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.

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.

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.