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（由申请人提供）：许多病毒使用非编码RNA元件来进行有效的感染和复制，但在病毒RNA的编码区中发现的元件较少，一个例子是竞争性抑制剂RNA（ciRNA）。 C 组肠道病毒（包括柯萨奇病毒和脊髓灰质炎病毒）的 C3 蛋白酶的编码区。该 RNA 303 核苷酸元件竞争性抑制 RNase L（一种因存在而被激活的 RNase）。一旦激活，RNase L 就会迅速降解 RNA，因此 ciRNA 可以抑制这种酶，这表明存在强大的选择压力来维持 ciRNA 序列以抑制该酶。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柯萨奇病毒感染的新疗法也很重要。柯萨奇病毒 A21 作为一种治疗黑色素瘤、乳腺癌和前列腺癌的药物正在进行临床试验。抑制 RNase L 的 ciRNA 的分子、生物物理和体内特征尚不清楚；了解 RNase L 和 ciRNA 之间的相互作用对于了解其作用机制非常重要。 RNA 能够直接抑制一种酶，该酶是为了确保其被破坏而产生的，该提议的目的之一是测试 ciRNA 在结合时折叠成独特结构的假设。抑制 RNAse L 的核糖核酸酶活性，确定 RNase L 和 ciRNA 之间的结合界面，并通过阐明结构相互作用来确定 ciRNA 和 RNase L 复合物内抑制的分子机制。为了确定该复合物的特征，我将描述其重要性。通过化学足迹探测、结合测定以及使用 RNase L 的野生型和突变型 ciRNA 的功能测定来确定不同的二级结构区域。由于该复合物的大小，我将重点关注结构的表征通过 X 射线晶体学分析 RNase L-ciRNA 复合物的关系 ciRNA 和 RNase L 之间复合物的结构将阐明抑制是否是封装活性位点的结果，从而诱导 R Nase L 活性位点几何构象的变化。该提案的第二个目标是追踪 RNase L 和 ciRNA 在感染过程中的定位，我计划使用单核细胞、上皮细胞和癌细胞的细胞培养物并感染这些细胞。与脊髓灰质炎病毒和柯萨奇病毒株一起证明 RNase L-ciRNA 复合物的生物学意义 RNase L 和 ciRNA 将被荧光标记，跟踪它们在发明过程中的运动，以更好地了解它们在感染中的作用。 CRIPR-Cas9 系统将用于进行 GPF 标记的 RNase L 和 RNA FISH 实验来追踪 ciRNA 元件，这些实验的组合将允许确定 RNase L 和 RNase L 的时间和地点。 ciRNA 共定位。