A genetic compensation response model for RIG loss

In previous studies, it has been reported that a few vertebrates lost the RIG-I gene during evolution (Liang and Su, 2021; Krchlíková et al., 2022; Chen et al., 2017). To investigate this further, we analyzed the genomes of various species, and determined the presence and absence of the RIG-I gene. As depicted in Figure 1A, the absence of the RIG-I gene does not appear to be uncommon in vertebrates, with at least three independent loss events observed in mammals, birds and fish, respectively. These gene-loss events occurred over a long evolutionary timescale. Based on the minimum age of the common ancestor of RIG-I-missing species (http://www.timetree.org/), the oldest recorded losses occurring in species like Erpetoichthys calabaricus, can be traced back to approximately 330 million years ago (MYA). Conversely, the detected loss in G. gallus took place less than 42 MYA. To gain a deeper understanding of the evolutionary dynamics of vertebrate RIG-I, we explored the conservation of the genes surrounding RIG-I. We analyzed the genome sequences of Homo sapiens, Callorhinchus milii, Danio rerio, T. belangeri, G. gallus, and M. miiuy (Figure 1B). Similar to the slow-evolving C. milii, humans possess RIG-I with TOPORS and ACO1 as its upstream and downstream genes, respectively. As previously reported (Xu et al., 2016; Krchlíková et al., 2022). T. belangeri, G. gallus, and M. miiuy do not have RIG-I. Interestingly, zebrafish has a RIG-I with its upstream gene being TOPORSb, seemingly a result of chromosome fragment duplication. However, one RIG-I gene was lost in the downstream region of TOPORSa, indicating that one of the RIG-I copies has been lost in zebrafish, similar to M. miiuy.

Given the important role of RIG-I in recognizing 5'ppp RNA, we selected two different models for experiments to detect the presence of other potential receptors for immune compensation in RIG-I-deficient species. Specifically, we extracted virus RNA from SCRV and VSV viruses and obtained their dephosphorylated RNA derivatives (SCRV-RNA-CIAP and VSV-RNA-CIAP) through Calf Intestinal Alkaline Phosphatase (CIAP) treatment. Next, these RNAs were transfected into MKC cells of M. miiuy and DF-1 cells of G. gallus respectively to detect the dimerization form of IRF3. Results showed that virus RNA are more potent than dephosphorylated virus RNA in activating IRF3 (Figure 1C and D). And further qPCR experiments showed that the induction ability of IFN-1 by SCRV-RNA is significantly higher than that of SCRV-RNA-CIAP (Figure 1E). These results indicate the presence of viral receptors in M. miiuy and G. gallus that can recognize 5’ppp-RNA. Next, we compared the structural domains of human RIG-I and M. miiuy MDA5 (mmiMDA5) and LGP2 (mmiLGP2) proteins, and observed that mmiLGP2 lacks the CARD domain (Figure 1F). We next co-transfected mmiLGP2 plasmids or si-mmiLGP2 with MDA5 plasmids into MKC cells. The results showed that mmiLGP2 can actively regulate mmiMDA5’s immune response to the SCRV virus (Figure 1G–I), which further indicates that it participates in antiviral immunity as an intermediate regulatory factor of MDA5 rather than a downstream signal transmitter (Saito et al., 2007). Therefore, these results prompted us to investigate whether MDA5, a structurally similar homolog to RIG-I, can substitute for RIG-I in recognizing 5'ppp-RNA in vertebrates without RIG-I (Figure 1J).

