Direct lysine dimethylation of IRF3 by the methyltransferase SMYD3 attenuates antiviral innate immunity [II]. Direct lysine dimethylation of IRF3 by the methyltransferase SMYD3 attenuates antiviral innate immunity [II]

Interferon regulatory factor 3 (IRF3) is the key transcription factor in the type I IFN signaling pathway, whose activation is regulated by multiple posttranslational modifica_x0002_tions. Here, we identify SMYD3, a lysine methyltransferase, as a negative regulator of IRF3. SMYD3 interacts with IRF3 and catalyzes the dimethylation of IRF3 at lysine 39. This modification reduces IRF3 phosphorylation, dimerization, and subsequent nuclear translocation, leading to the inhibition of downstream type I interferon production. In addition, Smyd3-deficient mice are more resistant to RNA and DNA viral infections. Zebrafish lacking smyd3 or treated with the inhibitor BCI121 are also more resistant to viral infection. Our findings reveal a role for SMYD3 in the regulation of antiviral innate immunity and provide insight into a unique modulation of IRF3 that affects its activation. Overall design: Whole RNA from MEF cells was purified using the RNeasy Mini Kit (QIAGEN, #74104). To ensure the quality of the information analysis, fastp software was used to remove the splice sequences, filter the low quality, N bases (indicating that the base information could not be determined), and obtain high quality clean data. The clean data and the number of bases and sequences of the clean data were counted. At the same time, the GC, Q20, Q30 contents of the clean data were calculated (Benagen Technology, Wuhan, China). Volcano Plot were generated using the ggplot2 software. Heatmaps were generated using the Multi Experiment Viewer (MeV) software. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses for the differentially expressed genes (DEGs) was performed using Cluster Profiler version 3.8.

干扰素调节因子3（Interferon regulatory factor 3, IRF3）是I型干扰素信号通路中的关键转录因子，其激活受多种翻译后修饰调控。本研究鉴定出赖氨酸甲基转移酶SMYD3为IRF3的负向调控因子：SMYD3可与IRF3相结合，并催化IRF3第39位赖氨酸的二甲基化修饰。该修饰会降低IRF3的磷酸化、二聚化及后续核转位过程，进而抑制下游I型干扰素的产生。此外，Smyd3基因敲除小鼠对RNA和DNA病毒感染的抵抗力显著增强；缺失smyd3基因或经抑制剂BCI121处理的斑马鱼，同样对病毒感染表现出更强的抗性。本研究结果揭示了SMYD3在抗病毒天然免疫调控中的重要作用，并为影响IRF3激活的独特调控机制提供了全新见解。

实验整体设计：本研究使用RNeasy Mini试剂盒（QIAGEN，货号#74104）提取小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblasts, MEF）的总RNA。为保障后续信息分析质量，采用fastp软件去除接头序列、过滤低质量序列及N碱基（指代无法确定的碱基信息），以获得高质量干净测序数据；随后统计干净数据的碱基总数与序列条数，并计算干净数据的GC含量、Q20与Q30值（武汉贝瑞基因科技有限公司）。使用ggplot2软件绘制火山图（Volcano Plot），采用多实验查看器（Multi Experiment Viewer, MeV）软件绘制热图；使用Cluster Profiler v3.8软件对差异表达基因（Differentially Expressed Genes, DEGs）进行基因本体（Gene Ontology, GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析。