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活化蛋白激酶（PKR）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。干扰素诱导的rna激活蛋白激酶（PKR）在先天免疫中起着关键作用。在体内PKR的主要激活剂被认为是长dsRNA (bbbb33 bp)，它可以桥接两个PKR单体并提高它们的有效浓度。与dsRNA的相互作用也减轻了激酶的抑制相互作用。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在体外具有明显的激活PKR的能力，但在体内这些rna是否是PKR的主要激活因子尚不清楚。该建议的中心假设是体内PKR的调控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‘-三磷酸依赖的方式激活PKR，这种方式被细胞5’端7mG和单磷酸的特征所阻断。由于5'-三磷酸发生在许多致病rna上，这表明PKR识别了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也通过内部核苷修饰来区分，这被证明可以消除PKR的激活。该提案的主要目的如下：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。建立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)。建立转录后RNA修饰和非沃森-克里克基序在调节PKR激活中的作用。鉴定允许细胞rna逃避PKR激活的修饰模式和非沃森-克里克基序。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。检测某些病毒二级结构RNA是否二聚化形成激活pcr的基序，而球状RNA三级结构折叠以掩盖激活pcr的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（CLIP）技术治疗（和不治疗）水泡性口炎病毒转染和感染。这些特定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、交联和分析性超离心来监测；rna -蛋白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作图来评估。

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.