| 其他摘要 | 免疫系统是机体对抗入侵微生物的重要武器，通过识别并清除入侵机体的病原微生物，使机体内环境保持稳定。先天性免疫系统是机体抗病毒感染的第一道防线。线粒体抗病毒蛋白 (MAVS)是第一个被发现的与先天免疫相关的线粒体蛋白，在抗病毒先天免疫过程中扮演着至关重要的角色。MAVS蛋白通过直接参与由视黄酸诱导基因I (RIG-I)和黑色素瘤相关基因5 (MDA5)介导的先天免疫反应信号通路起到抗病毒的作用。细胞内受体 RIG-I通过识别病毒早期复制中间产物，继而与 MAVS结合，激活 NFκB 和IRF3信号通路，诱导β-干扰素 (β-interferon, IFN-β)的表达，起到抑制病毒复制的作用。自2005年MAVS蛋白被四个课题组独立发现以来，十年来展开了针对MAVS的一系列研究，这些研究主要集中在解析MAVS蛋白的结构、功能以及亚细胞定位，通过这些研究我们从多方面认识了MAVS在抗病毒先天免疫方面的功能。真核生物的大多数基因表达在转录、转录后、翻译和翻译后等不同阶段都受到严格的调控，其中在转录后水平上的调控主要涉及到 mRNA 稳定性、翻译效率、以及亚细胞定位等环节。参与这些调控的各种顺式调控元件 (Cis-acting element)主要位于mRNA中的非翻译区，包括 5’UTR 和 3’UTR 区域。研究发现脊椎动物大多数基因的3’UTR片段比5’UTR 更长，且 3’UTR 平均长度在生物进化过程中有逐渐增加的趋势，这暗示3’UTR在基因表达调控中可能具有更重要的作用。真核生物中许多蛋白的亚细胞定位是通过信号肽序列完成的，但有些mRNA并不具有信号肽序列，它们的亚细胞定位则受到3’UTR 的调控。目前登记在GenBank数据库中的人类MAVS 基因的3’UTR区域长度为9976 bp，远远高于各类基因中3’UTR 的平均值 (1027.7 bp)，这暗示该基因3’UTR区域在基因表达调控中可能发挥着重要的生物学功能。本研究针对MAVS 3’UTR的功能开展细胞水平初步探索，我们构建了不同长度的MAVS 3’UTR表达载体，深入分析其在抗病毒先天免疫中的作用。研究发现MAVS 3’UTR各片段具有不同的功能，主要包括以下三个方面：① MAVS 3’UTR H1 (1-3386)片段中含有抑制其表达的顺式作用元件ARE (AU-rich element)，ARE通过与RNA结合蛋白相互作用来促进MAVS mRNA降解，从而抑制MAVS蛋白的表达，维持细胞的稳态，避免过度的免疫应答反应；② MAVS 3’UTR H5 (5955-7687)片段中存在亚细胞定位序列，可促进蛋白定位于线粒体附近，但其具体调控机制目前还不清楚，还有待进一步研究；③ MAVS 3’UTR中含有多个miRNA-27a结合位点，miRNA-27a通过与MAVS 3’UTR相互作用抑制MAVS的蛋白表达。在病毒感染时，病毒感染可以通过上调miRNA-27a表达进一步抑制MAVS蛋白，从而达到逃避宿主免疫监视的目的。综上所述，MAVS 3’UTR通过调控其蛋白表达及蛋白亚细胞定位等多重手段调节MAVS蛋白功能，在抗病毒先天免疫中发挥重要作用。; Immune system is the key weapon to defend against the microorganisms, it recognizes and cleans up the infected microorganisms to maintain the stability of internal environment. Innate immune system is the first defense line of our body to resist the virus infection. The mitochondrial antiviral signaling protein (MAVS), which is located on the outer membrane of mitochondria, plays an important role in antiviral innate immunity. MAVS is the adaptor protein for RIG-I and MDA5, both are members of RIG-I like receptors (RLRs) and serve as the main receptors to recognize RNA virus. RIG-I can combine with MAVS after being activated by RNA virus, and induce the production of IFN-β through the NFκB and IRF3 signaling pathways. In the past decade, the MAVS has been widely studied, with a focus on its structural domain, subcellular localization, and its complex function.The expression of most eukaryotic genes is strictly regulated at the transcriptional, post-transcriptional, translational and post-translational levels. The post-transcriptional regulation controls the stability of mRNA, the translation efficiency, and the subcellular localization, to name a few. The cis-acting elements that are involved in the post-transcriptional regulations are the untranslated regions (or UTR) of mRNA, including the 5’UTR and 3’UTR. It has been reported that the 3’UTRs of most genes in vertebrates are longer than the 5’UTRs, and the average length of the 3’UTRs are elongated during the evolution. All these observations suggested that the 3’UTR may play an important role in regulating gene expression. The subcellular localization of many eukaryotic proteins is controlled by the signal peptide. However, some mRNAs have no signal peptide. For these proteins, their subcellular localization is regulated by the 3’UTRs. According to the GenBank database, the length of the 3’UTR of human MAVS gene is 9976 bp, much longer than the reported average length (1027.7 bp) of the 3’UTR of most of human genes. Why human MAVS has such a long 3’UTR and what is its function in innate immunity are unresolved questions.In this study, we aimed to characterize the function of the MAVS 3’UTR. We cut the 3’UTR region into five different fragments (H1-H5) and inserted them into pCS-EGFP-N2 vector and psiCHECK2 vector, to define its potential role in controlling subcellular localization and to identify potential miRNA binding sites, respectively. We found that different fragments of the MAVS 3’UTR play different roles in regulating the subcellular localization and function of MAVS. First, there are two AU-rich elements (ARE) in the MAVS 3’UTR H1 fragment (region 1-3386 in the 3’UTR; numbering starts from the first nucleotide after the stop codon) that can suppress the expression of MAVS by promoting the degradation of its mRNA. Second, the MAVS 3’UTR H5 fragment (region 5955-7687) can affect the cellular localization of MAVS protein into mitochondria, thus affect the subsequent antiviral function of MAVS. Finally, there are four miRNA-27a binding sites in the MAVS 3’UTR. Further cellular analyses showed that miRNA-27a can inhibit the expression of MAVS and promote the replication of vesicular stomatitis virus (VSV). In summary, we provided the direct evidence that the MAVS 3’UTR plays an important role in antiviral innate immune response by regulating the expression and affecting the subcellular localization of MAVS. This observation offers a good example for the complex role of 3’UTR of key gene in innate immunity. |
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