CircYthdc2 generates polypeptides through two translation strategies to facilitate virus escape.

It is known that about 10 circular RNAs (circRNAs) can encode functional polypeptides in higher mammals. However, it is not clear whether the functional polypeptides that can be translated by circRNAs are only the products of the evolution of higher animals, or also widely exist in other lower organisms. In addition, it is also unclear whether the two ways of translating polypeptides using IRES and m6A in the one circRNA are exclusive or coexistent. Here, we discovered a novel circRNA derived from the 3'-5' RNA helicase Ythdc2 (Ythdc2) gene in lower vertebrate fish, namely circYthdc2, which can translate into a 170 amino acid polypeptide (Ythdc2-170aa) through IRES sequence or m6A modification, and is involved in antiviral immune of fish. Moreover, SCRV infection can promote circYthdc2 translate Ythdc2-170aa. Then, we found that both Ythdc2-170aa and Ythdc2 can promote the degradation of STING by promoting the ubiquitination modification of K11 and K48 link of STING, and weaken the host's antiviral innate immunity. Notably, when circYthdc2 is abundant, Ythdc2 preferentially degrades circYthdc2 and no longer promotes the degradation of STING. Further studies have shown that circYthdc2 is highly conserved from lower vertebrates to higher mammals, and human circYthdc2 can also encode the same polypeptide and play a similar function to that of fish circYthdc2. This discovery confirms for the first time that the ability of circRNA to encode functional proteins is evolutionarily conserved, and finds that the ways of polypeptide translation by the same circRNA were diverse, which is of great significance for further elucidating the function and evolution of circRNAs in vertebrates.

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.

circCBL and its host gene CBL collaboratively enhance the antiviral immunity and antibacterial immunity by targeting MITA in fish.

IMPORTANCE: Increasing evidence indicates that circular RNAs exert crucial functions in regulating gene expression in mammals. However, the function of circRNAs in lower vertebrates still needs further exploration. Our research results demonstrated that circRNA, namely circCBL, is involved in modulating antiviral and antibacterial immune responses in lower vertebrates. In addition, our study also found that circCBL can serve as a competing endogenous RNA to facilitate MITA expression, thereby modulating MITA-mediated innate immunity. Further research has proved that the host gene CBL also promotes the expression of MITA, enhancing antiviral and antibacterial immune responses. Our study not only elucidated the underlying biological mechanism of the circRNA-miRNA-mRNA axis in the innate immune response of lower vertebrates but also unveiled the synergistic antibacterial and antiviral mechanisms between circRNA and its host gene in lower vertebrates.

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.

METTL3-Mediated m6A Modification of TRIF and MyD88 mRNAs Suppresses Innate Immunity in Teleost Fish, Miichthys miiuy.

Methyltransferase (METTL3), the most important N6-methyladenosine (m6A) writer, plays a vital role in regulating immune-related signaling pathways. However, the underlying mechanism of METTL3 action remains largely unknown, especially in lower vertebrates. The results of this study show that METTL3 inhibits innate immune response and promotes the infection of miiuy croaker, Miichthys miiuy, by Siniperca chuatsi rhabdovirus and Vibrio anguillarum. Significantly, the function of METTL3 in inhibiting immunity depends on its methylase activity. Mechanistically, METTL3 increases the methylation level of trif and myd88 mRNA, rendering them sensitive to degradation by the YTHDF2/3 reader proteins. By contrast, we found that the YTHDF1 reader protein promotes the translation of myd88 mRNA. In summary, these results indicate that METTL3-mediated m6A modification of trif and myd88 mRNAs suppresses innate immunity by inhibiting the TLR pathway, unveiling a molecular mechanism by which RNA methylation controls innate immunity to pathogens in the 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.

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.

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.

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.

Cytokine Receptor-Like Factor 3 Negatively Regulates Antiviral Immunity by Promoting the Degradation of TBK1 in Teleost Fish.

