-b-120, and shRNASEK-b-226 were used to perform knockdown of RNASEK-b in target cells. All recombinant plasmids were confirmed via DNA sequencing (Tsingke, Beijing, China). The primers and shRNA used are listed in Table S1 .

Cells were seeded to six-well culture plates (Guangzhou Jet Bio-Filtration Co., Ltd., China) and 0.17 mm glass-bottom culture dishes (Wuxi NEST Biotechnology Co., Ltd., China) to grow. When reaching 60%–80% of confluence, cells were transfected with 2 or 1.5 μg of recombinant plasmids with 4 or 3 μl Lipofectamine 2000 (Invitrogen, USA, 11668019) or Lipo8000™ (Beyotime Biotechnology, China) Transfection Reagent for each sample, respectively. For the knockdown experiments, the negative control RNA oligonucleotides (termed NC) and specific siRNA against RNASEK-a-22 and -101 (termed siRNASEK-a-22 and -101) were designed by Suzhou GenePharma ( Table S1 ). When cells grew to 60%–80% confluence on six-well plates, 5 μl of 20 μmol/L siRNA was transfected using an equal volume of Lipofectamine 2000 or Lipo8000™ Transfection Reagent. Twenty-four hours post-transfection, transcriptional expression analysis, immunofluorescent staining, and subcellular localization were performed. Thirty-six hours after transfection, apoptosis-related assays and Western blot were conducted. The transfection methods were according to the instructions of the manufacturer. The siRNA used is listed in Table S1 .

CIK cells were used for subcellular localization and immunofluorescence. Cells were seeded to 0.17 mm glass-bottom culture dishes. When reaching 60%–80% of confluence, cells were transfected with pEGFP-RNASEK-a or -b; to investigate whether RNASEK-a/b localizes to endosomes, pEGFP-RNASEK-a and -b were individually co-transfected with the endosome markers pDsRed-Rab5/7. Twenty-four hours post-transfection, cells were fixed with 4% paraformaldehyde (Bio-Medical Assay Co., Ltd., China) for 10 min and dyed with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, 10 μg/ml; Sangon Biotech, China) for 20 min in darkness. In addition, MitoTracker^® Red CMXRos (Cell Signaling Technology, USA, 9082S), Lyso-Tracker Red (Beyotime Biotechnology, China, C1046), and ER-Tracker Red (Beyotime Biotechnology, China, C1041) were used to trace the mitochondria, lysosomes, and endoplasmic reticulum after cells were transfected with pEGFP-RNASEK-a or -b to observe the relationships between each other, the protocols of which were based on the instructions of the manufacturer. To examine whether RNASEK-a is colocalized with RNASEK-b, cells were transfected with p3×FLAG-RNASEK-a and pEGFP-RNASEK-b together. After 24 h of transfection, cells were fixed as above and permeabilized with 0.5% Triton X-100, then blocked with 5% bovine serum albumin, followed by incubation with the FLAG-tag antibody (1:5,000, Sigma, USA, F1804) (27) for 12 h (overnight) at 4°C. The next day, cells were incubated in the secondary antibody goat anti-mouse Cy3 for 1 h (1:200, BioLegend, USA, 405309) and counterstained with DAPI for 20 min without light. To assess the alteration of type I IFN protein expression, immunofluorescence was performed with type I IFN antibody (1:200, saved in our lab) (28) as that of FLAG-tag, in which the secondary antibody was substituted with donkey anti-rabbit Cy3 (1:200, BioLegend, USA, 406402). All finished preparations were photographed under 63× oil immersion objective lens (630× magnification) or 200× magnification of the Zeiss confocal microscope (confocal microscope LSM800, Zeiss, Germany) and analyzed by the Zen software (Zeiss, Germany).

Healthy grass carp were intraperitoneally injected with poly I:C (5 mg/kg, Sigma, USA) or 100 μl of live grass carp reovirus-097 (GCRV-097, 1 × 10⁻⁸ TCID₅₀; provided by Jian-Guo Su lab of Huazhong Agricultural University). Total RNA of each tissue (50 mg) was extracted from the brain, eye, intestine, gill, skin, spleen, liver, and kidney of grass carp at 6, 12, 24, 48, and 72 h post-stimulation, then cDNA was prepared as above. For the poly I:C group, only 6, 12, and 24 h were included because in our work we observed the expression of RNASEK-a and -b elevated after poly I:C stimulation and then declined almost to basal levels in most tested tissues, suggesting these two genes/proteins generated an early response to poly I:C in vivo.

