USP13 Cooperates with MARCH8 to Inhibit Antiviral Signaling by Targeting MAVS for Autophagic Degradation in Teleost.

Mitochondrial antiviral signaling protein (MAVS), as a central adapter protein in retinoic acid-inducible gene I-like receptor signaling, is indispensable for innate antiviral immunity. Yet, the molecular mechanisms modulating the stability of MAVS are not fully understood in low vertebrates. In this study, we report that the deubiquitinase ubiquitin-specific protease 13 (USP13) acts as a negative regulator of antiviral immunity by targeting MAVS for selective autophagic degradation in teleost fish. USP13 is induced by RNA virus or polyinosinic:polycytidylic acid stimulation and acts as a negative regulator to potentiate viral replication in fish cells. Mechanistically, USP13 functions as a scaffold to enhance the interaction between MAVS and the E3 ubiquitin ligase MARCH8, thus promoting MARCH8 to catalyze MAVS through K27-linked polyubiquitination for selective autophagic degradation. Taken together, to our knowledge, our study demonstrates a novel mechanism by which viruses evade host antiviral immunity via USP13 in fish and provides a new idea for mammalian innate antiviral immunity.

EFHD2 cooperates with E3 ubiquitin ligase Smurf1 to facilitate virus infection by promoting the degradation of TRAF6 in teleost fish.

The ubiquitin-proteasome system is one of the most important protein stability regulation systems. It can precisely regulate host immune responses by targeting signaling proteins. TRAF6 is a crucial E3 ubiquitin ligase in host antiviral signaling pathway. Here, we discovered that EF-hand domain-containing protein D2 (EFHD2) collaborated with the E3 ubiquitin ligase Smurf1 to potentiate the degradation of TRAF6, hence facilitating RNA virus Siniperca chuatsi rhabdovirus infection. The mechanism analysis revealed that EFHD2 interacted with Smurf1 and enhanced its protein stability by impairing K48-linked polyubiquitination of Smurf1, thereby promoting Smurf1-catalyzed degradation of TRAF6. This study initially demonstrated a novel mechanism by which viruses utilize host EFHD2 to achieve immune escape and provided a new perspective on the exploration of mammalian innate immunity.IMPORTANCEViruses induce host cells to activate several antiviral signaling pathways. TNF receptor-associated factor 6 (TRAF6) plays an essential role in these pathways. Numerous studies have been done on the mechanisms of TRAF6-mediated resistance to viral invasion. However, little is known about the strategies that viruses employ to antagonize TRAF6-mediated antiviral signaling pathway. Here, we discovered that EFHD2 functions as a host factor to promote viral replication. Mechanistically, EFHD2 potentiates Smurf1 to catalyze the ubiquitin-proteasomal degradation of TRAF6 by promoting the deubiquitination and stability of Smurf1, which in turn inhibits the production of proinflammatory cytokines and interferons. Our study also provides a new perspective on mammalian resistance to viral invasion.

STAM2 negatively regulates the MyD88-mediated NF-κB signaling pathway in miiuy croaker, Miichthys miiuy.

Signal transducing adapter molecule 2 (STAM2), a member of the Signal Transducing Adapter Molecule (STAM) family, is a protein with significant implications in diverse signaling pathways and endocytic membrane trafficking. However, the role of the STAM2, especially in fish, remains largely unknown. In this study, we discovered that STAM2 negatively regulates the NF-κB signaling pathway, and its inhibitory effect is enhanced upon LPS induction. Our study confirmed that STAM2 can enhance the degradation of myeloid differentiation primary-response protein 88 (MyD88), an upstream regulator of NF-κB pathway. Furthermore, the UIM domain of STAM2 is important for the inhibition of MyD88. Mechanistically, STAM2 inhibits the NF-κB signaling pathway by targeting the MyD88 autophagy pathway. In addition, we showed that STAM2 promotes the proliferation of Vibrio harveyi. In summary, our study reveals that STAM2 inhibits NF-κB signaling activation and mediates innate immunity in teleost via the autophagy pathway.

eIF3k inhibits NF-κB signaling by targeting MyD88 for ATG5-mediated autophagic degradation in teleost fish.