The cytokine receptor-like factor 3 (Crlf3) belongs to the orphan class I cytokine receptors and is identified as a neuroprotective erythropoietin receptor. In previous studies of Crlf3, few focused on its role in innate immunity. Therefore, this study explored the regulatory role of Crlf3 in innate immunity. TANK-binding kinase 1 (TBK1) is a vital adaptor protein for the activation of the RLRs-MVAS-IRF3 antiviral signaling axis; thus, its expression and activity must be tightly regulated to maintain immune homeostasis and avoid undesirable effects. Here, we report that Crlf3 is a negative regulator of type I interferon production. The expression of Crlf3 is induced by poly(I·C) or Siniperca chuatsi rhabdovirus (SCRV) treatment. Silencing of Crlf3 enhanced poly(I·C)- and SCRV-induced type I interferon production, whereas overexpression of Crlf3 suppressed type I interferon production. Mechanistically, Crlf3 interacted with TBK1 via its N domain and then inhibited type I interferon production by promoting TBK1 proteasomal degradation through K48-linked polyubiquitination. Our study shows that Crlf3 is a key factor for viral escape from innate antiviral immunity in fish and provides a new perspective on mammalian resistance to viral invasion. IMPORTANCE The expression of Crlf3 was upregulated with SCRV invasion, which proved that Crlf3 was involved in the regulation of the antiviral immune response. In this study, we found that the existence of Crlf3 promoted the replication of SCRV. Therefore, it is reasonable to believe that SCRV evades innate immune attack with the assistance of Crlf3. In addition, we report that Crlf3 negatively regulates interferon (IFN) induction by promoting the degradation of TBK1 in fish. We showed that Crlf3 is evenly distributed in the cytoplasm and interacts with TBK1. Further studies showed that Crlf3 specifically mediates K48-linked ubiquitination of TBK1 and promotes TBK1 degradation, resulting in a marked inhibition of retinoic acid-inducible gene I (RIG-I) downstream signaling.

FTO promotes innate immunity by controlling NOD1 expression via m6A-YTHDF2 manner in teleost.

N6-methyladenosine (m6A), the most abundant internal mRNA modification in eukaryotes, plays a vital role in regulating innate immunity. However, its underlying mechanism remains largely unknown, especially in lower vertebrates. The results of the present study show that fat-mass- and obesity-associated protein (FTO), also known as a m6A demethylase, improved the innate immunity and prevented Siniperca chuatsi rhabdo virus and Vibrio anguillarum infection in miiuy croaker. Significantly, FTO-promoted immunity was dependent on its m6A demethylase activity. In terms of mechanism, NOD1 has abundant methylation modification in its CDS and 3'UTR regions, and FTO can reduce its methylation level, thus avoiding its degradation by YTHDF2. In summary, our results indicate that the FTO-mediated m6A modification in NOD1 mRNA promotes innate immunity by activating the NOD-like receptor pathway, which provides a molecular mechanism for the regulation of immune response via RNA methylation in teleost.

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.

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.

Genomic organization, evolution and functional characterization of caspase-2 and caspase-8 in miiuy croaker (Miichthys miiuy).

As the central link and executor of cell apoptosis, the caspase protease family has received extensive attention in recent years. However, the genetic characteristics and immune functions of some caspases are still unknown in fish. In our study, we cloned the full-length caspase-2 (mmCasp2) and caspase-8 (mmCasp2) of miiuy croaker, then we analyzed characteristics and functions of these two genes which are upstream of the apoptosis cascade reaction. Mmcasp2 and mmCasp8 exhibited a conserved domain (CASc), and the different part is that the mmCasp2 has a CARD domain, while mmCasp8 have two DED domains. Sequence and evolution analysis results showed that caspase-2 is more conservative than caspae-8 in the process of evolution. Cellular localization analysis showed that the distribution of mmCasp2 and mmCasp2 was in cytoplasm. The real-time PCR analysis showed that these two caspases are constitutively expressed in different tissues, and the expression of mmCasp2 and mmCasp8 were up-regulated in the liver, spleen, and kidney after infection with V. anguillarum. Lastly, qRT-PCR and Luciferase assays analysis showed that mmCasp2 and mmCasp8 can inhibit the NF-кB pathway. In general, we systematically analyzed the structure, evolution and related functional experiments of the caspase-2 and caspase-8 in miiuy croaker, which will help further understand the role caspase family plays in the apoptosis and immune response.