CIK cells were seeded on six-well culture plates, then transfected with 5 μl poly I:C (1 mg/ml) or infected with 50 μl live GCRV-097 (1 × 10⁻⁸ TCID₅₀) using 5 μl Lipofectamine 2000 Transfection Reagent. Total RNA of 1 × 10⁶ cells was extracted at 0 h (without treatments) and 6, 12, 24, 48, and 72 h post-stimulation for each group. The obtained RNA was used to synthesize cDNA as above.

The prepared cDNA templates were utilized to perform expression analysis of RNASEK-a and -b in grass carp tissues and CIK cells by using quantitative real-time PCR (qRT-PCR). qRT-PCR was carried out in triplicate for each template with TB Green Premix Ex Taq II (TaKaRa Bio, China) on CFX Connect™ (Bio-Rad, USA). β-Actin was used as an internal control. All data were calculated with the 2^−ΔΔCT method to obtain relative normalized expression of genes, such as RNASEK-a, RNASEK-b, type I IFN, Bcl-2, and Bax. The primers used are listed in Table S1 .

CL or CO cells were inoculated in six-well culture plates to grow to 60%–80% of confluence, then transfected with expression plasmids and shRNA for overexpression and knockdown of target genes, respectively. Thirty-six hours post-transfection, cells were lysed in 100 μl of NP40 lysis buffer (Beyotime Biotechnology, China) with the addition of 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF, Sangon Biotech, China), 1 μg/ml leupeptin (Sangon Biotech, China), 1 μg/ml aprotinin (Sangon Biotech, China), and 1 mmol/L phosphate inhibitor cocktail (CWBIO, Beijing, China). Cell lysates were aspirated into a 1.5-ml tube and subjected to centrifugation at 12,000 r/min for 10 min at 4°C to remove cell debris. The acquired supernatant was boiled together with a quarter volume of 5× SDS-loading buffer containing 3.5% β-mercaptoethanol at 95°C for 10 min. The protein samples were separated through SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk for 1 h and incubated in the specific antibodies for 12 h (overnight) at 4°C. The next day, the membranes were incubated in horseradish peroxidase-conjugated secondary antibody for 1 h at a regular temperature. Finally, the membranes were exposed using a chemiluminescence imaging system (CLINX, China). Specific antibodies used here are against grass carp type I IFN (1:800, saved in our lab) (28), phosphorylated IRF3 (p-IRF3; 1:1,000, Beyotime Biotechnology, China, AF1594) (27), basal and phosphorylated IRF7 (p-IRF7; 1:1,000, HuaBio, China, ET1610-89) (29), eIF2α (1:500, saved in our lab) (30), phosphorylated eIF2α (p-eIF2α; 1:1,000, Abcam, UK, ab32157) (30), and GAPDH (loading control; 1:10,000, saved in our lab) (30). All commercial antibodies against p-IRF3, IRF7/p-IRF7, and p-eIF2α cross-react with grass carp, which is confirmed by transfection of pEGFP-IRF3, -IRF7, and -eIF2α into grass carp cells followed by comparison of the size of the basal and GFP recombinant proteins of each gene in our previous work (27, 29, 30).

CIK, CL, or CO cells were used to test cell apoptosis via four methods, namely, qRT-PCR, DNA ladder (DNA fragmentations of whole multiples of 180–200 bp), TdT-mediated dUTP nick-end labeling (TUNEL), or flow cytometry. CIK, CL, and CO cells were inoculated in six-well culture plates to reach 60%–80% of confluence, then transfected with recombinant plasmids or siRNA/shRNA for 36 h. Total RNA was extracted from CIK and CL cells to perform qRT-PCR. The ratio of Bax to Bcl-2 mRNA expression (Bax/Bcl-2 mRNA ratio) was used to assess cell apoptosis. Total DNA was extracted from CIK and CL cells and separated by agarose gel electrophoresis to observe the DNA ladder which reflects the degree of cell apoptosis. In addition, CIK cells were subjected to TUNEL staining by using an inverted fluorescence microscope (Zeiss, Germany) and One-Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, China, C1086) according to the instructions of the manufacturer, in which apoptotic cells are marked by TUNEL-positive green signals. For CO cells, the degree of apoptosis was determined by a flow cytometer (FACSVerse flow cytometer, BD, USA) and Annexin V, FITC Apoptosis Detection Kit (Dojindo, Japan, AD10), the protocols of which were based on the instructions of the manufacturer. Subsequently, the proportion of Annexin V-positive cells was analyzed for each sample through FlowJo vX.0.7 software.

