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Determining the specificity and biological functions of widespread host mRNA degradation by RNase L

确定 RNase L 广泛降解宿主 mRNA 的特异性和生物学功能

基本信息

批准号：
10116269
负责人：
James M Burke
金额：
$ 7.05万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-09 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10116269
关键词：
Affect Antiviral Agents Antiviral resistance Apoptosis Apoptotic Autoimmune Diseases Bioinformatics Biological Biological Process Cells Chronic Consensus Disease Double-Stranded RNA Elements Endoribonucleases Fluorescent in Situ Hybridization Gene Expression Genes Genetic Transcription Genetic Translation Grant High-Throughput Nucleotide Sequencing High-Throughput RNA Sequencing Infection Inflammation Inflammatory Innate Immune Response Innate Immune System Interferon-beta Interferons Lead Malignant Neoplasms Mammalian Cell Mediating Messenger RNA Modeling Molecular Multiple Sclerosis Mutagenesis Null Lymphocytes Pathway interactions Pattern Phosphorylation Production Proteins Research Resistance Rheumatoid Arthritis Ribonucleases Ribosomal RNA Ribosomes Role Specificity Stress Structure Systemic Lupus Erythematosus Testing Translating Translations Work base chronic infection cytokine experimental study human disease human pathogen immune system function innovation insight mRNA Decay mRNA Expression mRNA Transcript Degradation new technology novel pathogen promoter pseudotoxoplasmosis syndrome response ribosome profiling single cell analysis single molecule

项目摘要

Project Summary/Abstract The innate immune response is crucial for controlling infection by human pathogens. However, over-activation of the innate immune response can cause chronic inflammation that leads to human diseases, such as cancers and autoimmune disorders. To better understand and treat these diseases, developing a deeper understanding of how the innate immune system functions is paramount. In particular, the mechanisms that lead to global host shut-off of translation in response to double-stranded RNA (dsRNA), while allowing the expression of dsRNA- induced antiviral and pro-inflammatory mRNAs has remained an incompletely understood aspect of the innate immune response. Assessment of the potent antiviral endoribonuclease, ribonuclease L (RNase L), at the single-cell level revealed that it is the primary driver of translational arrest and functions by promoting rapid and widespread turnover of mRNAs. This is a significant shift in the understanding of dsRNA-induced translational arrest, as it would permit translation of mRNAs that are not degraded by RNase L. Consistent with this, the mRNA of the potent antiviral interferon-b (IFN-b) cytokine escapes RNase L-mediated mRNA turnover, potentially allowing for translation of the IFN-b mRNA. Based on these preliminary findings, this application proposes to test the hypothesis that widespread RNase L- mediated mRNA turnover functions to preferentially promote translation of antiviral mRNAs that are resistant to RNase L-mediated mRNA turnover. These findings may provide novel insights into RNase L-mediated translational arrest and antiviral gene expression that will have translational importance for understanding and treating human disease associated with dysregulation of the innate immune response. Aim 1: High-throughput sequencing and single-molecule fluorescent in situ hybridization (smFISH) will be used to identify mRNAs in addition to the IFN-b mRNA that are resistant to RNase L-mediated mRNA turnover. Aim 2: Targeted mutagenesis, chimeric mRNAs, and heterologous promoters, will be used to determine the mechanistic basis by which RNase L resistant mRNA escape RNase L-mediated mRNA turnover. Aim 3: Single-cell analysis of mRNA expression and protein translation in conjunction with ribosomal profiling will be performed to determine if RNase L-mediated mRNA promotes the translation of RNase L resistant mRNAs. Completion of these aims will determine the breadth of mRNAs resistant to RNase L-driven mRNA turnover, determine the mechanism(s) by which mRNAs escape RNase L-mediated mRNA turnover, and provide a novel mechanism by which RNase L regulates antiviral gene expression during the innate immune response. !

项目摘要/摘要先天免疫反应是控制人类病原体感染的关键。然而，过度激活先天免疫反应的一部分会引起慢性炎症，从而导致人类疾病，如癌症和自身免疫性疾病。为了更好地了解和治疗这些疾病，加深对这些疾病的认识了解先天免疫系统如何发挥作用是至关重要的。特别是，导致全球宿主的机制关闭翻译以响应双链RNA(DsRNA)，同时允许dsRNA- 诱导的抗病毒和促炎的mRNAs仍然是先天的一个不完全了解的方面。免疫反应。在单细胞水平上对高效抗病毒核糖核酸酶L(核糖核酸酶L)的评估被揭示它是翻译逮捕和职能的主要驱动力，通过促进快速和广泛的人员流动 MRNAs。这是对dsrna诱导的翻译停滞的理解的一个重大转变，这是它所允许的。翻译不被核糖核酸酶降解的mRNAs。与此一致，有效的抗病毒的mRNAs 干扰素-b细胞因子逃避核糖核酸酶L介导的mR NA翻转，潜在地允许翻译干扰素-b基因的表达。基于这些初步发现，本申请提出了检验普遍存在的核糖核酸酶L- 介导的mRNA周转功能优先促进抗病毒mRNAs的翻译核糖核酸酶L介导的信使核糖核酸翻转。这些发现可能为研究L介导的核糖核酸酶提供新的见解翻译抑制和抗病毒基因表达将对理解和治疗与先天免疫反应失调相关的人类疾病。目标1：高吞吐量将使用测序和单分子荧光原位杂交(SmFISH)来鉴定除了干扰素-bmRNA外，还有抵抗核糖核酸酶L介导的mR NA翻转。目标2：目标明确突变、嵌合的mRNAs和异源启动子将被用来确定机制基础，通过其中核糖核酸酶L抗性的信使核糖核酸酶L介导的信使核糖核酸翻转。目标3：信使核糖核酸的单细胞分析将结合核糖体分析进行表达和蛋白质翻译，以确定RNase L介导的m RNA促进核糖核酸酶L抗性m RNA的翻译。完成这些目标将确定抵抗核糖核酸酶L驱动的mRNA周转的mRNAs的广度，确定其机制(S) 哪些mRNAs逃脱了核糖核酸酶L介导的信使核糖核酸翻转，并为核糖核酸酶L 在先天免疫反应中调节抗病毒基因的表达。好了！