MDA5 promotes host antiviral innate immunity

To further investigate the alternative immune function of MDA5, we first investigated the M. miiuy MDA5-mediated signaling pathway in response to SCRV virus and double-stranded mimetic poly(I:C)-HMW infection. Two siRNA molecules (si-MDA5-1 and si-MDA5-2) were designed to assess the function of MDA5. si-MDA5-1 demonstrated higher inhibitory efficiency compared to si-MDA5-2 (Figure 2A). Therefore, si-MDA5-1 (referred to as si-MDA5) was selected for subsequent experiments. Subsequently, we constructed the MDA5 expression plasmid and confirmed its protein overexpression efficiency and cytoplasmic localization (Figure 2B and C). Considering that mammalian MDA5 activates NF-κB and IRF3/IRF7 to induce IFNs, we investigated whether M. miiuy MDA5 could impact these pathways. Dual-luciferase reporter assays demonstrated that knockdown of MDA5 significantly inhibits NF-κB, IRF3, IRF7, and IFN-1 reporter genes (Figure 2D). In addition, we investigated the role of MDA5 in regulating the expression of IFN-stimulated genes (ISGs), which are crucial effectors. As shown in (Figure 2E–G), we observed that knockdown or overexpression of MDA5 can significantly inhibit or promote the expression levels of IFN-1, Mx1, ISG15, and Viperin upon SCRV infection or poly(I:C)-HMW stimulation. To visually represent the biological significance of MDA5 in SCRV-induced host cells, we utilized the virus plaque and qPCR methods to examine whether it could influence viral replication. As demonstrated in (Figure 2H–K), knockdown of MDA5 led to a significant increase in viral plaque formation and promoted SCRV replication in SCRV-infected cells, while overexpression had the opposite effect. Next, we explored that how MDA5 conducts signal transduction, and results showed that MDA5 is capable of binding not only to MAVS but also to STING (Figure 2L and M). In our previous laboratory investigations, we have substantiated the induction effect of STING on IFN under SCRV infection or poly(I:C) stimulation (Chu et al., 2021). Under the infection of SCRV virus, co-transfection of STING plasmids or si-STING with MDA5 plasmids can significantly increase or decrease the induction of IFN-1, indicating the important role of STING in MDA5-mediated immune transmission in response to RNA virus (Figure 2N and O). Finally, through the determination of IRF3 dimer and IFN induction, our results showed that the induction of antiviral innate immunity by SCRV-RNA depends on MDA5 (Figure 2P and Q). Overall, these findings indicated that MDA5 plays a positive role in regulating the antiviral responses of M. miiuy.

RD domain is required for MDA5 to recognize SCRV RNA

We investigated whether M. miiuy MDA5, in the absence of RIG-I, can directly bind to the RNA of SCRV. To address this question, we precipitated the MDA5 protein from SCRV-infected MKC cells that were transfected with MDA5-Flag and pcDNA3.1-Flag. The bound RNA was then amplified using SCRV-specific primers or β-actin primers as a control. Our results showed that MDA5 interacted with SCRV RNA, while β-actin mRNA did not associate with MDA5 (Figure 3A). The C-terminal regulatory domain (RD) of human RIG-I binds to viral RNA in a triphosphate-dependent manner, to determine if the RD domain of M. miiuy MDA5 has similar functions, we generated truncated mutations called MDA5-ΔRD and western blot analysis confirmed the expression of MDA5 and MDA5-ΔRD plasmids (Figure 3B). We subsequently noted that MDA5-ΔRD exhibited an inability to interact with SCRV RNA, which was supported by the loss of its immunological capability in inhibiting SCRV viral replication (Figure 3C–E). We also examined the effects of MDA5 and MDA5-ΔRD on antiviral factors in both uninfected, SCRV-infected, and poly(I:C)-HMW-stimulated MKC cells. In the absence of infection, overexpression of both MDA5 and MDA5-ΔRD stimulated the expression of antiviral genes (Figure 3F–I). However, when cells were infected or stimulated with SCRV or poly(I:C)-HMW, only the overexpression of MDA5, not MDA5-ΔRD, significantly increased the expression of antiviral genes (Figure 3F–I). In summary, these findings provided evidence that MDA5 may recognize SCRV RNA through its RD domain.

