Regulation of PKR by Novel RNA Motifs

新型 RNA 基序对 PKR 的调节

• 批准号: 8035428
• 负责人: PHILIP C BEVILACQUA
• 金额: $ 27.81万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035428
• 关键词: Binding; Biochemical; Biological; Biological Assay; Biological Process; Chemicals; Complex; Dimerization; Double-Stranded RNA; Event; Fluorescence Polarization; Genomics; HIV; Hepatitis Delta Virus; Human; Immune response; Immunoprecipitation; In Vitro; Infection; Interferons; Kinetics; Lead; Maps; Masks; Measurement; Mediating; Messenger RNA; Modification; Molecular; Molecular Biology Techniques; Molecular Models; Monitor; Mutagenesis; N-terminal; Natural Immunity; Nucleosides; Organism; Pattern; Phosphotransferases; Proteins; RNA; RNA Folding; RNA-Protein Interaction; Regulation; Research; Ribonuclease III; Role; Structure; Techniques; Technology; Testing; Thermodynamics; Transcript; Transfection; Translation Initiation; Vesicular stomatitis Indiana virus; Viral; Viral Proteins; adenosine deaminase; analytical ultracentrifugation; base; crosslink; eIF-2 Kinase; gel electrophoresis; human DICER1 protein; immune RNA; in vivo; inhibitor/antagonist; insight; interest; molecular modeling; molecular recognition; monomer; novel; pathogen; programs; protein function; public health relevance; research study; response; stem; tripolyphosphate; viral RNA

DESCRIPTION (provided by applicant): The objective of this application is to uncover roles for RNA structure in regulating biological activity of the RNA-activated protein kinase, PKR. Viral and cellular RNAs fold into diverse secondary and tertiary structures and interact with proteins to alter the innate immune response. A key player in innate immunity is the interferon-induced RNA-activated protein kinase, PKR. The major activator of PKR in vivo has been proposed to be long dsRNA (>33 bp), which can bridge two PKR monomers and increase their effective concentration. Interaction with dsRNA also relieves an inhibitory interaction in the kinase. Subsequently, PKR carries out trans-autophosphorylation that activates it to phosphorylate eIF21, which inhibits the initiation of translation. Despite the obvious ability of dsRNA to activate PKR in vitro, it remains unclear whether such RNAs are major activators of PKR in vivo. The central hypothesis of this proposal is that regulation of PKR in vivo is mediated by novel RNA motifs with unconventional structures. In particular, evidence is presented that ssRNA activates PKR in a 5'-triphosphate-dependent fashion, which is blocked by cellular 5'-end signatures of 7mG and monophosphate. Because a 5'-triphosphate occurs on many pathogenic RNAs, this suggests a novel pathogen-associated molecular pattern (PAMP) that is recognized by PKR. Moreover, additional evidence suggests that self RNA is also distinguished by internal nucleoside modifications, which are shown to abrogate PKR activation. The central aims of the proposal are as follows: 1.) Determine the mechanism by which ssRNA activates PKR in a 5'-triphosphate-specific fashion, often with the assistance of short stem-loops. Develop a molecular model for interaction of the 5'-triphosphate and short stem-loop with PKR, as well as a mechanistic framework for activation. 2.) Establish roles for posttranscriptional RNA modifications and non-Watson-Crick motifs in modulating PKR activation. Identify patterns of modifications and non-Watson-Crick motifs that allow cellular RNAs to evade PKR activation. 3.) Determine roles for short viral RNA secondary structures and globular tertiary motifs in modulating PKR activation. Test if certain viral secondary structure RNAs dimerize to form PKR-activating motifs, while globular RNA tertiary structures fold to mask PKR-activating RNA secondary structures. 4.) Identify viral and endogenous RNAs that regulate PKR in vivo. Use cross-linking and immunoprecipitation (CLIP) technologies with (and without) vesicular stomatitis virus transfections and infections. These Specific Aims will be accomplished by a variety of biochemical and molecular biology techniques including RNA and protein mutagenesis, in vitro and in vivo PKR and eIF21 activation assays, kinetics and thermodynamic measurements, and CLIP experiments. Binding assays will be conducted by fluorescence polarization and ITC; protein dimerization will be monitored by pull-down assays, crosslinking, and analytical ultracentrifugation; RNA-protein interactions will be assayed by chemical crosslinking and mutagenesis; and RNA tertiary structure will be assessed by native gel electrophoresis and structure mapping. PUBLIC HEALTH RELEVANCE: Innate immunity offers a host early protection from foreign organisms and viruses, and the protein PKR is an important part of this response in humans. This proposal aims to understand how novel molecular patterns in pathogenic RNA are recognized by PKR as different from self RNA, which cause the innate immune response to be initiated. Viral RNAs with potentially pathogenic patterns from human immunodeficiency virus (HIV) and hepatitis delta virus (HDV) will be studied.

