Evolutionarily conserved Otub1 suppresses antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker, Miichthys miiuy.

Increasing evidence has been shown that OTUB1, a member of OTU deubiquitinases, is of importance in regulating the immune system. However, its molecular identification and functional characterization in teleosts are still rarely known. In this work, we cloned the otub1 of miiuy croaker (Miichthys miiuy), analyzed its sequence, structure, and evolution at genetic and protein levels, and determined its function in the antiviral immune response. The complete open reading frame (ORF) of miiuy croaker otub1 is 843 bp in length, encoding 280 amino acids. Miiuy croaker Otub1 has an OTU domain at the carboxyl terminus, which is a common functional domain that exists in OTU deubiquitinases. Molecular characteristics and evolution analysis results indicated that miiuy croaker Otub1, especially its functional domain, is highly conserved during evolution. The luciferase reporter assays showed that miiuy croaker Otub1 could significantly inhibit the poly(I:C) and Irf3-induced IFN1 and IFN-stimulated response element (ISRE) activation. Further experiments showed that miiuy croaker Otub1 decreases Irf3 protein abundance by promoting its proteasomal degradation. These data suggest that the evolutionarily conserved Otub1 acts as a suppressor in controlling antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker.

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.

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.

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.

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.

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.

METTL16, an evolutionarily conserved m6A methyltransferase member, inhibits the antiviral immune response of miiuy croaker (Miichthys miiuy).

Methyltransferase like-16 (METTL16) is an m6A RNA methylation transferase that is known to methylate U6 snRNA and pre-mRNA of S-adenosylmethionine synthase but has been poorly studied in fish. In this study, METTL16 was identified in miiuy croaker (Miichthys miiuy). We first performed bioinformatics analysis of the miiuy croaker METTL16 (mmiMETTL16). MmiMETTL16 and other vertebrates METTL16 have a relatively conserved MTD structural domain and gene structure, suggesting that their methylase activity may also be conservative. In healthy miiuy croaker, mmiMETTL16 was commonly expressed in the tested tissues. Expression of mmiMETTL16 in kidney, liver, and spleen tissues was significantly increased after poly(I:C) stimulation. Consistently, mmiMETTL16 was sensitive to poly(I:C) stimulation in miiuy croaker kidney cell (MKC), suggesting that METTL16 might participate in antiviral immunity. For further functional experiments, immunofluorescence of mmiMETTL16 presents in the nucleus in kidney cells. In addition, the overexpression of mmiMETTL16 could significantly increase the overall m6A level of MKC cells, which shows that the function of METTL16 as methyltransferase is conservative in miiuy croaker. Last, mmiMETTL16 can inhibit the expression of TNF-α, IFN-1, Mx1, and ISG15, suggesting that mmiMETTL16 can suppress the immune response caused by viral stimulation. In summary, studies on mmiMETTL16 will contribute to future studies on the role of METTL16 and potential mechanisms of the m6A regulation network in the teleost immune system.

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.

Long non-coding RNA LTCONS8875 regulates innate immunity by up-regulating IRAK4 in Miichthys miiuy (miiuy croaker).

In recent years, many studies have shown that long non-coding RNAs (lncRNAs) can regulate many biochemical processes, such as cell growth, proliferation, and immune response, which have attracted great attention. There are relatively many studies on lncRNA in mammals, while the research on lncRNA in lower vertebrates has just begun. In this study, we found a lncRNA, lncRNA LTCONS8875, related to innate immune response in Miichthys miiuy (miiuy croaker). Our results showed that lncRNA LTCONS8875 can up-regulate the expression of IRAK4 at the mRNA and protein levels, and significantly increase the production of inflammatory factors under LPS stimulation. Our research also confirmed that lncRNA LTCONS8875 plays an active role in regulating inflammation, cell proliferation, and cell viability. In summary, this research results showed that lncRNA LTCONS8875 can as an active regulatory role of innate immunity in miiuy croaker by up-regulating the expression of IRAK4, providing some insights for understanding the network mechanism of non-coding regulation of fish immunity.

Long noncoding RNA TARL promotes antibacterial activity and prevents bacterial escape in Miichthys miiuy through suppression of TAK1 downregulation.

Noncoding RNA (ncRNA) is an important regulatory factor that plays a major role in innate immunity. However, most studies on ncRNA have focused on mammals, resulting in a knowledge gap on ncRNA in lower vertebrates such as teleost fish. In this study, we identified a new long noncoding RNA (lncRNA), termed TAK1-related lncRNA (TARL), which can play a positive role in the antibacterial immunity of Miichthys miiuy to Vibrio anguillarum and V. harveyi. We also found a novel microRNA miR-2188-3p that could target TAK1 and inhibit the host antibacterial response and promote bacterial escape. We further found that the antibacterial effect inhibited by miR-2188-3p could be reversed with TARL. Moreover, V. anguillarum and V. harveyi are the two most susceptible Gram-negative pathogens of aquaculture fish, and the economic losses caused by these two bacteria are immeasurable every year. This study is the first to report on the ability of lncRNA to prevent the escape of V. anguillarum and V. harveyi in fish through the competing endogenous RNA (ceRNA) mechanism. Our results not only elucidate the ceRNA mechanism of the lncRNA in antibacterial immune responses but also provide new insights into the impact of lncRNA on host immunity and bacterial escape.

