Regulation of PKR by Novel RNA Motifs

新型 RNA 基序对 PKR 的调控

• 批准号: 7774329
• 负责人: PHILIP C BEVILACQUA
• 金额: $ 28.11万
• 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/7774329
• 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; 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; 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）：本申请的目的是揭示RNA结构在调节RNA激活蛋白激酶PKR的生物活性中的作用。病毒和细胞 RNA 折叠成不同的二级和三级结构，并与蛋白质相互作用以改变先天免疫反应。先天免疫中的一个关键角色是干扰素诱导的 RNA 激活蛋白激酶 PKR。体内 PKR 的主要激活剂是长 dsRNA (>33 bp)，它可以桥接两个 PKR 单体并增加其有效浓度。与 dsRNA 的相互作用也减轻了激酶中的抑制性相互作用。随后，PKR 进行反式自磷酸化，激活其磷酸化 eIF21，从而抑制翻译的起始。尽管 dsRNA 在体外具有明显激活 PKR 的能力，但仍不清楚此类 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'-三磷酸依赖性方式激活 PKR，该方式被 7mG 和单磷酸的细胞 5' 末端特征阻断。由于 5'-三磷酸出现在许多致病性 RNA 上，这表明 PKR 识别出一种新的病原体相关分子模式 (PAMP)。此外，其他证据表明，自身 RNA 的特点还在于内部核苷修饰，这些修饰被证明可以消除 PKR 激活。该提案的中心目标如下： 1.) 确定 ssRNA 以 5'-三磷酸特异性方式激活 PKR 的机制，通常在短茎环的帮助下。开发 5'-三磷酸和短茎环与 PKR 相互作用的分子模型，以及激活的机制框架。 2.) 确定转录后 RNA 修饰和非 Watson-Crick 基序在调节 PKR 激活中的作用。识别允许细胞 RNA 逃避 PKR 激活的修饰模式和非 Watson-Crick 基序。 3.) 确定短病毒RNA二级结构和球状三级基序在调节PKR激活中的作用。测试某些病毒二级结构 RNA 是否二聚形成 PKR 激活基序，而球状 RNA 三级结构是否折叠以掩盖 PKR 激活 RNA 二级结构。 4.) 鉴定体内调节 PKR 的病毒和内源 RNA。在有（和没有）水疱性口炎病毒转染和感染的情况下使用交联和免疫沉淀 (CLIP) 技术。这些具体目标将通过各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以及 CLIP 实验。结合测定将通过荧光偏振和 ITC 进行；将通过下拉分析、交联和分析超速离心来监测蛋白质二聚化； RNA-蛋白质相互作用将通过化学交联和诱变进行测定； RNA 三级结构将通过非变性凝胶电泳和结构图谱进行评估。 公共卫生相关性：先天免疫为宿主提供了针对外来生物和病毒的早期保护，而蛋白 PKR 是人类这种反应的重要组成部分。该提案旨在了解 PKR 如何将病原性 RNA 中的新分子模式识别为不同于自身 RNA，从而引发先天免疫反应。将研究来自人类免疫缺陷病毒（HIV）和丁型肝炎病毒（HDV）的具有潜在致病模式的病毒RNA。