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

• 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
• 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

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 先天免疫反应对于控制人类病原体感染至关重要。但是，过度激活 先天免疫反应会导致慢性炎症，导致人类疾病，例如癌症 和自身免疫性疾病。更好地理解和治疗这些疾病，建立更深入的理解 先天免疫系统功能的最重要。特别是导致全球主机的机制 响应双链RNA（dsRNA）的翻译关闭，同时允许dsRNA-的表达 诱导的抗病毒和促炎性mRNA仍然是一个不完全理解的先天性的 免疫反应。 在单细胞水平上评估有效的抗病毒核酸酶核糖核酸酶L（RNase L） 通过促进快速而广泛的营业额，它是转化逮捕和功能的主要驱动力 mrnas。这是对DSRNA诱导的翻译逮捕的理解的重大转变，因为这将允许 RNase L.不降解的mRNA的翻译。 干扰素B（IFN-B）细胞因子逃脱RNase L介导的mRNA转换，有可能允许翻译 IFN-B mRNA。 基于这些初步发现，该应用提出了测试广泛的RNase l-的假设 介导的mRNA转离功能优先促进抗病毒mRNA的翻译 RNase L介导的mRNA更新。这些发现可能为RNase L介导的新颖见解提供了新的见解 翻译停滞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：高通量 测序和单分子荧光原位杂交（Smfish）将用于识别MRNA 除了对RNase L介导的mRNA更新具有抗性的IFN-B mRNA。目标2：针对性 诱变，嵌合mRNA和异源启动子将用于确定机械基础 这是RNase L耐药mRNA逃脱RNase L介导的mRNA转换。目标3：mRNA的单细胞分析 将进行表达和蛋白质翻译与核糖体分析结合的表达和蛋白质翻译，以确定RNase是否是否 L介导的mRNA促进了RNase L耐药mRNA的翻译。这些目标的完成将 确定抗RNase L驱动mRNA的mRNA宽度，确定机制 mRNA逃脱了RNase L介导的mRNA转换，并提供了一种新型机制 在先天免疫反应期间调节抗病毒基因表达。 呢