Optimal activation of NF-κB signaling is crucial for the initiation of inflammatory responses and eliminating invading bacteria. Bacteria have likewise evolved the ability to evade immunity; however, mechanisms by which bacteria dysregulate host NF-κB signaling are unclear. In this study, we identify eukaryotic translation initiation factor eIF3k, a nonessential member of the eIF3 translation initiation complex, as a suppressor of the NF-κB pathway. Mechanistically, we show that eIF3k expression induced by Vibrio harveyi enhances E3 ligase Nrdp1-mediated K27-linked ubiquitination of MyD88, an upstream regulator of NF-κB pathway activation. Furthermore, we show that eIF3k acts as a bridge linking ubiquitin-tagged MyD88 and ATG5, an important mediator of autophagy. We demonstrate that the MyD88-eIF3k-ATG5 complex is transported to the autophagosome for degradation, and that innate immune signaling is subsequently terminated and does not attack invading V. harveyi. Therefore, our study identifies eIF3k as a specific inhibitor of the MyD88-dependent NF-κB pathway and suggests that eIF3k may act as a selective autophagic receptor that synergizes with ATG5 to promote the autophagic degradation of MyD88, which helps V. harveyi to evade innate immunity. We conclude that V. harveyi can manipulate a host's autophagy process to evade immunity in fish and also provide a new perspective on mammalian resistance to bacterial invasion.

Long Noncoding RNA MIR2187HG Suppresses TBK1-Mediated Antiviral Signaling by Deriving miR-2187-3p in Teleost Fish.

Long noncoding RNAs (lncRNAs) function as microregulatory factors that influence gene expression after a variety of pathogenic infections, and they have been extensively studied in the past few years. Although less attention has been paid to lncRNAs in lower vertebrates than in mammals, current studies reveals that lncRNAs play a vital role in fish stimulated by pathogens. Here, we discovered a new lncRNA, termed MIR2187HG, which can function as a precursor of a small RNA, miR-2187-3p, with regulatory functions in the miiuy croaker (Miichthys miiuy). Upon Siniperca chuatsi rhabdovirus (SCRV) virus infection, the expression levels of MIR2187HG were remarkably enhanced. Elevated MIR2187HG expression can act as a pivotally negative regulator that participates in the innate immune response of teleost fish to inhibit the intracellular TANK-binding kinase 1 (TBK1)-mediated antiviral signaling pathways, which can effectively avoid excessive immunity. In addition, we found that SCRV could also utilize MIR2187HG to enhance its own numbers. Our results not only provide evidence regarding the involvement of the lncRNAs in response to viruses in fish but also broaden our understanding of the function of lncRNAs as precursor microRNAs (miRNAs) in teleost fish for the first time. IMPORTANCE SCRV infection upregulates MIR2187HG levels, which in turn suppresses SCRV-triggered type I interferon production, thus promoting viral replication in miiuy croaker. Notably, MIR2187HG regulates the release of miR-2187-3p, and TBK1 is a target of miR-2187-3p. MIR2187HG could acquire from miR-2187-3p the function of inhibiting TBK1 expression and subsequently modulate TBK1-mediated NF-κB and IRF3 signaling. The collective results suggest that the novel regulation mechanism of TBK1-mediated antiviral response during RNA viral infection was regulated by MIR2187HG. Therefore, a new regulation mechanism for lncRNAs to regulate antiviral immune responses in fish is proposed.

PSMD13 inhibits NF-κB pathway by targeting TAK1 for K63-linked ubiquitination in miiuy croaker (Miichthys miiuy).

Transforming growth factor-ß activated kinase 1 (TAK1) is an adaptor molecular in the TLR-mediated NF-κB pathway which has been implicated in the regulation of a wide range of physiological and pathological processes. Proteasome 26S subunit, non-ATPase (PSMD) 13 is essential for the structural maintenance and function of the 26S proteasome. However, the mechanism of PSMD13 in innate immune regulation is not clear. In this study, the expression of PSMD13 mRNA was significantly increased under Vibrio harveyi stimulation, and PSMD13 inhibited the NF-κB pathway by targeting TAK1. Mechanically, PSMD13 significantly inhibited the K63-linked ubiquitination of TAK1, thereby inhibiting the expression of TAK1. Moreover, this discovery enriches the research of the PSMD family in regulating the innate immune response and provides a new idea for the study of the mammalian innate immune regulation mechanism.

