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（RNase III）酶，其产生小的干扰， 来自dsRNA前体的RNA（siRNA）和microRNA（miRNA）。RNA沉默 这些小RNA是抵抗病毒感染的重要防御系统。切粒机损失 导致抗病毒免疫力丧失。 以前的研究在C. elegans显示C.线虫RNase Dicer 凋亡的半胱天冬酶-3产生稳定的Dicer C末端片段，该片段现在起着 脱氧核糖核酸酶（DNase）在发育性细胞凋亡途径中的作用。但更多 在这项研究之后的十多年中，是否特异性的Dicer的RNase到DNase的转化 蛋白质水解在包括人类在内的任何其他动物中发生仍然未知。是否切丁 脱氧核糖核酸酶在抗病毒免疫中有什么作用还不清楚。 当细胞被病毒感染时，尽管它们有抗病毒系统，它们中的许多细胞仍会经历 程序性细胞死亡（PCD或凋亡），这有助于中止生产和释放 从细胞中分离出后代病毒。越来越多的证据表明，一些蛋白质既有“白天”， 健康细胞中的“工作”和经历PCD的细胞中的“死亡工作”。因此，进化可能 在同一个分子中将日常工作和死亡工作联系起来，以确保细胞死亡是适当的， 与多种正常细胞过程相联系并受其调节。 在这个提议中，我们假设人类Dicer在RNA沉默中作为RNA酶有日常工作 途径和死亡工作作为一种DNA酶在PCD途径。具体来说，我们将测试假设 通过促凋亡caspase-3切割人Dicer产生稳定的C-末端 Dicer片段（Dcr-C），在PCD途径中作为DNA酶（DNA切口酶）发挥作用， 人体细胞我们还将检验Dicer转化为Dcr-C及其DNA 切口酶活性对于抗病毒免疫至关重要。我们令人兴奋的初步研究表明， Dcr-C在试管和细胞中显示DNA切口酶活性，支持我们的假设。如果 如果成功，将形成采用生物化学和细胞方法的拟议研究。 为将来在PCD和抗病毒中Dicer RNase到DNase转化的动物研究奠定了基础 哺乳动物的免疫力了解Dcr的生理作用和分子机制- C-DNA切口酶将揭示PCD途径和抗病毒系统中新的调控机制 包括RNA沉默和PCD之间通过Dicer的新联系，这两者对于 抗病毒防御

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.