| Innate immunity is the host’s first line of defense against pathogen invasion,and pathogen recognition mediated by pattern recognition receptors(PRRs)is critical for the initiation of innate immune responses.PRRs induce subsequent immune responses by recognizing pathogen-associated molecular patterns(PAMPs)and activating different signaling pathways.m~6A methylation is one of the most common RNA modifications in eukaryotes and can affect multiple aspects of RNA processing,including mRNA splicing,translation,localization,decay,and stability.m~6A labeling is dynamic and has been shown to affect a variety of biological processes,such as embryonic stem cell differentiation,early development,circadian rhythms,cell signaling,and is implicated in multiple aspects of cancer.As the second m~6A methyltransferase to be discovered,METTL16 is a conserved U6 sn RNA methyltransferase as well as a SAM synthase MAT2A pre-mRNA methyltransferase.Recently,there are also increasing data showing that m~6A methylation-related genes can regulate multiple immune pathways,but there are few reports about METTL16.In this paper,miiuy croaker as the research object,some bioinformatics analysis of the METTL16 gene was carried out,and the function of METTL16,especially in the innate immune pathway,was discussed.Here are the main content from the study:1.Using the homologous species METTL16 gene sequence,through the BLAST local miiuy croaker transcriptome and the genome database,obtain the METTL16 gene sequence information of miiuy croaker.The length of the METTL16 open reading frame(ORF)of miiuy croaker is 1685 bp,encoding a total of 561 amino acids.According to the prediction,there is a domain MTD in the N-terminal of METTL16.At the same time,the amino acid sequences of other species were analyzed,and it was found that the METTL16 genes in various species all had the N-terminal domain MTD,and the amino acid sequence similarity was as high as 92.96%.By comparing the three-dimensional structure of the MTD domain,it was found that the species were highly similar,which once again confirmed the structural conservation of METTL16.In addition,the METTL16 protein sequences of different species were used to draw an evolutionary tree, in which tetrapods were clustered into one group and fish were clustered into one group.Based on the genomic data of different species,the linear analysis of METTL16 gene showed that the gene types and directions on both sides of METTL16 gene were the same in all species.2.The expression pattern of METTL16 was analyzed by qRT-PCR.METTL16 is expressed in all healthy tissues of miiuy croaker.By detecting the changes of METTL16expression in the immune tissues of miiuy croaker before and after poly(I:C)stimulation,the results showed that the expression of METTL16 in the liver,spleen and kidney tissues increased with time,confirming that the expression of METTL16 responds to poly(I:C)stimulate.To further confirm the involvement of METTL16 in the antiviral immune response,the expression of METTL16 in kidney cells of miiuy croaker before and after poly(I:C)stimulation and SCRV infection was measured,and the results showed a significant increase in METTL16 expression.The involvement of METTL16 in antiviral immune response was demonstrated.3.The eukaryotic expression plasmid of METTL16 in miiuy croaker was constructed,and METTL16 was located in the nucleus as determined by immunofluorescence assay,indicating that METTL16 functions in the nucleus of cells.4.To further investigate the role of METTL16 in the antiviral immune response,the expression of inflammatory cytokines before and after METTL16 overexpression was detected using qRT-PCR,and the results showed that METTL16 was able to inhibit the expression of TNF-α,IFN-1,Mx1 and ISG15,and METTL16 also significantly inhibited the above inflammatory after poly(I:C)stimulation cytokine expression.In summary,METTL16 can suppress the innate immune response caused by the virus,laying the foundation for future studies on the function of METTL16. |
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