Dicer DNA nickase activity and its role in anti-viral immunity in human cells

摘要 DICER是一种多结构域核糖核酸酶III(RNaseIII)，可产生少量干扰 来自dsRNA前体的RNA(SiRNAs)和microRNAs(MiRNAs)。RNA沉默介导的 这些小RNA是抵御病毒感染的重要防御系统。失去了迪切尔 导致抗病毒免疫力丧失。 先前对线虫的研究表明，PRO-2对线虫核糖核酸酶的切割作用 凋亡的caspase-3产生稳定的Dester的C-末端片段，现在作为一种 脱氧核糖核酸酶(DNase)在发育过程中的作用。然而，更多的 经过十多年的这项研究，无论是将核糖核酸酶转化为DNA酶的DICER由特定的 蛋白分解在任何其他动物中都会发生，包括人类。迪格尔是否 DNA酶在抗病毒免疫中的作用也尚不清楚。 当细胞被病毒感染时，尽管它们的抗病毒系统，它们中的许多经历 程序性细胞死亡(PCD或细胞凋亡)，有助于中止产生和释放 从细胞中分离出后代病毒。越来越多的证据表明，有些蛋白质既有一天也有一天- 在健康细胞中的工作和在经历PCD的细胞中的死亡工作。因此，进化可能具有 将日间工作和死亡工作联系在一起，以确保细胞死亡适当 与多个正常的细胞过程相连并受其调节。 在这个提议中，我们假设人类Dester在RNA沉默中有作为核糖核酸酶的日常工作 途径和死亡工作作为PCD途径中的DNA酶。具体地说，我们将检验这个假设 促凋亡的caspase-3对人DICER的切割产生稳定的C-末端 脱氧核糖核酸脱氢酶(Dcr-C)片段，在PCD途径中起DNA酶(DNase)的作用 人类细胞。我们还将检验这样一种假设，即DICER向DCR-C及其DNA的转换 尼克酶活性对抗病毒免疫至关重要。我们令人振奋的初步研究表明 DCR-C在试管和细胞中显示出DNA镍酶活性，支持我们的假说。如果 如果成功，拟议的使用生化和细胞方法的研究将形成 猪冠状核糖核酸酶转化为脱氧核糖核酸酶及抗病毒作用的动物研究基础 哺乳动物的免疫力。了解DCR-2的生理作用和分子机制 C DNA尼克酶将揭示PCD途径和抗病毒系统的新调节机制 包括一种新的联系之间的RNA沉默和PCD通过迪格尔，这两个是至关重要的 防病毒防御。

Abstract Dicer is a multi-domain ribonuclease III (RNase III) enzyme that produces small interfering RNAs (siRNAs) and microRNAs (miRNAs) from dsRNA precursors. RNA silencing mediated by these small RNAs is an important defense system against viral infection. Loss of Dicer causes loss of anti-viral immunity. Previous study in C. elegans showed that cleavage of the C. elegans RNase Dicer by pro- apoptotic caspase-3 produces a stable C-terminal fragment of Dicer that now functions as a deoxyribonuclease (DNase) in the developmental apoptosis pathway. However, even more than a decade after this study, whether the RNase-to-DNase conversion of Dicer by specific proteolysis occurs in any other animals including human remains unknown. Whether Dicer DNase has any role in anti-viral immunity also remains unknown. When cells are infected by viruses despite their anti-viral system, many of them undergo programmed cell death (PCD or apoptosis), which helps to abort the production and release of progeny viruses from the cells. Growing evidence indicates that some proteins have both ‘day- jobs’ in healthy cells and ‘death-jobs’ in cells undergoing PCD. Thus, evolution may have linked day-jobs and death-jobs in the same molecule to ensure that cell death is appropriately linked to and regulated by multiple normal cellular processes. In this proposal, we hypothesize that human Dicer has day-jobs as an RNase in RNA silencing pathway and death-jobs as a DNase in PCD pathway. Specifically, we will test the hypothesis that cleavage of the human Dicer by pro-apoptotic caspase-3 produces a stable C-terminal fragment of Dicer (Dcr-C) that functions as a DNase (DNA nickase) in the PCD pathway in human cells. We will also test the hypothesis that the conversion of Dicer to Dcr-C and its DNA nickase activity are crucial for anti-viral immunity. Our exciting preliminary studies showed that Dcr-C exhibits DNA nickase activity in test tube and in cells, supporting our hypotheses. If successful, the proposed studies employing biochemical and cellular approaches will form foundations for future animal studies of Dicer RNase-to-DNase conversion in PCD and anti-viral immunity in mammals. Understanding the physiological roles and molecular mechanisms of Dcr- C DNA nickase will reveal new regulatory mechanisms in the PCD pathway and anti-viral system including a novel link between RNA silencing and PCD via Dicer, both of which are crucial for anti-viral defense.