Post-transcriptional Gene Regulation by Cytoplasmic Poly(ADP-ribose) Polymerases

 描述（由申请人提供）：基因表达由每种生物体中的DNA和RNA结合蛋白介导。基因表达的调节通常由这些核酸结合蛋白的蛋白质修饰介导。该建议集中在一种未被开发但在治疗上重要的蛋白质修饰，称为聚（ADP-核糖）（PAR）。PAR在细胞核中的DNA修复和转录中的作用是众所周知的。最近，我们发现PAR还修饰了细胞质中的几个转录后mRNA基因调控因子。我们的数据是一致的，最近的蛋白质组学研究表明，聚（ADP-核糖基）化（PAR化）的蛋白质组富含RNA结合蛋白，这表明PAR在RNA代谢中起着比以前更广泛的调节作用。在本提案中，我们将重点关注PAR如何调节microRNA功能。MicroRNA是一类约22个核苷酸的非编码RNA，其调节许多基本细胞过程，包括应激反应。虽然已经有很多关于microRNA生物发生的特征，但对microRNA活性如何调节知之甚少。关键数据：最近的数据，包括我们的研究表明，microRNA的活动受到核心microRNA结合蛋白Argonaute（AGO）的PAR化的抑制。这种抑制受PAR聚合酶13（PARP- 13）调节，其中其过表达降低microRNA活性。有趣的是，PARP-13是无催化活性的;因此，必须涉及其他具有催化活性的PARP。这种涉及多于一种PARP的PAR化机制代表了一种新的范例。在这个提议中，我们将研究PARP-13如何与细胞质的、催化活性的PARP相互作用以PARylate AGO（Aim 1），确定AGO上的PAR聚合物如何降低microRNA活性（Aim 2），并确定AGO的哪些结构域被PAR修饰（Aim 3）。我们将使用一种新的质谱技术来鉴定AGO PAR化位点。到目前为止，PAR化位点的鉴定一直是该领域的一个挑战，因此这项研究允许首次系统分析蛋白质底物各个位点的功能作用。值得注意的是，PARP-13也被称为锌抗病毒蛋白（ZAP）-一种宿主因子，在过表达时抑制辛德毕斯病毒、埃博拉病毒、B型肝炎病毒和HIV的复制。因此，AGO PARylation介导的microRNA活性抑制可能参与宿主的抗病毒反应，我们将与约翰霍普金斯的Diane Griffin博士合作，在辛德毕斯病毒感染（Aim 3c）的背景下进行探索。团队：为了确保成功，该项目与两个关键合作者（我们正在申请一个资助模块）进行：AGO生物化学专家Leemor Joshua-Tor博士（冷泉港实验室）和蛋白质组学专家Shao-En Ong博士（华盛顿大学）。其他顾问包括菲利普夏普博士（麻省理工学院）和卡尔诺维纳（哈佛）的微RNA生物学，特德道森博士（约翰霍普金斯）和保罗张（麻省理工学院）的PAR生物学，和皮埃尔库伦博士（约翰霍普金斯）的生物化学。

 DESCRIPTION (provided by applicant): Gene expression is mediated by DNA- and RNA-binding proteins in every organism. Regulation of gene expression is commonly mediated by protein modifications of these nucleic acid binding proteins. This proposal focuses on an under-explored but therapeutically important protein modification called poly(ADP- ribose) (PAR). PAR has been well known for its roles in DNA repair and transcription in the nucleus. Recently, we discovered that PAR also modifies several post-transcriptional mRNA gene regulators in the cytoplasm. Our data are consistent with recent proteomics studies showing that poly(ADP-ribosyl)ated (PARylated) proteomes are enriched with RNA-binding proteins, suggesting that PAR plays a much broader regulatory role in RNA metabolism than previously appreciated. In this proposal, we will focus on how PAR regulates microRNA functions. MicroRNAs are a class of ~22 nucleotide non-coding RNAs that regulate many fundamental cellular processes, including stress responses. Although much has been characterized about microRNA biogenesis, little is known about how microRNA activities are regulated. Key data: Recent data including ours indicate that microRNA activities are inhibited by the PARylation of the core microRNA-binding protein Argonaute (AGO). Such inhibition is regulated by PAR polymerase 13 (PARP- 13) where its overexpression reduces microRNA activities. Intriguingly, PARP-13 is catalytically inactive; therefore, other catalytically active PARP(s) must be involved. Such a PARylation mechanism involving more than one PARP represents a new paradigm. In this proposal, we will investigate how PARP-13 interacts with a cytoplasmic, catalytically active PARP to PARylate AGO (Aim 1), determine how PAR polymers on AGO reduce microRNA activities (Aim 2) and identify which domains of AGO are modified by PAR (Aim 3). We will use a novel mass spectrometry technique to identify AGO PARylation sites. Until now, the identification of PARylation sites has been a challenge for the field and thus this study allows the first systematic analysis of the functional roles of individual sites of a protein substrate. O note, PARP-13 is also known as zinc antiviral protein (ZAP) - a host factor that inhibits the replication of Sindbis virus, Ebola virus, Hepatitis B virus and HIV upon overexpression. Therefore, AGO PARylation-mediated inhibition of microRNA activities may be involved in host antiviral responses, which we will explore in the context of Sindbis virus infection (Aim 3c) in collaboration with Dr. Diane Griffin at Johns Hopkins. The Team: To ensure success, this project is performed with two key collaborators (both of whom we are requesting for one funding module): AGO biochemistry expert Dr. Leemor Joshua-Tor (Cold Spring Harbor Laboratory) and proteomics expert Dr. Shao-En Ong (University of Washington). Other consultants include Drs. Phillip Sharp (MIT) and Carl Novina (Harvard) on microRNA biology, Drs. Ted Dawson (John Hopkins) and Paul Chang (MIT) on PAR biology, and Dr. Pierre Coulombe (Johns Hopkins) on biochemistry.