Vinculin B inhibits NF-κB signaling pathway by targeting MyD88 in miiuy croaker, Miichthys miiuy.

Myeloid differentiation factor 88 (MyD88) is the canonical adaptor for inflammatory signaling pathways downstream from members of the Toll-like receptor (TLR) and interleukin-1 (IL-1) receptor families, which activates the NF-κB signaling pathway and regulates immune and inflammatory responses. In this study, we found that Vinculin B (Vclb) is an inhibitor in the NF-κB signaling pathway, and its inhibitory effect was enhanced by LPS induction. Furthermore, Vclb inhibits NF-κB activation by targeting MyD88, thereby suppressing the production of inflammatory cytokines. Mechanistically, Vclb inhibits the NF-κB signaling pathway by targeting MyD88 ubiquitin-proteasome pathway. In summary, our study reveals that Vclb inhibits NF-κB signaling activation and mediates innate immunity in teleosts via the ubiquitin-proteasome pathway of MyD88.

Identification of a novel fusion gene NLRC3-NLRP12 in miiuy croaker (Miichthys miiuy).

Fusion gene is a new gene formed by the fusion of all or part of the sequences of two genes, it is caused by chromosome translocation, middle deletion or chromosome inversion. Numerous studies in the past have continuously shown that gene fusions are tightly associated with the occurrence and development of various diseases, especially cancer. Many fusion genes have been identified in humans. However, few fusion genes have been identified in fish. In this study, a novel NLRC3-NLRP12 fusion gene was identified in the Miichthys miiuy (miiuy croaker) by quantitative real-time PCR (qRT-PCR), PCR, and Sanger sequencing. This fusion gene is fused by two genes related to NLRs (nucleotide binding domain and oligomerization domain like receptors). We found that the expression of the NLRC3-NLRP12 fusion gene was significantly upregulated after infection with Vibrio anguillarum (V. anguillarum) or stimulation with lipopolysaccharide (LPS). In addition, the NLRC3-NLRP12 fusion gene was strongly induced by V. anguillarum infection, peaking within the kidney and liver at 12 h post infection. Further functional experiments showed that overexpression of NLRC3-NLRP12 significantly inhibited nuclear factor kappa-B (NF-κB) activation. This study suggests that the newly discovered NLRC3-NLRP12 fusion genes may play an important role in innate immunity in miiuy croaker.

Circular RNA circPlce1 regulates innate immune response in miiuy croaker, Miichthys miiuy.

In recent years, more and more researchers have devoted to the study of circular RNAs (circRNAs) in noncoding RNAs. As an important regulator in a variety of biological processes, circRNAs are relatively abundant in the study of mammals, while research in lower vertebrates is still lacking. In this study, we found a circRNA, circPlce1, related to innate immune response in Miichthys miiuy (miiuy croaker). The experimental results confirmed that circPlce1 could promote the production of antiviral genes and inflammatory response under the stimulation of poly (I: C) and LPS. We also confirmed that circPlce1 can promote NF-κB and IRF3 pathways through luciferase reporter assay experiment. In addition, we also found that circPlce1 can promote cell proliferation and improve cell viability. In conclusion, our results showed that circPlce1 plays an active role in regulating inflammatory response, cell proliferation and cell viability, providing a foundation for the study of the biological function of circRNAs in the innate immune response in teleost fish.

Long noncoding RNA LTCONS6801 up-regulates TBK1 mediated antiviral innate immunity in miiuy croaker, Miichttys miiuy.