MDA5 recognizes 5’ppp-RNA in M. miiuy and G. gallus

To provide specific evidence of MDA5 recognition of 5’ppp-RNA in M. miiuy, we synthesized 67 nt 5’ppp-SCRV from the SCRV virus and through in vitro transcription using T7 polymerase. As a positive control capable of binding to the mmiMDA5 receptor, 112 bp dsRNA from the MDA5 gene sequence was also generated. Gel analysis and nuclease sensitivity confirmed the generation of RNA products of the expected size (Figure 4A). The RNA pull-down experiment showed that 5’ppp-SCRV can be recognized by mmiMDA5, similar to 112 bp blunt end dsRNA (Figure 4B). Next, we transfected wild-type 5’ppp-SCRV and dephosphorylated mutant SCRV RNAs (5’-OH-SCRV and 5’pppGG-SCRV) into MKC cells to detect the dimer of IRF3, and found that the activation ability of 5'ppp-RNA on IRF3 depends on its triphosphate structure (Figure 4C). in addition, knockdown of mmiMDA5 blocked the induction of IRF3 by 5’ppp-SCRV (Figure 4D). Furthermore, biotin-labeled 5’ppp-RNA captured mmiMDA5, while the biotin-labeled mutant types (5’-OH-SCRV and 5’pppGG-SCRV) did not (Figure 4E). Additionally, we purified recombinant MDA5 and MDA5-ΔRD of M. miiuy to conduct electrophoresis mobility shift assay (EMSA) (Figure 4F). When recombinant mmiMDA5 was subjected to EMSA using a biotin-labeled 5’ppp-SCRV probe, a clear complex was detected (Figure 4G). This complex formation was inhibited by adding an excess of cold 5’ppp-SCRV but not 5’-OH-SCRV and 5’pppGG-SCRV, demonstrating the direct interaction between mmiMDA5 and 5’ppp-SCRV (Figure 4G). However, 5’ppp-SCRV cannot bind to the MDA5-ΔRD (Figure 4H). Next, we investigated whether G. gallus MDA5 (ggaMDA5) shares the ability to recognize 5’ppp-RNA seen in fish. We synthesized 67 nt 5’ppp-VSV from the VSV virus and found that it is similar to 5’ppp-SCRV, binding to ggaMDA5 and activating IRF3 in a triphosphate-dependent manner (4I-4M). Overall, our findings demonstrated that MDA5 detection of RNA in two RIG-I deficient vertebrates (M. miiuy and G. gallus) is guided by the RNA 5'-triphosphate end and is disrupted when RNA is capped at the 5'-triphosphate end or dephosphorylated.

Altered m⁶A modification and expression of MDA5 upon SCRV infection

As stated in the previous results, MDA5 has evolved to possess immune recognition abilities that take over the role of RIG-I, significantly enhancing the host’s ability to resist viral infections. However, evolution is a continuous battle between hosts and viruses, viruses often evolve complex escape mechanisms to evade immune surveillance and destruction, in which the m⁶A modification mechanism is an important way. To investigate the impact of SCRV infection on the overall methylation dynamics of fish hosts, we utilized methylated RNA immunoprecipitation sequencing (MeRIP-seq, GenBank accession number: PRJNA819945) to measure changes in m⁶A modification of host transcripts during SCRV infection (Figure 5A). From the results shown in Figure 5B, we identified 3060 differentially m⁶A methylated peaks (p-value<0.05) and 2082 differentially expressed genes (p-value<0.05) between the non-infected and SCRV-infected group, indicating that infection significantly alters the m⁶A modification of specific host transcripts. It is worth noting that hyper-methylated m⁶A peaks were also observed in the MDA5 mRNA transcripts during SCRV infection (fold change >1.5, p<0.05), and the expression level of MDA5 was higher in the infection group compared to the control group (p<0.05). Further analysis of the m⁶A-seq data showed that MDA5 contained m⁶A sites in the exon1 region which were markedly higher in the infection group than that in the control group (Untreated VS SCRV: p=0.01; Figure 5C). Next, we tested the concrete m⁶A modification sites of MDA5 mRNA. We used the SRAMP database to predict potential m⁶A sites in MDA5-exon1 (Zhou et al., 2016). Four potential sites were predicted and two specific primers were designed (Figure 5D), then m⁶A quantitative real-time PCR and semiquantitative PCR were performed. In line with the m⁶A-seq data, the results further confirmed that m⁶A sites exactly exist in MDA5 exon1 (Figure 5E). After being infected with SCRV or poly(I:C)-HMW, the methylation level of MDA5 increased in MKC cells, further confirming the sequencing data (Figure 5F). We further inserted the MDA5-exon1 into pmirGLO, and results showed that the infection of SCRV can reduce the luciferase activity of pmirGLO-MDA5-exon1, and the use of cycloleucine (CL, an amino acid analogue that can inhibit m⁶A modification) hindered the degradation of MDA5-exon1 by SCRV, indicating that SCRV can regulate MDA5 expression through a methylation mechanism (Figure 5G). MDA5 is classified as an interferon-stimulated gene (ISG). Consistently, in spleen and kidney tissues infected with SCRV, the expression level of MDA5 demonstrates fluctuation. (Figure 5H). We further explored the mRNA and protein levels of MDA5 in MKC cells infected with SCRV and poly(I:C)-HMW and found that their expression all first significantly increased and then returned to the normal level (Figure 5I and J). Overall, our results suggested that the m⁶A modification of MDA5 is increased, and the expression level of MDA5 shows fluctuating changes upon SCRV infection.