Genome-wide identification and analysis of chemokine receptor superfamily in miiuy croaker, Miichthys miiuy.

The chemokine receptor (ChemR) superfamily, which is divided into 4 subfamilies (CXCR, CCR, XCR, and CX3CR), is the main receptors of chemokines in innate immune responses. In the current study, we have identified 27 ChemRs in miiuy croaker: 13 CCR genes, 11 CXCR genes, and 3 XCR genes. Multiple characteristics of these genes, including phylogeny, gene structures, conserved motifs, chromosome locations, evolutionary mechanism, and expression levels upon the bacterial challenge were analyzed. Gene structure and location analysis showed that all ChemR genes contain fewer introns (≤4) and they are unevenly distributed on the 12 chromosomes. And the XCR subfamily of miiuy croaker don't have the DRY motif of ChemR. Phylogenetic and synteny analysis showed that these genes experienced tandem and segmental duplication event in several species, and tandem duplication might be the main expansion way in miiuy croaker. The major ChemRs of each orthologous group in vertebrates were selected for molecular evolution analysis, the results of which indicated that compared with vertebrates, ChemRs of teleost fishes may have a relatively high evolutionary dynamic. In addition, a total of 21 positively selected codons were detected in vertebrate ChemRs under Model 8. RNA-Seq analysis and qRT-PCR verification demonstrated that CXCR3.2, CXCR5, and XCR1 genes were up-regulated significantly upon the Vibrio harveyi infection. These results provide valuable information for investigating the evolutionary relationships of chemokine receptor superfamily in miiuy croaker and laid the basis for further functional analysis.

New 4-N-phenylaminoquinoline derivatives as antioxidant, metal chelating and cholinesterase inhibitors for Alzheimer's disease.

A series of new 4-N-phenylaminoquinoline derivatives were designed, synthesized, and their anticholinesterase activities, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, metal-chelating ability were tested. Among them, compounds 11j, 11k and 11l had comparable inhibition activities to reference drug galantamine both in AChE and in BChE. Especially, compound 11j revealed the most potent inhibition to eeAChE and eqBChE with IC50 values of 1.20 µM and 18.52 µM, respectively. Furthermore, both kinetic analysis of AChE inhibition and molecular docking study indicated that compound 11j was mixed-type inhibitor, binding simultaneously to the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE, and propidium iodide displacement assay showed significant displacement of propidium iodide with compound 11k (25.80%) from PAS of eeAChE. More importantly, compound 11l displayed excellent DPPH radical scavenging activity (84% at 1 mg/mL), and its EC50 value was 0.328 µM. In addition, compounds 11a, 11j, 11k and 11l exhibited obvious biometal chelating abilities toward Al3+, Fe2+, Cu2+ and Zn2+ ions. Taken together, 4-N-phenylaminoquinoline derivatives targeting multiple pathogenetic factors deserve further investigation for treatment of AD.

Design, synthesis, evaluation and molecular modeling study of 4-N-phenylaminoquinolines for Alzheimer disease treatment.

Dual binding site acetylcholinesterase (AChE) inhibitors and butyrylcholinesterase (BChE) inhibitors have recently emerged as two classes of new anti-Alzheimer agents to positively modify the disease's course. In this work, a new series of 4-N-phenylaminoquinolines was synthesized and evaluated for their abilities to inhibit AChE and BChE. Compound 11b showed significant inhibitory activities on AChE and BChE with IC50 values of 0.86 and 2.65 µM, respectively, a lot better than that of reference drug galanthamine. Furthermore, docking study showed that compound 11b interacted simultaneously not only with active and peripheral sites of AChE, but also with all five regions of BChE active site. These findings suggest that these derivatives could be regarded as promising starting points for further drug discovery developments.