All values were analyzed as mean ± SD. Statistical analysis was performed by the two-sample t-test and shown with figures produced by GraphPad Prism Version 8. The significance level used was P <0.05.

For both grass carp RNASEK-a and -b, the CDS consists of 306 bp encoding a polypeptide of 101 amino acids with two transmembrane domains and four Cys residues ( Figure S1A ). To investigate the conservation of the RNASEK family members, multiple sequence alignment was conducted according to their amino acid sequences. Both grass carp RNASEK-a and -b are highly conserved across 10 selected species, consisting of 8 mammals and 2 fish, especially with the matched genes from zebrafish ( Figure S1B ). For further analysis of the evolutionary status of RNASEK family members, the phylogenetic tree was constructed. As shown in Figure S2 , RNASEK exists in nearly all metazoans. Mammalian ones are extremely consistent with each other. RNASEK-a and -b paralogs are distinguished only in bony fish. RNASEK-b orthologs are located on only one branch, displaying a closer genetic relationship than RNASEK-a ones which are divided into three different branches. Taken together, the RNASEK family is conservative from invertebrates to mammals, suggesting that it plays a key role in biological processes.

The comparison of RNASEK gene structure was performed previously (3). However, in recent years, a plethora of RNASEK family genes were newly discovered in various taxa, including fish RNASEK-a and -b. To understand the gene structure of the RNASEK family more completely, we searched for RNASEK homologs from 22 animal species (including mammals, bony fish, birds, amphibians, turtles, and arthropods) by using NCBI (https://www.ncbi.nlm.nih.gov/) to compare their intron/exon structure. In general, the genome of RNASEK is composed of three exons with the exception of two exons for RNASEK of Phasianus colchicus and Chelonoidis abingdonii as well as four exons for RNASEK-a of Seriola dumerili, Monopterus albus, and Micropterus salmoides. The length of the RNASEK genome is highly divergent, ranging from 421 to 12,730 bp, and the reason for this is a higher degree of variability in introns than exons. From primitive animals to human, the size, positions, and numbers of exons are relatively conserved, of which exons 1, 2, and 3 individually contain 78, 77, and 142 bp for all examined mammalian RNASEK and 78, 83, and 145 bp for all listed fish RNASEK-b and one half of the fish RNASEK-a. As for the remaining three fish RNASEK-a, exons 1, 2, 3, and 4 individually contain 75, 80, 124–127, and 18 bp, respectively. Other animal RNASEK genomes have two or three exons with high conservation in the same taxon, and in particular, the size of exon 1 is highly conserved in all listed species (ranging from 69 to 85 bp) ( Figure S3 ). However, the length of intronic regions of the RNASEK genome varies much from 66 to 11,781 bp, which is related to taxonomic groups. For instance, there is a slight difference from each other in mammalian ones, from 250 to 3,714 bp in fish RNASEK-b, from 102 to 466 bp in fish RNASEK-a that consisted of three exons, from 494 to 3,650 bp in another half of fish RNASEK-a, approximately 66 bp in Drosophila melanogaster RNASEK, and from 649 to 11,781 bp in that of Petromyzon marinus ( Figure S3 ). Thus, the variations of RNASEK homolog genes, to a large degree, are attributed to the differences in their intronic architecture. Additionally, fish RNASEK-a has two forms of composition but only one for RNASEK-b, which is coincident with the results from the above phylogenetic tree.