Genomic organization, evolution and functional characterization of embryonic lethal abnormal vision like protein 1 (ELAVL1) in miiuy croaker (Miichthys miiuy).

Embryonic lethal vision-like protein 1 (ELAVL1), an AU-rich elements (AREs) binding protein involved in the regulation of inflammatory transcript stability, which has not been reported in fish. In this study, we identified the ELAVL1 gene in Miichthys miiuy (mmiELAVL1), and then analyzed its structure and evolution, furthermore described its expression pattern in miiuy croaker. The results showed that mmiELAVL1 and other vertebrate ELAVL1 genes all have three highly conserved RNA recognition motif (RRM) protein domains, and the structure and protein structure are evolutionarily conserved, indicating that their functions may also conservative. In healthy miiuy croaker, mmiELAVL1 was commonly expressed in the tested tissues, and mmiELAVL1 is mainly localized in the nucleus of kidney cells. In addition, mmiELAVL1 responds to poly(I:C) and SCRV stimulation and promotes antiviral genes, indicating its active role in immune process. In summary, this study will facilitate future studies on the role and underlying mechanisms of ELAVL1 in fish immune responses.

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.

microRNA-27c negatively regulates NF-κB and IRF3 signaling pathway via targeting MITA in miiuy croaker.

As a non-coding RNA with regulatory functions, microRNAs(miRNAs) can regulate gene expression and participate in a variety of physiological and pathological processes. In recent years, although there have been many studies on miRNA, the regulation mechanisms of miRNA in teleost fish have not been fully elucidated. In this study, it was first predicted that MITA is the target of miR-27c through bioinformatics, and it was confirmed by dual fluorescence experiments. Then we found that miR-27c can inhibit the expression of MITA at the mRNA and protein levels, thereby promoting the NF-κB or IRF3 pathway. It is speculated that miR-27c plays an important role in the innate immunity of teleost fish. This study will help to further understand miRNAs regulatory mechanism in teleost fish.

Efficient blue TADF-type organic afterglow material via boric acid-assisted confinement.

A heat treatment method is developed to produce blue afterglow materials, achieving a photoluminescence quantum yield of 65% and an emission lifetime of 0.18 s (afterglow: >2 s). The afterglow is attributed to TADF of norfloxacin, activated by the confinement effect of boric acid.

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.

Rho-GDP-dissociation inhibitor-γ negatively regulates NF-κB signaling by promoting the degradation of TAK1 in miiuy croaker (Miichthys miiuy).

Transforming growth factor-beta activated kinase 1 (TAK1) is an adaptor molecular in TLR-mediated NF-κB signaling pathway and plays indispensable roles in innate immunity. As the most typical innate immune pathway, the strict regulation of NF-κB signaling pathway is particularly important. Rho-GDP-dissociation inhibitor-γ (Rho-GDIγ) is a member of the Rho protein family that regulates many important physiological processes. In this study, we demonstrated the mechanism of suppressing TAK1 expression in the teleost and found that Rho-GDIγ negatively regulated the NF-κB signaling pathway mediated by TAK1. We determined that TAK1 could directly interact with Rho-GDIγ. It is interesting that Rho-GDIγ promotes TAK1 degradation through the ubiquitin proteasome pathway. This study brings a new experimental basis to the teleost fish innate immune signaling pathway. Moreover, this discovery may provide new insights into innate immune regulation mechanism in mammals.

Long Noncoding RNA MIR122HG Inhibits MAVS-Mediated Antiviral Immune Response by Deriving miR-122 in Miiuy Croaker (Miichthys miiuy).

Long noncoding RNAs (lncRNAs) function as micro regulators to impact gene expression after multiple pathogen infections, which have been largely studied in the last few years. Although lncRNA studies on lower vertebrates have received less attention than those on mammals, current studies suggest that lncRNA plays an important role in the immune response of fish to pathogen infections. Here, we studied the effect of MIR122HG as the host gene of miR-122 and indirectly negatively regulate MAVS-mediated antiviral immune responses in miiuy croaker (Miichthysmiiuy). We found that poly(I:C) significantly increases the host MIR122HG expression. The increased MIR122HG expression inhibited the production of the antiviral immune-related genes IFN-1, ISG15 and Viperin upon SCRV treatment. In addition, MIR122HG can act as a pivotally negative regulator involved in the MAVS-mediated NF-κB and IRF3 signaling pathways, which can effectively avoid an excessive immune response. Additionally, we found that MIR122HG can promote the replication of SCRV. Our study provides evidence about the involvement of lncRNAs in the antiviral immune response of fish and broadens the understanding of the function of lncRNAs as a precursor miRNA in teleost fish.

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.