m⁶A-modification weakens MDA5 mRNA stability and antiviral ability

SCRV infection can increase the methylation level of MDA5, and this mechanism is expected to have a regulatory effect on MDA5. To investigate this, the regulatory effects of METTL3 and METTL14, the main components of the methyltransferase complex, on MDA5 were explored. Three siRNAs targeting METTL3 and METTL14 were designed, and si-METTL3-2 and si-METTL14-3 showed higher knockdown efficiency (Figure 6A). METTL3 and METTL14-overexpressing plasmids were also constructed, and the interaction between them confirmed their cooperative role in m⁶A modification (Figure 6B). Subsequently, the global m⁶A levels were measured in cells after knockdown or overexpression of METTL3 and METTL14. Consistent with expectations, the knockdown of METTL3 and METTL14 significantly decreased the global m⁶A level, while their overexpression significantly increased the overall methylation level (Figure 6C). To investigate their role in the process of virus infection, we used the virus plaque and qPCR methods to explore whether it could affect SCRV replication. As shown in (Figure 6D and E), overexpression of METTL3 or METTL14 significantly enhances viral plaque formation and promotes SCRV replication in SCRV-infected cells. And as m⁶A writers, METTL3 and METTL14 can directly regulate the methylation level of MDA5 and induce MDA5 degradation by reducing its mRNA stability (Figure 6F–I). We further inserted the MDA5-exon1 into pmirGLO and mVenus-C1 vector, and results showed that si-METTL3&14 can reduce the luciferase activity of pmirGLO-MDA5-exon1, and METTL3&14 could suppress the levels of GFP, further indicating the presence of methylation sites in MDA5-exon1 (Figure 6—figure supplement 1A and D). To investigate the potential m⁶A sites on MDA5-exon1, we constructed mutant MDA5-exon1 reporter plasmids (Figure 5D and Figure 6—figure supplement 1B). As expected, our results showed that the luciferase activity of wild-type pmirGLO-MDA5-exon1 reporter, but not of mutant pmirGLO-MDA5-exon1 reporter, was markedly increased upon METTL3&14 knockdown (Figure 6—figure supplement 1C). Furthermore, we found that the use of cycloleucine hindered the degradation of MDA5 by METTL3 and METTL14, indicating that these proteins regulate MDA5 expression through a methylation mechanism (Figure 6J and K). Additionally, MDA5 expression was degraded by METTL3&14 in both SCRV-infected and uninfected groups (Figure 6L). To investigate the impact of this mechanism on viral infection, we co-transfected METTL3&14 and MDA5 plasmids and found that METTL3&14 partially suppressed the antiviral immune benefit of MDA5 (Figure 6M–O). Taken together, our data demonstrate that METTL3&14-mediated m⁶A methylation of MDA5 inhibits its mRNA stability and expression levels, thus suppressing the antiviral capabilities of MDA5.