To assess the expression profiles of grass carp RNASEK-a and -b via qRT-PCR in CIK cells, we cultured them in the context of stimulation of poly I:C and GCRV-097 for the indicated time periods (0, 6, 12, 24, 48, and 72 h). Both RNASEK-a and -b transcripts were significantly elevated after each stimulation and peaked at 24 h (up to 2.25–9.04-fold versus 0 h), and then declined ( Figures 1A–D ), the trends of which were similar with those of type I IFN ( Figure S4 ).

To perform the expression analysis of RNASEK-a and -b in grass carp tissues by qRT-PCR, healthy grass carp were intraperitoneally injected with poly I:C and GCRV-097 for various time periods (0, 6, 12, 24, 48, or 72h). RNASEK-a mRNA expression was the highest in the liver and nearly the lowest in the brain and eye for both stimulation ( Figures 1E, F ). In the poly I:C group, RNASEK-a transcripts increased after treatments except for the skin and almost peaked at 6, 12, or 24 h for different tissues ( Figure 1E ). In the GCRV group, RNASEK-a mRNA expression was upregulated post-stimulation in the kidney, brain, and eye and peaked at 12, 6, and 48 h, respectively ( Figure 1F ). The expression patterns of RNASEK-b were investigated in the same way as those of RNASEK-a. RNASEK-b expression reached the highest in the brain and very low in the kidney, spleen, intestine, gill, and skin. In the poly I:C and GCRV groups, the transcriptional levels of RNASEK-b elevated post-treatments apart from the skin (GCRV group), which peaked at 6, 12, 24, or 48 h in different tissues ( Figures 1G, H ). Besides, both genes were widely expressed in all tested tissues and their expression levels declined from the peak ( Figures 1E–H ).

In summary, dsRNA poly I:C and GCRV can upregulate RNASEK-a and -b expression in vitro and in vivo.

To dissect the function of grass carp RNASEK-a and -b, we performed subcellular localization of them in CIK cells. We first traced the RNASEK-a and -b through separately overexpressing the recombinants pEGFP-RNASEK-a and -b in CIK cells. As shown in Figures 2A, B , RNASEK-a and -b (green) existed only in the cytoplasm. Afterward, we expressed p3×FLAG-RNASEK-a (red) and pEGFP-RNASEK-b (green) together and observed that they were colocalized with each other (yellow) in the cytoplasm ( Figure 2C ). To investigate the fine localization of RNASEK-a and -b, pEGFP-RNASEK-a and -b were individually transferred into the cells together with the recombinants pDsRed2-Rab5 (early endosomal marker, red) or -Rab7 (late endosomal marker, red). Interestingly, both RNASEK-a and -b had colocalization (yellow) with these two endosome markers ( Figures 2D, E, I, J ). Furthermore, CIK cells were stained with MitoTracker (red), Lyso-Tracker (red), and ER-Tracker (red) after transfection of pEGFP-RNASEK-a and -b, demonstrating that both RNASEK-a and -b mainly localized to the endoplasmic reticulum but rarely to the mitochondria and lysosomes ( Figures 2F–H, K–M ). In brief, grass carp RNASEK-a and -b have similar subcellular localization, which is localized mainly to the endosomes and endoplasmic reticulum but rarely to the mitochondria and lysosomes in the cytoplasm.

To investigate whether RNASEK-a and -b are involved in innate immune response within CIK cells, we tested their effect on type I IFN mRNA and protein expression via qRT-PCR and immunofluorescence, respectively. Overexpression experiments were performed by transfection of pcDNA3.1-RNASEK-a and -b into cells for 24 h. As shown in Figures 3A, B , RNASEK-a and -b overexpression individually upregulated type I IFN transcripts by 2.49- and 2.11-fold compared with the controls. Furthermore, RNASEK-a and -b overexpression significantly enhanced the average fluorescence intensity of type I IFN protein (red), reaching 1.32- and 1.83-fold versus the control, respectively ( Figures 3I, M ). RNASEK-a knockdown was completed by transfection of siRNA against RNASEK-a-22 and -101 into cells, which individually downregulated type I IFN transcripts by 1.33 and 1.49 times in comparison with the control ( Figure 3C ). For immunofluorescence, these two siRNAs separately decreased the fluorescence intensity of type I IFN by 1.99- and 1.40-fold versus the controls ( Figures 3J, K, N, O ). RNASEK-b knockdown was finished by using shRNA transfection, including GP-shRNASEK-b-22, shRNASEK-b-120, and -226. The last two shRNA-mediated knockdown of RNASEK-b individually inhibited type I IFN mRNA expression by 3.39 and 1.59 times than the control ( Figure 3D ). In addition, GP-shRNASEK-b-22 transfection reduced the fluorescence intensity of type I IFN by 1.20-fold in comparison with the control ( Figures 3L, P ). Collectively, both RNASEK-a and -b promote type I IFN expression at transcriptional and protein levels in CIK cells.

