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

细胞质聚（ADP-核糖）聚合酶的转录后基因调控

• 批准号: 8886016
• 负责人: Anthony K L Leung
• 金额: $ 34.72万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8886016
• 关键词: Ablation; Adenosine Diphosphate Ribose; Antiviral Agents; Antiviral Response; Binding; Binding Proteins; Biochemical; Biochemistry; Biogenesis; Biological Assay; Biology; Cell Nucleus; Cell physiology; Cells; Clinical Trials; Collaborations; Cytoplasm; Cytoplasmic Granules; DNA; DNA Repair; Data; Development; Ebola virus; Embryo; Ensure; Enzymes; Funding; Gene Expression; Gene Expression Regulation; Genetic Transcription; HIV; Hepatitis B Virus; Human; In Vitro; Individual; Integration Host Factors; Laboratories; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolism; MicroRNAs; Modeling; Mus; Nuclear; Nucleotides; Organism; Pharmacologic Substance; Physiological; Play; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymerase; Polymers; Polynucleotides; Post-Translational Protein Processing; Protein Binding; Protein Isoforms; Proteins; Proteome; Proteomics; RNA; RNA Binding; RNA-Binding Proteins; Regulator Genes; Relative (related person); Reporter; Role; Sindbis Virus; Site; Stress; Surface; System; Tankyrase; Techniques; Testing; Therapeutic; Universities; Untranslated RNA; Viral; Viral Physiology; Virus Diseases; Washington; Zinc; biological adaptation to stress; inhibitor/antagonist; innovation; novel; nucleic acid binding protein; overexpression; public health relevance; response; success

 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.

 描述(申请人提供)：基因表达是由DNA和RNA结合蛋白在每个生物体中介导的。基因表达的调节通常是通过这些核酸结合蛋白的蛋白质修饰来实现的。这项建议的重点是一种未被探索但具有重要治疗意义的蛋白质修饰，称为聚(ADP-核糖)(PAR)。PAR在细胞核DNA修复和转录中的作用已广为人知。最近，我们发现PAR还可以修饰细胞质中几个转录后的mRNA基因调控因子。我们的数据与最近的蛋白质组学研究相一致，这些研究表明，聚(ADP-核糖基)化(PARylated)蛋白质组富含RNA结合蛋白，表明PAR在RNA代谢中发挥的调节作用比先前认识的要广泛得多。在这个提案中，我们将重点研究PAR如何调节microRNA的功能。MicroRNAs是一类~22个核苷酸的非编码RNA，调节许多基本的细胞过程，包括应激反应。虽然关于microRNA的生物发生已经有了很多特征，但关于microRNA活动是如何调节的却知之甚少。关键数据：最近的数据，包括我们的数据表明，microRNA的活性被核心microRNA结合蛋白ArgAerte(AGO)的PAR化抑制。这种抑制由PAR聚合酶13(PARP-13)调节，PARP-13的过度表达降低了microRNA的活性。有趣的是，PARP-13是催化不活跃的；因此，必须涉及其他具有催化活性的PARP(S)。这种涉及多个PARP的PAR化机制代表了一种新的范式。在这项提案中，我们将研究PARP-13如何与细胞质中具有催化活性的PARP相互作用以形成PARylate AGO(目标1)，确定AGO上的PAR聚合物如何降低microRNA活性(目标2)，并确定PAR修饰AGO的哪些结构域(目标3)。我们将使用一种新的质谱学技术来鉴定AGO的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，因此本研究首次系统地分析了蛋白质底物个别位点的功能作用。O请注意，PARP-13也称为锌抗病毒蛋白(ZAP)--一种宿主因子，可在过度表达时抑制辛德比斯病毒、埃博拉病毒、乙肝病毒和艾滋病毒的复制。因此，AGO PAR化介导的对microRNA活性的抑制可能参与宿主的抗病毒反应，我们将在Sindbis病毒感染(AIM 3c)的背景下与约翰霍普金斯大学的Diane Griffin博士合作探索这一点。团队：为了确保成功，这个项目是与两个关键的合作者(我们都在申请一个资助模块)一起执行的：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)。其他顾问包括麻省理工学院的Phillip Sharp博士和哈佛大学的Carl Novina博士，PAR生物学的Ted Dawson博士和Paul Chang博士，以及Johns Hopkins的Pierre Coulombe博士。