Detailed m⁶A regulatory mechanism of MDA5

The m⁶A mechanism is jointly regulated by writers, erasers, and readers. The m⁶A modification of mRNA recruits m⁶A-binding proteins (readers) which have the function of recognizing m⁶A sites and accelerating mRNA decay. In this study, we hypothesized that YTHDF readers (YTHDF1, YTHDF2, and YTHDF3) may recognize m⁶A-modified MDA5 and destabilize it. We examined the binding ability of YTHDF proteins to MDA5 mRNA and found that YTHDF2 and YTHDF3 exhibited stronger binding compared to YTHDF1 (Figure 7A). We then designed two siRNAs targeting YTHDF1, YTHDF2, and YTHDF3 respectively, and selected si-YTHDF1-1, si-YTHDF2-si-2, si-YTHDF3-2 with better knockdown effects for subsequent experiments (Figure 7B). Results showed that the knockdown or overexpression of YTHDF2 and YTHDF3 led to the increase or decrease of the expression levels of MDA5, whereas YTHDF1 did not appear to influence MDA5 expression (Figure 7C and D, Figure 7—figure supplement 1A and C). In addition, overexpression of YTHDF2&3 can increase the luciferase activity of wild-type pmirGLO-MDA5-exon1 reporter, but not of mutant pmirGLO-MDA5-exon1 reporter (Figure 7—figure supplement 1B). We then found that si-METTL3&14 can counteract the negative impact of YTHDF2&3 on MDA5 mRNA, protein, and stability (Figure 7E and F). Since the m⁶A mechanism is reversible, we then studied the regulation of MDA5 by two major erasers. Results showed that FTO, not ALKBH5, can upregulate MDA5 expression (Figure 7G). Moreover, the overexpression of FTO can reverse the inhibitory effect of YTHDF2&3 on MDA5 (Figure 7H and I). Taken together, these data suggested that through METTL3&14-m⁶A-YTHDF2&3 regulatory network, m⁶A can mediate the degradation of MDA5, and this process can be countered by FTO (Figure 7J).

Fish and challenge

M. miiuy (~50 g) was obtained from Zhoushan Fisheries Research Institute, Zhejiang Province, China. Fish was acclimated in aerated seawater tanks at 25℃ for 6 weeks before experiments. SCRV virus was isolated from mandarin fish as described previously (Liu et al., 2023). The experimental procedure for SCRV infection was performed as described (Chu et al., 2020). Briefly, In the SCRV injection group, fish was challenged with 200 μl SCRV at a multiplicity of infection (MOI) of 5 through intraperitoneal. As a comparison, 200 μl of physiological saline was used to challenge the individuals. Afterwards, fishes were respectively sacrificed at different time point and the kidney and spleen tissues were collected for RNA extraction.

Cell culture

M. miiuy kidney cells (MKCs) and spleen cells (MPCs) are cell lines derived from the kidney and spleen tissues of M. miiuy (Geng et al., 2022), with passages ranging from 20 to 40 times. MKC and MPC cells were cultured in L-15 medium (HyClone) supplemented with 15% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin at 26℃. Fish EPC cells (Epithelioma papulosum cyprini cells) were maintained in medium 199 (Invitrogen) supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 26℃ in 5% CO₂. HEK293T cells were cultured in DMEM (HyClone) supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37℃ in 5% CO₂. Chicken embryo fibroblast cells (DF-1) were cultured in DMEM (HyClone) supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 39℃ in 5% CO₂.

Oligonucleotides

The sequences of the 67 nt 5’ppp-SCRV or 5’ppp-VSV were derived from the 5’ and 3’UTRs of the VSV or SCRV genome as previously described (Goulet et al., 2013), and the sequence of 112 bp dsRNA was taken from the first 100 bp of the MDA5 gene of miiuy croaker flanked by 5’-gggaga and tctccc-3’. These RNAs were synthesized by in vitro transcription using the Ribo RNAmax-T7 kit (RiboBio). Biotin-labeled RNAs were transcribed in vitro using a Ribo RNAmax-T7 Biotin Labeling Transcription Kit (RiboBio). For generating duplexes, RNA oligonucleotides were mixed in hybridization buffer (20 mM Tris-HCl [pH 8.0], 1.5 mM MgCl₂, and 1.5 mM DTT), boiled for 1 min, and incubated at 37℃ for 1 hr. For removal of 5’-triphosphates, 20 mg of RNA was treated with 20 U Calf Intestinal Alkaline Phosphatase (CIAP, Invitrogen) for 2 hr at 37℃ in the presence of RNase inhibitor (Beyotime) and extracted with phenolchloroform. RNA molecules with a terminal 5’ m7G cap were synthesized by the incorporation of m7G cap analogue (Yeason) in the transcription reactions. RNA was analysed on a denaturing 17% polyacrylamide, 7 M urea gel following digestion with 50 ng/μl of RNase A (Beyotime) or 100 mU/μl of DNase I (Beyotime) for 30 min.

