Ago2 and a miRNA reduce Topoisomerase 1 for enhancing DNA cleavage in antibody diversification by activation-induced cytidine deaminase.
Ago2 and a miRNA reduce Topoisomerase 1 for enhancing DNA cleavage in antibody diversification by activation-induced cytidine deaminase.
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DOI：
10.1073/pnas.2216918120
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发表时间：
2023-05-02
影响因子：
11.1
通讯作者：
Honjo, Tasuku
中科院分区：
文献类型：
--
作者：
Kobayashi, Maki;Wakaguri, Hiroyuki;Shimizu, Masakazu;Higasa, Koichiro;Matsuda, Fumihiko;Honjo, Tasuku
AID （Activation-induced cytidine deaminase，激活诱导胞苷脱氨酶）在感染和免疫后通过免疫球蛋白基因重组产生类开关重组（class switch recombination， CSR）和体细胞超突变（somatic hypermutation， SHM）等抗体多样化过程中起着至关重要的作用。AID降低拓扑异构酶1 （Top1）蛋白，使DNA双链变为非b结构，增强DNA切割；然而，AID导致Top1下降的潜在机制尚不清楚。我们发现，在艾滋病依赖的ago2结合中，Top1 3'UTR在抑制Top1合成和增加DNA切割方面发挥着不可或缺的作用。此外，miR-92a-3p需要Top1 3'UTR来促进DNA切割并降低Top1。我们的研究结果表明，这种miRNA-Ago2与Top1 3'UTR结合，并以aids依赖的方式减少Top1，增强了Top1介导的DNA切割，这是抗体多样化所必需的。激活诱导胞苷脱氨酶（AID）是免疫球蛋白（Ig）基因通过类开关重组（CSR）和体细胞超突变（SHM）印迹免疫记忆的必需酶。艾滋病依赖性拓扑异构酶1 （Top1）的减少促进了Ig基因多样化时发生的DNA切割，而艾滋病诱导Top1减少的机制尚不清楚。在这里，我们阐明了microRNA-Ago2复合物在艾滋病依赖性Top1减少中的作用。Ago2结合到Top1 3'UTR上，带有两个aids依赖性的Ago2结合位点（5 ' -和3'dABs）。B淋巴瘤细胞中Top1 3'UTR敲除（3'UTRKO）导致IgH基因DNA断裂效率降低，同时伴有CSR和SHM频率的降低。此外，在3'UTRKO细胞中，艾滋病依赖性的Top1蛋白减少和ago2与Top1 mRNA的结合下调。蔗糖梯度高翻译部分的Top1 mRNA以aid依赖性和Top1 3 ' utr介导的方式减少，导致Top1蛋白合成减少。AID和Ago2都定位于mrna结合蛋白部分，并相互作用。此外，我们在Top1 mRNA中发现了一些可能与5 ' -和3'dAB结合的候选mirna。其中miR-92a-3p敲低诱导3'UTRKO细胞表型为野生型细胞，而对3'UTRKO细胞无影响。综上所述，Ago2-miR-92a-3p复合物将以艾滋病依赖的方式被募集到Top1 3'UTR中，并在转录后减少Top1蛋白的合成。这些结果导致非b -DNA结构的增加，增强Ig基因中Top1的DNA切割，并有助于免疫记忆的形成。
AID (Activation-induced cytidine deaminase) is essential for antibody diversification, such as class switch recombination (CSR) and somatic hypermutation (SHM) upon infection and immunization through immunoglobulin gene recombination. AID decreases the Topoisomerase 1 (Top1) protein to alter the DNA duplex into a non-B structure and enhance DNA cleavage; however, the underlying mechanisms of the Top1 decrease by AID are poorly understood. We found the indispensable role of Top1 3′UTR in the AID-dependent Ago2-binding for suppressing Top1 synthesis and increasing DNA cleavage. Furthermore, miR-92a-3p requires Top1 3′UTR to promote DNA cleavage and decrease Top1. Our findings suggest that this miRNA-Ago2 binds to Top1 3′UTR and reduces Top1 in an AID-dependent manner, enhancing Top1-mediated DNA cleavage, which is required for antibody diversification. Activation-induced cytidine deaminase (AID) is the essential enzyme for imprinting immunological memory through class switch recombination (CSR) and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. AID-dependent reduction of Topoisomerase 1 (Top1) promotes DNA cleavage that occurs upon Ig gene diversification, whereas the mechanism behind AID-induced Top1 reduction remains unclear. Here, we clarified the contribution of the microRNA-Ago2 complex in AID-dependent Top1 decrease. Ago2 binds to Top1 3′UTR with two regions of AID-dependent Ago2-binding sites (5′- and 3′dABs). Top1 3′UTR knockout (3′UTRKO) in B lymphoma cells leads to decreases in DNA break efficiency in the IgH gene accompanied by a reduction in CSR and SHM frequencies. Furthermore, AID-dependent Top1 protein reduction and Ago2-binding to Top1 mRNA are down-regulated in 3′UTRKO cells. Top1 mRNA in the highly translated fractions of the sucrose gradient is decreased in an AID-dependent and Top1 3′UTR–mediated manner, resulting in a decrease in Top1 protein synthesis. Both AID and Ago2 localize in the mRNA-binding protein fractions and they interact with each other. Furthermore, we found some candidate miRNAs which possibly bind to 5′- and 3′dAB in Top1 mRNA. Among them, miR-92a-3p knockdown induces the phenotypes of 3′UTRKO cells to wild-type cells whereas it does not impact on 3′UTRKO cells. Taken together, the Ago2-miR-92a-3p complex will be recruited to Top1 3′UTR in an AID-dependent manner and posttranscriptionally reduces Top1 protein synthesis. These consequences cause the increase in a non-B-DNA structure, enhance DNA cleavage by Top1 in the Ig gene and contribute to immunological memory formation.
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影响因子：
16.6
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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