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. !

项目总结/摘要 先天免疫应答对于控制人类病原体的感染至关重要。然而，过度激活 先天免疫反应的一部分会引起慢性炎症，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， 先天免疫系统的功能至关重要。特别是，导致全球宿主的机制 响应于双链RNA（dsRNA）而关闭翻译，同时允许dsRNA的表达， 诱导的抗病毒和促炎基因仍然是一个不完全理解的方面的先天性 免疫反应 在单细胞水平上评估有效的抗病毒核糖核酸内切酶，核糖核酸酶L（RNase L）， 它是翻译停滞的主要驱动力，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一个重大转变，因为它允许 不被RNase L降解的mRNA的翻译。与此一致的是，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周转，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周转功能，优先促进抗病毒mRNA的翻译， RNA酶L介导的mRNA周转。这些发现可能为RNase L介导的 翻译停滞和抗病毒基因表达，这将对理解和 治疗与先天免疫应答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 此外，IFN-b mRNA对RNase L介导的mRNA周转具有抗性。目标2：目标明确 诱变，嵌合mRNA和异源启动子，将用于确定机制基础， 其RNA酶L抗性mRNA逃避RNA酶L介导的mRNA周转。目的3：mRNA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RNA酶是否 L介导的mRNA促进RNase L抗性mRNA的翻译。实现这些目标将 确定对RNase L驱动的mRNA周转具有抗性的mRNA的宽度，通过以下方式确定机制： 这些mRNA逃避了RNase L介导的mRNA周转，并提供了一种新的机制， 在先天免疫反应中调节抗病毒基因的表达。 !