The development of sequencing technology has further accelerated the research of noncoding RNA (ncRNA). A large number of studies have shown that long noncoding RNA (lncRNA) in ncRNA can regulate gene expression in various ways and then affect various physiological and biochemical processes of the host. In this study, we found a novel lncRNA in Miichthys miiuy, named LTCONS6801, which is beneficial to TANK-binding kinase 1 (TBK1) and its mediated pathway to promote the host immune function. First, we found that lncRNA LTCONS6801 can enhance cell activity through cell viability detection and cell proliferation detection. Besides, after poly (I: C) stimulation, overexpression of lncRNA LTCONS6801 promoted the expression of antiviral gene and TBK1. We found that lncRNA LTCONS6801 further affects NF-κB and IRF3 signaling pathways by regulating the expression of TBK1. In short, lncRNA LTCONS6801 is an lncRNA that can positively regulate the host innate immune response by regulating the expression of TBK1. Our study enriches the theory and insight of lncRNA regulating antiviral immune pathway and clarifies the important role of lncRNA in antiviral immunity of teleost fish.

Identification and functional regulation of three alternative splicing isoforms of the fthl27 gene in miiuy croaker, Miichthys miiuy.

Alternative splicing is an important basic mechanism for eukaryotes to control gene expression. Different forms of alternative splicing may lead to the production of protein subtypes with different functions, leading to the expansion of protein diversity in organisms, affecting cell production and metabolism, and is even related to the occurrence of many diseases. Many studies have shown that ferritin is usually associated with inflammation, vascular proliferation, and tumors, which is the focus of immunological research. It not only plays a role in iron metabolism and storage in the body, but also plays an important regulatory role in pathways related to immune and inflammatory regulation. However, there are few studies on alternative splicing events of the ferritin gene nowadays. Therefore, this study identified three different splicing isoforms in its ferritin gene fthl27 of Miichthys miiuy through Sanger sequencing, qRT-PCR, and other experimental techniques, and we found that three different splicing isoforms of the ferritin gene fthl27 in M. Miiuy cells showed an upregulation trend after being stimulated by Lipopolysaccharide (LPS) and poly (I: C). The experiment also found that the three isoforms may have different regulatory effects on the expression of inflammatory factors and antiviral immune factors, playing an important role in the innate immune response of fish.

Inducible MicroRNA-132 Inhibits the Production of Inflammatory Cytokines by Targeting TRAF6, TAK1, and TAB1 in Teleost Fish.

The innate immune response is the first line of defense against pathogen infection. Eradication of pathogen infection requires appropriate immune and inflammatory responses, but excessive inflammation may cause inflammatory and autoimmune diseases. MicroRNAs (miRNAs) are a group of small noncoding RNAs, and accumulating evidence has shown that in mammals, they can act as negative regulators that participate in the regulation of inflammation and immune responses. However, the miRNA-mediated immune regulation networks in the inflammatory responses of lower vertebrates are largely unknown. In this study, we report an miRNA, miR-132, identified from miiuy croaker, that acts as a negative regulator in the host's bacterium-induced inflammatory response. We found that miR-132 expression was dramatically increased upon infection by the Gram-negative bacterium Vibrio harveyi and lipopolysaccharide (LPS). Inducible miR-132 inhibits the production of inflammatory cytokines by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), transforming growth factor-activated protein kinase 1 (TAK1), and TAK1 binding protein 1 (TAB1), thus avoiding an excessive inflammatory response. Furthermore, we demonstrate that miR-132 modulates the inflammatory response through a TRAF6-, TAK1-, and TAB1-mediated NF-κB signaling pathway. These results collectively reveal that miR-132 plays a negative regulatory role in the host antibacterial immune response, which will help to gain insight into the intricate network of host resistance to pathogen infection in lower vertebrates.

microRNA-20-1 and microRNA-101a Suppress the NF-κB-Mediated Inflammation Production by Targeting TRAF6 in Miiuy Croaker.

