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：
9757551
负责人：
James M Burke
金额：
$ 6.16万
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/9757551
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. !

项目概要/摘要先天免疫反应对于控制人类病原体的感染至关重要。然而，过度激活先天免疫反应的减少会引起慢性炎症，从而导致人类疾病，例如癌症和自身免疫性疾病。为了更好地认识和治疗这些疾病，加深了解先天免疫系统如何发挥作用至关重要。特别是导致全球主机的机制响应双链 RNA (dsRNA) 关闭翻译，同时允许 dsRNA- 的表达诱导的抗病毒和促炎 mRNA 仍然是先天性的一个不完全了解的方面免疫反应。在单细胞水平上评估有效的抗病毒内切核糖核酸酶核糖核酸酶 L (RNase L) 它是翻译逮捕的主要驱动力，并通过促进快速和广泛的周转来发挥作用 mRNA。这是对 dsRNA 诱导的翻译停滞理解的重大转变，因为它允许不被 RNase L 降解的 mRNA 的翻译。与此相一致的是，有效抗病毒药物的 mRNA 干扰素-b (IFN-b) 细胞因子逃避 RNase L 介导的 mRNA 更新，可能允许翻译 IFN-b mRNA。基于这些初步发现，本申请提出检验广泛存在的 RNase L- 介导的 mRNA 转换功能优先促进具有抗性的抗病毒 mRNA 的翻译 RNase L 介导的 mRNA 周转。这些发现可能为 RNase L 介导的研究提供新的见解翻译停滞和抗病毒基因表达对于理解和理解具有翻译重要性治疗与先天免疫反应失调相关的人类疾病。目标 1：高通量测序和单分子荧光原位杂交（smFISH）将用于鉴定除了对 RNase L 介导的 mRNA 周转有抵抗力的 IFN-b mRNA 之外。目标 2：有针对性诱变、嵌合 mRNA 和异源启动子将用于确定机制基础其中 RNase L 抗性 mRNA 逃避 RNase L 介导的 mRNA 周转。目标 3：mRNA 的单细胞分析将进行表达和蛋白质翻译以及核糖体分析，以确定 RNase 是否 L 介导的 mRNA 促进 RNase L 抗性 mRNA 的翻译。完成这些目标将确定对 RNase L 驱动的 mRNA 周转具有抗性的 mRNA 的广度，通过以下方式确定机制这些 mRNA 逃避 RNase L 介导的 mRNA 转换，并提供了一种新的机制，通过 RNase L 在先天免疫反应过程中调节抗病毒基因的表达。！