Plasmids construction

To construct Flag or Myc-tagged MDA5 (GeneBank: PP179381.1), MAVS, STING, METTL3, METTL14, FTO, ALKBH5, YTHDF1, YTHDF2, and YTHDF3 expression plasmids, their CDS sequences of the M. miiuy were amplified by PCR and then ligated into a pcDNA3.1 vector (Invitrogen), respectively. Then, MDA5-△RD were generated by PCR on the basis of MDA5 plasmid. To construct MDA5-exon1 reporter vector, the exon1 region of M. miiuy MDA5 gene was amplified using PCR with the gene-specific primers and ligated into a pmir-GLO luciferase reporter vector (Promega). Meanwhile, M. miiuy MDA5-exon1 were inserted into the mVenus-C1 (Invitrogen), which included the sequence of enhanced GFP. For mutant reporter vectors (MDA5-exon1-mut), adenosine (A) in the predicted m⁶A motif was replaced by an uracil (U) by using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme) with specific primers. To construct Flag-tagged MDA5 expression plasmid of G. gallus, the CDS sequence of MDA5 was amplified by PCR and then ligated into a pcDNA3.1 vector (Invitrogen). The correct construction of the recombinant plasmids was verified through Sanger sequencing and extracted using an Endotoxin-Free Plasmid DNA Miniprep Kit (Tiangen, China) before using plasmids. The primers were listed in Supplementary file 1.

Protein purification and analysis

For protein purification, MDA5 plasmids with 6×His tag was constructed based on pcDNA3.1. (His6)-Flag-tagged MDA5 of M. miiuy and G. gallus were transiently overexpressed in 293T cells and lysed in a binding buffer (500 mM NaCl, 20 mM Tris/HCl [pH 7.4], 20 mM Imidazole, and 1% Triton X-100) including protease inhibitor cocktail (Roche). The lysate was incubated over night at 4℃ with anti-His beads (Solarbio). Anti-His beads were washed subsequently with washing buffer (500 mM NaCl, 20 mM Tris/HCl [pH 7.4], 20 mM Imidazole, and 1% Triton X-100). MDA5 proteins were eluted by an addtion of elution buffer (500 mM NaCl, 20 mM Tris/HCl [pH 7.4], 500 mM Imidazole, and 1% Triton X-100) to the beads. Purity of recombinant MDA5 was determined by SDS-PAGE separation.

RNA interference

The MDA5-specific small interfering RNA (si-MDA5-1 and si-MDA5-2) sequences were 5’- CGGACUACAUGCAGCGUAATT-3’ and 5’-GACCAAUGAGAUUUCUAUGTT-3’, respectively. The LGP2-specific small interfering RNA (si-LGP2) sequence was 5’-GGUUCACCUUGUAGAGCAATT-3’. The STING-specific small interfering RNA (si-STING) sequence was 5’-ACGACAGCAUCCAUUUCUATT-3’. The METTL3-specific small interfering RNA (si-METTL3-1, si-METTL3-2, and si-METTL3-3) sequences were 5’-CAUCACAAACGAACUCAACTT-3’, 5’- AACGUGGGCAAACUCUUUUTT-3’ and 5’-GAGAUUCUGGAACUACUUATT-3’, respectively. The METTL14-specific small interfering RNA (si-METTL14-1, si-METTL14-2, and si-METTL14-3) sequences were 5’-GCUGGAAAUCGAGGAGAUATT-3’, 5’- CCGTACGAAGAGGTGTACATT-3’ and 5’-GAGCCUCCCUUGGAAGAAUTT-3’, respectively. The YTHDF1-specific small interfering RNA (si-YTHDF1-1 and si-YTHDF1-2) sequences were 5’-AAAGACUUUGACUGGAACUTT-3’ and 5’-CAGUCGAUCAGAGACCUAATT-3’, respectively. The YTHDF2-specific small interfering RNA (si-YTHDF2-1 and si-YTHDF2-2) sequences were 5’-GCAGGGUGUUUAUCAUCAATT-3’, 5’- CUAUGCUCCCAGCUCAAUUTT-3’, respectively. The YTHDF3-specific small interfering RNA (si-YTHDF3-1 and si-YTHDF3-2) sequences were 5’-GGUGGACUACAAUGCCUAUTT-3’ and 5’-UCUACAGUAACAGCUAUGGTT-3’, respectively. The scrambled control RNA sequences were 5’ - UUCUCCGAACGUGUCACGUTT - 3’. They were purchased from GenePharma (Shanghai, China).