Upon recognition of pathogen components by pattern recognition receptors, cells could be activated to produce inflammatory cytokines and type I interferons. The inflammation is tightly modulated by the host to prevent inappropriate inflammatory responses. MicroRNAs (miRNAs) are noncoding small RNAs that can inhibit gene expression and participate in various biological functions, including maintaining a balanced immune response in the host. To maintain the balance of the immune response, these pathways are closely regulated by the host to prevent inappropriate reactions of the cells. However, in lower vertebrates, the miRNA-mediated inflammatory response regulatory networks remain largely unknown. Here, we report that two miRNAs, i.e., miR-20-1 and miR-101a, were identified as negative regulators in teleost inflammatory responses. Initially, we found that both miR-20-1 and miR-101a dramatically increased after lipopolysaccharide (LPS) stimulation and Vibrio harveyi infection. Upregulated miR-20-1 and miR-101a inhibited LPS-induced inflammatory cytokine production by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), thus avoiding excessive inflammation. Moreover, miR-20-1 and miR-101a regulate the inflammatory responses through the TRAF6-mediated NF-κB signaling pathway. Collectively, these data indicate that miR-20-1 and miR-101a act as negative regulators by regulating the TRAF6-mediated NF-κB signaling pathway and participate in host antibacterial immune responses, which will provide new insights into the intricate networks of the host-pathogen interactions in the lower vertebrates.

Long non-coding RNA LTCONS7822 positively regulates innate immunity by targeting MITA in miiuy croaker (Miichthys miiuy).

Accumulated studies have shown that long non-coding RNA (lncRNA) is considered a critical regulatory factor in mammals, with a length greater than 200 nucleotides, and it can participate in gene imprinting, dose compensation, transcription enhancement, and antisense regulation. Most of the above studies are carried out in mammals, and there are very few studies on lncRNA of lower vertebrates. Here, we report a novel lncRNA, LTCONS7822, which can play a positive regulatory effect on antiviral immunity in miiuy croaker, Miichthys miiuy. Our results show that the levels of lncRNA LTCONS7822 were significantly increased after poly (I:C) stimulation. Further study, we found that lncRNA LTCONS7822 could positively regulate MITA at the post-transcriptional level. In addition, the dual-luciferase reporter assay analysis showed that the positive regulatory effect of lncRNA LTCONS7822 on NF-κB and IRF3 signaling pathways presented the dose and time-dependent manner. Western blotting experiments proved that lncRNA LTCONS7822 has a positive regulatory effect on MITA. Collectively, our study provided new information to enrich the immune regulation network of lncRNA in teleost fish.

The long noncoding RNA LTCONS5539 up-regulates the TRAF6-mediated immune responses in miiuy croaker (Miichthys miiuy).

With the further study of long noncoding RNAs (lncRNAs), an increasing number of biological studies have demonstrated that lncRNAs are involved in various physiological processes, including cell proliferation, apoptosis, invasion, development and disease states. However, unlike mammals, little is known about the role of lncRNAs in the innate immunity of teleost fish. Here, we identify a lncRNA, named LTCONS5539, as critical role in the antiviral and antibacterial response of miiuy croaker and the results showed that lncRNA LTCONS5539 plays a critical regulatory role on TRAF6. Firstly, we found that LPS and poly(I:C) can up-regulate the expression of lncRNA LTCONS5539. Elevated lncRNA LTCONS5539 is capable of increasing the production of inflammatory factors and antiviral genes. Furthermore, the over-expression of lncRNA LTCONS5539 increases the expression of TRAF6 which was confirmed by qPCR and western blotting. On these foundations, we also proved that lncRNA LTCONS5539 modulates innate immunity through TRAF6-mediated immune responses through dual luciferase reporter assay. These results will help to further understand the immunomodulatory mechanisms of lncRNA in teleost fish.

microRNA-122 regulates NF-κB signaling pathway by targeting IκBα in miiuy croaker, Miichthys miiuy.

The inhibitory protein IκBα plays a key role in the inflammatory process and immune response by regulating the activity of the transcription factor NF-κB. microRNA (miR) is a small non-coding RNA that can regulate many biochemical processes, such as cell growth, proliferation, and immune response. In this study, it was first predicted that IκBα is the target of miR-122 through bioinformatics, and it was confirmed by dual fluorescence experiments. Then we found that miR-122 can inhibit the expression of IκBα at the mRNA and protein levels, thereby promoting the p65-activated NF-κB pathway. It is speculated that miR-122 plays an important role in the innate immunity of teleost fish. This study will help to further understand miRNAs regulatory mechanism in teleost fish.

