The m6A Reader YTHDF2 Modulates Antiviral and Antibacterial Activity by Suppressing METTL3 Methylation-Modified STING in Fish.

At present, N6-methyladenosine (m6A) modification has been proven to participate in a wide range of gene expression regulation, such as stability, translation, splicing, and output, among others, which has attracted much attention. Unlike mammals, however, the role of m6A in innate immunity of lower invertebrates has not yet been studied. In this study, we found that the total m6A level of Miichthys miiuy increased during Siniperca chuatsi rhabdovirus and Vibrio anguillarum infection, suggesting that m6A may play an important role in the immune process against pathogens in fish. In addition, our study shows that stimulator of IFN genes (STING) plays a dual immune function against viruses and bacteria in fish, and through degrading STING by identifying its m6A methylation site modified by methyltransferase-like 3 (METTL3), YTH domain family 2 (YTHDF2) can weaken the IRF3 and NF-κB-driven signaling pathway, thus weakening the innate immunity and promoting the infection of Siniperca chuatsi rhabdovirus and V. anguillarum to the M. miiuy. Although there have been reports on m6A modification of STING in mammals, it is still unclear whether there is also m6A modification in lower vertebrates, especially in fish. Therefore, our study provides a reference for filling the gap of m6A modification between fish and mammals.

Exon-Intron Circular RNA circRNF217 Promotes Innate Immunity and Antibacterial Activity in Teleost Fish by Reducing miR-130-3p Function.

Circular RNA (circRNA) is produced by splicing head to tail and is widely distributed in multicellular organisms, and circRNA reportedly can participate in various cell biological processes. In this study, we discovered a novel exon-intron circRNA derived from probable E3 ubiquitin-protein ligase RNF217 (RNF217) gene, namely, circRNF217, which was related to the antibacterial responses in teleost fish. Results indicated that circRNF217 played essential roles in host antibacterial immunity and inhibited the Vibrio anguillarum invasion into cells. Our study also found a microRNA miR-130-3p, which could inhibit antibacterial immune response and promote V. anguillarum invasion into cells by targeting NOD1. Moreover, we also found that the antibacterial effect inhibited by miR-130-3p could be reversed with circRNF217. In mechanism, our data revealed that circRNF217 was a competing endogenous RNA of NOD1 by sponging miR-130-3p, leading to activation of the NF-κB pathway and then enhancing the innate antibacterial responses. In addition, we also found that circRNF217 can promote the antiviral response caused by Siniperca chuatsi rhabdovirus through targeting NOD1. Our study provides new insights for understanding the impact of circRNA on host-pathogen interactions and formulating fish disease prevention to resist the severely harmful V. anguillarum infection.

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.

Circular RNA circSamd4a Regulates Antiviral Immunity in Teleost Fish by Upregulating STING through Sponging miR-29a-3p.

Circular RNAs (circRNAs) are a subgroup of endogenous noncoding RNA that is covalently closed rings and widely expressed. In recent years, there is accumulating evidence indicating that circRNAs are a class of important regulators, which play an important role in various biological processes. However, the biological functions and regulation mechanism of circRNAs in lower vertebrates are little known. In this study, we discovered a circRNA Samd4a (circSamd4a) that is related to the antiviral immune response of teleost fish. It can act as a key regulator of the host's antiviral response and play a key role in inhibiting Sininiperca chuatsi rhabdovirus replication. Further studies have shown that circSamd4a may act as a competing endogenous RNA, which can enhance the STING-mediated NF-κB/IRF3 signaling pathway by adsorbing miR-29a-3p, thereby enhancing the antiviral immune response. Therefore, circSamd4a plays an active regulatory role in the antiviral immune response of bony fish. Our research results provide a strong foundation for circular RNA to play a regulatory role in the antiviral immune response of 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.

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.

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.

CircMIB2 therapy can effectively treat pathogenic infection by encoding a novel protein.

The mRNA therapy is widely used in the treatment of diseases due to its efficient characteristics, and the COVID-19 vaccine is the application of mRNA therapy. However, due to the instability of mRNA, mRNA vaccines often need lots of modifications to ensure its stability. Recent research shows that circRNA with stable RNA structure can encode protein, which provides a new direction for mRNA therapy. Here, we discovered a novel circRNA (circMIB2) derived from E3 ubiquitin-protein ligase MIB2 (MIB2) gene in lower vertebrate fish, which can translate into a 134 amino acid protein (MIB2-134aa) through m6A modification, and is involved in innate immunity. MIB2-134aa is completely consistent with the amino acid sequence of the two domains of host gene MIB2 protein; host gene MIB2 can target TRAF6 through the two domains and inhibit the innate immune response by promoting the ubiquitination degradation of the K11-link of TRAF6, MIB2-134aa also targets TRAF6 through these same domains. Interestingly, MIB2-134aa greatly reduced the degradation of TRAF6 by its host gene MIB2. More importantly, we found that circRNA therapy of circMIB2 can significantly inhibit the colonization of Vibrio anguillarum in zebrafish, and it provides a new direction for the treatment of pathogenic diseases of 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.

microRNA-144 modulates the NF-κB pathway in miiuy croaker (Miichthys miiuy) by targeting IκBα gene.

MicroRNAs (miRNA) are non-coding RNAs that regulate many biochemical processes, such as cell growth, proliferation and immune responses. In this study, we investigated miR-144 as a regulator of IκBα that promotes the activation of NF-κB signaling pathway. And IκBα interact with p65 blocks nuclear translocation of NF-κB and anchors NF-κB in cytoplasmic quiescent cells in an inactive form. The seed region of miR-144 can regulate gene expression by binding to the 3' UTR of IκBα and repress IκBα expression at the post-transcriptional level. More importantly, miR-144 can promote the activation of p65 by inhibiting IκBα, thus affecting the NF-κB signaling pathway. Thus, preventing excessive inflammatory responses from causing autoimmune diseases will help to further understand the immunoregulatory mechanisms of miRNAs in fish after invasion by pathogens.