RNA that prevents RNase activity: Biochemical and biophysical studies of the ciRNA-RNase L complex

阻止 RNase 活性的 RNA：ciRNA-RNase L 复合物的生化和生物物理研究

基本信息

批准号：
9121438
负责人：
Daniel R Eiler
金额：
$ 5.61万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9121438
关键词：
Active Sites Affect Affinity Antiviral Agents Antiviral Response Apoptotic Aseptic Meningitis Benign Prostatic Hypertrophy Beryllium Binding Biochemical Biological Biological Assay Breast Melanoma Cell Culture Techniques Cell Line Cells Chemicals Chronic Fatigue Syndrome Cleaved cell Clinical Trials Code Common Cold Complex Conjunctivitis Coxsackie Viruses Coxsackievirus Infections Cytoplasm Data Depressed mood Development Diabetes Mellitus Dinucleoside Phosphates Double-Stranded RNA Elements Encapsulated Encephalitis Ensure Enterovirus Environment Enzymes Epithelial Event Genome Genomics Geometry Gray unit of radiation dose Hand, Foot and Mouth Disease High Prevalence Human poliovirus Immune system In Vitro Incidence Infection Interferon Type I Label Link Malignant Neoplasms Malignant neoplasm of prostate Maps Medical Modeling Molecular Movement Mutation Myocarditis Names Nucleotides Oncolytic Paralysed Peptide Hydrolases Play Poliomyelitis Property Proteins Publishing RNA RNA Binding RNA Folding RNA Probes RNA Sequences RNA Viruses Research Research Project Grants Resistance Ribonucleases Role Specificity Structure System Testing Transfer RNA Untranslated RNA Vaccines Viral Genome Viral Pathogenesis Viral Proteins Virus Virus Diseases X-Ray Crystallography base biophysical analysis cancer cell cancer therapy cell type design dimer fibrosarcoma human disease human tissue in vivo inhibitor/antagonist invention monocyte mutant novel therapeutics novel vaccines nuclease overexpression pressure prevent public health relevance research study stoichiometry viral RNA

项目摘要

DESCRIPTION (provided by applicant): Many viruses use non-coding RNA elements to manipulate cellular machinery for effective infection and replication. Less frequently, elements are found in the coding region of a viral RNA. An example is the competitive inhibitor RNA (ciRNA) within the coding region of the C3 protease of group C enteroviruses, including coxsackievirus and poliovirus. This RNA 303 nucleotides element competitively inhibits RNase L, an RNase that becomes activated by the presence of double-stranded RNA in the cytoplasm. Once activated, RNase L rapidly degrades RNA and thus is a powerful antiviral enzyme. The ciRNA can serve to inhibit this enzyme, and this suggests there is strong selective pressure to maintain the ciRNA sequence to depress the antiviral response and facilitate successful infection. Understanding the detailed molecular events that occur during infection is important for the development of new therapies against poliovirus and coxsackievirus infection. These molecular event could also be important for a novel therapy were coxsackievirus A21 is in clinical trials as a therapy against melanoma, breast, and prostate cancers. The molecular, biophysical, and in vivo features of the ciRNA that inhibits RNase L are unknown; understanding the interactions between RNase L and ciRNA is important in understanding how an RNA is able to directly inhibit an enzyme that is made to ensure its destruction. Aim one of this proposal is t test the hypothesis that ciRNA folds into a unique structure when it binds making it able to inhibi RNAse L's ribonuclease activity, determine the binding interface between RNase L and ciRNA, and determine the molecular mechanism of inhibition within the ciRNA and RNase L complex by elucidating the structural interactions. To determine the features of this complex, I will characterize the importance of different secondary structural regions by chemical footprint probing, binding assays, and functional assays with wild-type and mutant forms of ciRNA with RNase L. Due the size of this complex I will focus on to characterize the structural relationship of the RNase L-ciRNA complex by X-ray crystallography. A structure of a complex between ciRNA and RNase L would illuminate whether inhibition is the result of encapsulating the active site, inducing a conformational change in the active site geometry of R Nase L directly, or another mechanism. The second aim of this proposal is to track the localization of RNase L and ciRNA during infection. I plan to use cell culture of monocytes, epithelial, and cancer cells and infect these cells with poliovirus and coxsackievirus strains to demonstrate the biological significance of the RNase L-ciRNA complex. RNase L and ciRNA will be fluorescently labeled track their movements during to invention to better understand their role within infection. The cell lines listed are being genetically edited by the CRIPR-Cas9 system to make GPF tagged RNase L and RNA FISH experiments will be used to track the ciRNA element. The combination of these experiments will allow to determine when and where RNase L and ciRNA co-localize.

描述（由适用提供）：许多病毒使用非编码RNA元件来操纵细胞机械以进行有效的感染和复制。在病毒RNA的编码区域中发现元素。一个例子是C组肠病毒C3蛋白的编码区域内的竞争性抑制剂RNA（CIRNA），包括Coxsackivievirus和Poliovirus。该RNA 303核苷酸元件竞争性抑制RNase L，RNase L是一种RNase，它因细胞质中的双链RNA而被激活。一旦激活，RNase L迅速降解RNA，因此是一种强大的抗病毒酶。 CIRNA可以用来抑制这种酶，这表明存在强大的选择性压力来维持CIRNA序列以降低抗病毒药反应并促进成功感染。了解感染过程中发生的详细分子事件对于开发针对脊髓灰质炎病毒和Coxsackievivirus感染的新疗法很重要。这些分子事件对于一种新的疗法也可能很重要，因为Coxsackievivirus A21是针对黑色素瘤，乳腺癌和前列腺癌的临床试验。抑制RNase L的CIRNA的分子，生物物理和体内特征是未知的。了解RNASE L和CIRNA之间的相互作用对于理解RNA如何直接抑制以确保其破坏的酶。目的之一是t检验了以下假设：CIRNA结合使其与hibi rNase L的核糖核酸酶活性折叠成独特的结构，确定RNase L和CIRNA之间的结合界面，并确定通过阐明结构相互作用的CIRNA和RNase L复合物中抑制的分子机制。为了确定这种复合物的特征，我将通过化学足迹探测，结合测定和具有野生型和突变形式的CIRNA的ciRNA的二次结构区域的重要性来表征带有RNase L的功能测定。由于该复合物的尺寸，我将重点介绍X型晶体结晶的RNase L-CiRNA复合物的结构关系。 CIRNA和RNase L之间的复合物的结构将阐明抑制是否是封装活性位点，直接诱导R nase L的活性位点几何形状的构象变化的结果，还是直接的其他机制。该提案的第二个目的是跟踪感染过程中RNase L和CIRNA的定位。我计划使用单核细胞，上皮和癌细胞的细胞培养，并用脊髓灰质炎病毒和coxsacchievieviviviviviviviorus菌株感染这些细胞，以证明RNase L-CiRNA复合物的生物学性。 RNase L和CIRNA将被荧光标记在发明过程中跟踪其运动，以更好地了解它们在感染中的作用。 CRIPR-CAS9系统一般对列出的细胞系进行编辑，以使GPF标记为RNase L，RNA Fish实验将用于跟踪CIRNA元素。这些实验的组合将允许确定RNase L和CIRNA共定位的何时何地。