MicroRNA-103 and microRNA-190 negatively regulate NF-κB-mediated immune responses by targeting IL-1R1 in Miichthys miiuy.

Accumulating evidence has demonstrated that microRNAs (miRNAs) regulate various physiological and pathological processes at the transcriptional level, thus called novel regulators in immune response. In this study, we used bioinformatics and functional experiments to determine the role of miR-103 and miR-190 in the regulation of IL-1R1 gene involved in the immune and inflammatory responses in miiuy croakers. First, we predicted the target genes of miR-103 and miR-190 through bioinformatics and found that IL-1R1 is a direct target gene of miR-103 and miR-190. This was further confirmed by the dual-luciferase reporter assay that the over-expression of miR-103, miR-190 mimics and the pre-miR-103, pre-miR-190 plasmids inhibit the luciferase levels of the wild-type of IL-1R1 3'UTR. miR-103 and miR-190 inhibitors increase the luciferase levels of IL-1R1-3'UTR. Additionally, we found that miR-103 and miR-190 could negatively regulate the mRNA expression of IL-1R1. Importantly, we demonstrated that miR-103 and miR-190 significantly inhibit the NF-κB signaling pathway by targeting IL-1R1 upon LPS stimulation. Collectively, these results provide strong evidence for an important regulatory mechanism of miR-103 and miR-190 targeting the IL-1R1 gene, thereby preventing excessive inflammatory immune responses from causing autoimmunity.

PCNA negatively regulates MITA through the autophagy pathway in miiuy croaker (Miichthys miiuy).

Interferon-mediated innate immune response is the first line of defense against foreign pathogen infection. Overexpression of MITA can activate the expression of interferon and promote the innate immune response of the body to the virus. These innate immune responses are tightly controlled to prevent the host from over-immunizing itself. In this study, we reported that structurally highly conserved PCNA negatively regulates MITA. PCNA overexpression can promote MITA degradation and block the expression of interferon, while the autophagy inhibitor 3-MA significantly inhibits MITA degradation, indicating that PCNA can degrade MITA through the autophagy pathway. PCNA inhibits interferon production by targeting MITA and avoids excessive immune response. In summary, our results indicate that PCNA is involved in the immune response by degrading MITA through the autophagy pathway, which will provide new ideas for further studies on the regulatory mechanism of immune signaling pathways in lower vertebrates.

NOP56 negatively regulates MyD88-mediated NF-κB signaling in miiuy croaker, Miichthys miiuy.

MyD88 is a critical adaptor in the TLRs signaling pathway, which can activate NF-κB signaling pathway and promote proinflammatory cytokines production. However, the molecular mechanisms that modulate MyD88 expression, especially in teleost, remain largely unknown. In this study, we showed that NOP56 serve as a negative regulator of the MyD88-mediated NF-κB signaling pathway. NOP56 overexpression inhibited the protein expression of MyD88. Whereas, siRNA knockdown of NOP56 had opposite effect. Furthermore, we found that the NOSIC domain is responsible for the suppressive effect of NOP56 in MyD88 expression at protein level. Therefore, we identified NOP56 as a negative regulator of MyD88-mediated NF-κB signaling by inhibiting MyD88 expression and provided new insight into the regulation mechanism in teleost fish.

Circular RNA circRara promote the innate immune responses in miiuy croaker, Miichthys miiuy.

With the in-depth study of circRNA, more and more biological studies have shown that circRNAs play an important role in mammals, such as cell proliferation, apoptosis, invasion, development and disease state. However, the regulatory mechanism of circRNA in lower vertebrates remains unclear. Here, we found a new circular RNA and named it circRara. We carried out the experimental study on its antiviral and antibacterial response, cell proliferation and activity. The results showed that circRara had a positive regulatory effect on the antiviral and antibacterial response, cell proliferation and activity in miiuy croaker. First, we found that the expression of circRara could be up-regulated under the stimulation of LPS and poly (I: C), but not the expression of linear Rara. In addition, the increase of circRara can increase the production of inflammatory factors and antiviral genes, which was confirmed by double luciferase reporter gene experiment and qPCR. These results will help to further understand the immunomodulatory mechanism of circRNA in teleost fish.