描述（由申请人提供）：本应用的目的是发现RNA结构在调节RNA激活蛋白激酶的生物学活性中的作用。病毒和细胞RNA折叠成多种二级和三级结构，并与蛋白质相互作用以改变先天免疫反应。先天免疫的关键参与者是干扰素引起的RNA激活蛋白激酶PKR。 PKR in act in Vivo的主要激活因子已被认为是长dsRNA（> 33 bp），可以桥接两个PKR单体并增加其有效浓度。与DSRNA的相互作用还可以缓解激酶中的抑制作用。随后，PKR进行反式自磷酸化，从而激活其磷酸化EIF21，从而抑制了翻译的启动。尽管DSRNA在体外激活PKR的能力明显，但尚不清楚此类RNA是否是体内PKR的主要激活剂。该提议的中心假设是，体内PKR的调节是由具有非常规结构的新型RNA基序介导的。特别是，有证据表明，ssRNA以5'-三磷酸依赖性的方式激活PKR，该方式被7mg和单磷酸盐的细胞5'-End特征阻塞。由于5'-三磷酸盐发生在许多致病性RNA上，因此这表明PKR识别出一种与病原体相关的分子模式（PAMP）。此外，其他证据表明，自我RNA也以内核苷修饰为区分，这些修饰被证明可以消除PKR激活。该提案的中心目的如下：1。）确定ssRNA以5'-三磷酸盐特异性方式激活PKR的机制，通常通常在短的茎环的帮助下。开发一种分子模型，用于5'-三磷酸盐和短茎环与PKR的相互作用，以及激活的机械框架。 2.）在调节PKR激活中，建立了转录后RNA修饰和非Watson-Crick图案的作用。识别修饰的模式和非沃特森式基序，以允许细胞RNA逃避PKR激活。 3.）确定短病毒RNA二级结构和球形三级基序在调节PKR激活中的作用。测试某些病毒二级结构RNA是否二聚以形成PKR激活基序，而球状RNA三级结构则折叠以掩盖PKR激活RNA二级结构。 4.）鉴定体内调节PKR的病毒和内源性RNA。使用交联和免疫沉淀（剪辑）技术（且没有）囊炎病毒病毒转染和感染。这些具体目标将通过多种生化和分子生物学技术来实现，包括RNA和蛋白质诱变，体外和体内PKR和EIF21激活测定，动力学和热力学测量方法以及夹子实验。结合测定将通过荧光极化和ITC进行。蛋白质二聚化将通过下拉测定，交联和分析性超速离心来监测； RNA - 蛋白质相互作用将通过化学交联和诱变来测定。 RNA三级结构将通过天然凝胶电泳和结构映射评估。 公共卫生相关性：先天免疫提供了宿主的早期保护，以防止外国生物体和病毒，而蛋白质PKR是人类这种反应的重要组成部分。该建议旨在了解PKR在致病性RNA中的新分子模式如何与自我RNA不同，这会导致先天免疫反应启动。将研究具有人类免疫缺陷病毒（HIV）和乙型肝炎病毒（HDV）的潜在致病模式的病毒RNA。