Cells transfection

Transient transfection of cells with siRNA was performed in 24-well plates using Lipofectamine RNAiMAX (Invitrogen), and cells were transfected with DNA plasmids was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. For functional analyses, the overexpression plasmid (500 ng per well) or control vector (500 ng per well) and siRNA (100 nM) were transfected into cells in culture medium and then harvested for further detection.

RNA extraction and quantitative real-time PCR

Total RNA was extracted using the TRIzol Reagent (Invitrogen) and the cDNA was synthesized using the FastQuant RT Kit (Tiangen) which contains DNase treatment of RNA to eliminate genomic contamination, following the manufacturer’s instructions. The expression profiles of each gene were conducted by using the SYBR Premix Ex Taq (Takara), as previously described. The quantitative real-time PCR was conducted in an Applied Biosystems QuantStudio 3 (Thermo Fisher Scientific). β-actin was used as internal controls. Primer sequences are listed in Supplementary file 1.

Western blotting

Cellular lysates were generated by using 1×SDS PAGE loading buffer. Proteins were extracted from cells and measured with the BCA Protein Assay kit (Vazyme), then subjected to SDS-PAGE (10%) gel and transferred to PVDF (Millipore) membranes by semidry blotting (Bio-Rad Trans Blot Turbo System). The membranes were blocked with 5% BSA. Protein was blotted with different antibodies. The antibody against MDA5 was diluted at 1: 500 (Beyotime, Cat# AF7164); The antibody against IRF3 was diluted at 1: 500 (Boster, Cat# BA4351-2); anti-Flag, anti-Myc, anti-HA and anti-Tubulin monoclonal antibody were diluted at 1: 1,000 (Beyotime); and HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Abbkine) at 1: 5,000. The results were the representative of three independent experiments. The immunoreactive proteins were detected by using WesternBright ECL (Advansta). The digital imaging was performed with a cold CCD camera.

Dual-luciferase reporter assays

For luciferase assays, MKC cells were co-transfected MDA5 expression plasmid, together with NF-κB, IRF3, IRF7, or IFN-1 luciferase reporter genes, and the pRL-TK Renilla luciferase reporter plasmid. For the detection of m⁶A site, the wild or mutant of MDA5-exon1 luciferase reporter was co-transfected si-METTL3, si-METTL14, si-YTHDF1, si-YTHDF2, si-YTHDF3 or si-Ctrl into MKC cells. After 48 hr transfection, the cells were collected and lysed for the reporter luciferase activities measured by using a dual-luciferase reporter assay system (Promega). All the luciferase activity values were gained against the Renilla luciferase control.

RNA binding protein immunoprecipitation (RIP)

To identify whether MDA5 or MDA5-△RD can directly bind to SCRV viral RNA, MKC cells (~2.0 × 10⁷) were transfected with pcDNA3.1-Flag, pcDNA3.1-MDA5-Flag or pcDNA3.1-MDA5-△RD-Flag and then infected with SCRV at an MOI of 5 for 24 hr. In addition, to identify whether YTHDF1, YTHDF2, and YTHDF3 can directly bind to MDA5, MKC cells were co-transfected with pcDNA3.1-Flag, pcDNA3.1-YTHDF1/2/3-Flag. For RNA immunoprecipitation (RIP) assays, MKC cells were harvested after 48 hr transfection, and RIP assays were carried out with Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) and anti-Flag antibody (Abcam) following the manufacturer’s protocol. Then, the expression of target gene SCRV-M, SCRV-G, β-actin, or MDA5 were detected by qRT-PCR analysis.

RNA pull-down assay

Cytoplasmic extracts were prepared from 2×10⁷ MKC cells transfected with mmiMDA5-Flag (MDA5 of miiuy croaker) plasmids and 5×10⁷ DF1 cells transfected with gga-MDA5-Flag (MDA5 of chicken) plasmids. The extracts were incubated with biotinylated 5’ppp-RNA or dsRNA and subjected to pull-down with streptavidin agarose beads (Geneseed), followed by subsequent Coomassie bluestaining or SDS-PAGE analysis and immunoblotting with anti-Flag antibody (Beyotime).

