Regulation of PKR by Novel RNA Motifs

描述(由申请人提供)：本申请的目的是揭示RNA结构在调节RNA激活的蛋白激酶(PKR)生物学活性中的作用。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，改变先天免疫反应。在先天免疫中起关键作用的是干扰素诱导的RNA活化蛋白激酶，即PKR。在体内，PKR的主要激活剂是长dsRNA(&gt；33bp)，它可以连接两个PKR单体，提高它们的有效浓度。与dsRNA的相互作用也解除了激酶中的抑制相互作用。随后，PKR进行反式自动磷酸化，激活它使eIF21磷酸化，从而抑制翻译的启动。尽管dsRNA在体外具有明显的激活PKR的能力，但目前还不清楚这些RNA是否是体内PKR的主要激活剂。这一建议的中心假设是，体内对PKR的调控是由具有非常规结构的新RNA基序介导的。特别是，有证据表明，单链RNA以5‘-三磷酸依赖的方式激活PKR，这种激活被7 mg和单磷酸的细胞5’端签名所阻断。由于5‘-三磷酸存在于许多致病RNA上，这提示了一种新的病原体相关分子模式(PAMP)，该模式被PKR识别。此外，更多的证据表明，自身RNA也通过内部核苷修饰来区分，这被证明可以取消PKR的激活。该提案的中心目标如下：1.)确定单链RNA以5‘-三磷酸特异的方式激活PKR的机制，通常是在短茎环的辅助下。建立了5‘-三磷酸和短茎环与PKR相互作用的分子模型，并建立了活化的机理框架。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，而球形RNA三级结构折叠以掩盖PKR激活RNA二级结构。4.)识别体内调节PKR的病毒和内源性RNA。使用有(或没有)水泡性口炎病毒感染和感染的交联和免疫沉淀(CLIP)技术。这些特定的目标将通过各种生化和分子生物学技术来实现，包括RNA和蛋白质突变、体外和体内PKR和eIF21激活分析、动力学和热力学测量以及CLIP实验。结合分析将通过荧光偏振和ITC进行；蛋白质二聚化将通过下拉试验、交联和分析性超速离心法进行监测；RNA-蛋白质相互作用将通过化学交联和诱变进行分析；RNA三级结构将通过天然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对外来生物和病毒的早期保护，而PKR蛋白是人类这种反应的重要组成部分。这一建议旨在了解致病RNA中新的分子模式是如何被PKR识别为不同于自身RNA的，从而启动先天免疫反应。将研究具有人类免疫缺陷病毒(HIV)和丁型肝炎病毒(HDV)潜在致病模式的病毒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.