For further validation of the above results, CO cells were used to assess type I IFN protein through Western blot. pEGFP-RNASEK-a and -b were transfected into cells to finish overexpression. As shown in Figures 3E, G , RNASEK-a and -b overexpression elevated type I IFN protein by 3.56- and 1.66-fold compared with the control, respectively. Given the high expression of RNASEK-b and very low abundance for RNASEK-a in CO cells, we merely performed RNASEK-b knockdown with shRNASEK-b-120 and -226. They individually downregulated type I IFN by 2.51 and 3.22 times than the control ( Figures 3F, H ). In short, both RNASEK-a and -b promote type I IFN expression in CO cells, consistent with that in CIK cells.

In grass carp, endosomal TLR–IRF3/IRF7 axis has been reported to induce type I IFN expression (17), which allows us to consider whether RNASEK-a and -b promote type I IFN production via activating IRF3/IRF7 because of their localization. For validation of this hypothesis, overexpression experiments were carried out with the transfection of pcDNA3.1-RNASEK-a and -b into CIK cells. As expected, RNASEK-a and -b overexpression individually upregulated the protein expression of p-IRF3 by 1.95- and 1.91-fold as well as p-IRF7 by 1.92 and 1.74 times versus the controls ( Figure 4 ). Collectively, grass carp RNASEK-a and -b separately activate IRF3 and IRF7.

Apoptosis plays a key role in innate immunity, which allows us to think about whether RNASEK-a and -b affect apoptosis in fish. We selected CIK, CL, or CO cells as models to validate our hypothesis by using qRT-PCR, DNA ladder, TUNEL, or flow cytometry methods. In CIK cells, pcDNA3.1-RNASEK-a and -b were used to overexpress the matched genes, which separately upregulated the Bax/Bcl-2 mRNA ratio by 1.45- and 1.66-fold in comparison with the controls ( Figures 5A, B ), indicating that they can enhance apoptosis. In CL cells, overexpression of RNASEK-a and -b was individually performed with pEGFP-RNASEK-a and -b transfection, increasing the Bax/Bcl-2 mRNA ratio by 1.29 and 1.43 times versus the controls, respectively ( Figures 5C, D ). As for the DNA ladder in CIK ( Figure 5E ) and CL ( Figure 5F ) cells, the transfection of both pEGFP-RNASEK-a and -b produced more DNA fragmentations (indicated by red arrows), one characteristic of cell apoptosis, than the controls ( Figures 5E, F ). Besides, pcDNA3.1-RNASEK-a and -b transfection separately increased the number of apoptotic CIK cells (TUNEL-positive green signals, pointed by red arrows) by 1.93 and 2.39 times compared with the controls ( Figures 5G, H, I, J ). Furthermore, we overexpressed these two genes in CO cells via transfection of pcDNA3.1-RNASEK-a and -b, which separately elevated the proportion of Annexin V-positive apoptotic cells by 1.20 and 1.21 times than the control ( Figures 5K, L ). Altogether, grass carp RNASEK-a and -b individually promote cell apoptosis.

As already known, increased p-eIF2α expression promotes cell apoptosis in grass carp (30). To investigate whether the pro-apoptosis effects of both RNASEK-a and -b on apoptosis are associated with p-eIF2α, we performed overexpression experiments with transfection of pcDNA3.1-RNASEK-a and -b into CIK and CL cells. As shown in Figure 6 , RNASEK-a and -b significantly elevated p-eIF2α protein expression by 5.14- and 4.46-fold in CIK cells as well as 4.51 and 5.14 times in CL cells compared with the controls, respectively. Taken together, grass carp RNASEK-a and -b individually activate eIF2α.