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

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！

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. !