EMSA assay

Recombinant MDA5 proteins were mixed with biotin-labeled oligonucleotides in a reaction mixture (10 µl: 20 mM Tris-HCl pH 8.0, 1.5 mM MgCl₂, 1.5 mM DTT). After incubation at room temperature for 15 min, the reaction mixtures were conducted on 6% non-denaturing polyacrylamide gels and the EMSA was performed using Chemiluminescent EMSA Kit (Beyotime).

MeRIP-seq assays and analysis

Total RNA was isolated using TRIzol reagent. m⁶A immunoprecipitation and library preparation were performed following the manufacturer’s protocol. Poly (A) RNA is purified from 50 μg total RNA using Dynabeads Oligo (dT) (Thermo Fisher) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB) under 86℃ for 7 min. Then the cleaved RNA fragments were incubated for 2 hr at 4℃ with m⁶A-specific antibody (Synaptic Systems) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). For sequencing data analysis, we used HISAT2 to map reads to the reference genome of M. miiuy. Mapped reads of IP and input libraries were provided for R package exomePeak, which identifies m⁶A peaks with bed or bigwig format that can be adapted for visualization on the IGV software. The differentially expressed mRNAs were selected with log2 (fold change)>1 or log2 (fold change) <-1 and p-value <0.05 by R package edgeR. The circus map of m⁶A peaks in untreated and SCRV-infected spleen tissues of M. miiuy was showed by TBtools (Chen et al., 2020).

MeRIP-qPCR

Total RNA was extracted using TRIzol reagent (Invitrogen). Methylated m⁶A RNA immunoprecipitation (Me-RIP) was performed according to the protocol of EpiQuik CUT&RUN m⁶A RNA Enrichment Kit (Epigentek). qPCR analysis of the methylated RNA was performed to detect methylated MDA5 mRNA levels. Relative m⁶A level for each transcript was calculated as the percent of input in each condition normalized to that of the respective positive control spike-in. Fold change of enrichment was calculated with mock samples normalized to 1 (McIntyre et al., 2020).

Total m⁶A quantification assay

Total RNA was extracted by the TRIzol Reagent (Invitrogen). Then, an EpiQuik m⁶A RNA Methylation Quantification Kit (EpiGentek) was used to detect the total m⁶A level according to the manufacturer’s protocol. Briefly, positive control (PC), negative control (NC), and 200 ng isolated mRNA were added to each well with the capture antibody. Next, the RNA was incubated with m⁶A antibody in wells, and m⁶A content was quantified at a wavelength of 450 nm.

RNA stability assays

Cells were treated with actinomycin D (5 μg/mL) and then collected at different time points. RNA was extracted by TRIzol reagent (Invitrogen), and the mRNA levels were measured using qRT-PCR.

Database mining and sequence analysis

The species tree is constructed by submitting species names to the NCBI (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi). In order to determine whether there is RIG-I in vertebrates, we use H. sapiens and D. rerio RIG-I protein sequences to TBLASTN the whole genome sequence of the species on the ensemble website (http://www.ensemble.org/). In addition, in order to prevent the possibility of incomplete species genome, we also used RIG-I protein sequences of H. sapiens and D. rerio to compare the transcriptome and genome sequenced of the designated species on NCBI website (http://www.ncbi.nlm.nih.gov/Genbank/). For further gene synteny analysis, RIG-I of H. sapiens, C. milii, D. rerio were used as anchor sites. In order to identify the MDA5 and LGP2 from M. miiuy, we used the homologues of zebrafish reported previously as queries to seek for the transcriptome (Che et al., 2014) and a chromosome-scale genome database Xu et al., 2024 of M. miiuy by using local TBLASTN and BLASTN programs, then the protein structures were predicted by SMART website (http://smart.embl-heidelberg.de/).

Statistical analysis

Data are expressed as the mean ± SE from at least three independent triplicated experiments. A two-sided Student’s t test was used to evaluate the data. The relative gene expression data was acquired using the 2 ^-∆∆CT method, and comparisons between groups were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple comparison tests. A value of p<0.05 was considered significant.