Elucidating the Quality Control Pathways that Cope with Mutant Ribosomal RNA in Mammalian Cells

阐明应对哺乳动物细胞中突变核糖体 RNA 的质量控制途径

• 批准号: 10663816
• 负责人: Zachary Stolp
• 金额: $ 6.95万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10663816
• 关键词: Affect; Anticodon; Bacteria; Biological Assay; CRISPR interference; Catalysis; Cell Line; Cell Survival; Cells; Codon Nucleotides; Complex; Cytoplasmic Granules; Data; Data Set; Defect; Detection; Disease; Dissection; Energy consumption; Engineering; Ensure; Event; Failure; Fluorescence Microscopy; Foundations; Future; Genetic Code; Genetic Screening; Half-Life; Health; Homologous Gene; Human; In Vitro; Knock-out; Knowledge; Label; Link; Luciferases; Malignant Neoplasms; Mammalian Cell; Messenger RNA; Molecular Machines; Monitor; Mutation; Nature; Nerve Degeneration; Northern Blotting; Oxidative Stress; Pathway interactions; Peptides; Play; Process; Production; Protein Biosynthesis; Proteins; Quality Control; RNA; RNA Degradation; RNA, Ribosomal, 18S; Reporter; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Spinach - dietary; Stress; System; Techniques; Technology; Testing; Time; Transfer RNA; Translating; Translations; Ultraviolet Rays; Work; Yeasts; aptamer; career; coping; coping mechanism; detection method; developmental disease; experimental study; genome wide screen; human disease; knock-down; mutant; novel; ribosome profiling; ribosomopathy; screening; small molecule; tool; translational genetics; whole genome

PROJECT SUMMARY Protein production is absolutely essential and faulty translation has been linked to a wide-range of diseases including cancer, neurodegeneration and a class of predominantly developmental disorders known as ribosomopathies. In order to ensure faithful translation of the genetic code, cells need to monitor the integrity of all components of the translation apparatus, including mRNAs, tRNAs, and ribosomes. Although the quality control mechanisms that monitor mRNAs and tRNAs are relatively well characterized, very little is known about quality control of the ribosome itself. The eukaryotic ribosome is a complex molecular machine consisting of 4 ribosomal RNAs (rRNA) and 80 proteins, and all these components are subjected to mutations, oxidative stress, and UV radiation. Notably, the rRNA carries out the core enzymatic functions of the ribosome during translation. Despite the importance of rRNA to ribosomal function and human health, the quality control mechanisms that cope with the presence of defective rRNA have not been studied in mammalian cells. Previous work in yeast has discovered that rRNAs harboring mutations are preferentially degraded, confirming the existence of quality control mechanisms targeting faulty rRNA. However, the components involved in the detection and degradation of these rRNAs remain poorly characterized. This knowledge gap is primarily due to technological constraints, including limited rRNA detection methods and appropriate reporter systems geared for genome-wide screens. Recent technological advances have primed the field for unbiased and mechanistic studies. Due to the critical nature of ribosomal quality control to human health, I seek to define the quality control mechanisms that mammalian cells impart on rRNA. I have engineered a mammalian fluorescent rRNA reporter that can be paired with both unbiased genetic screening and hypothesis-driven approaches. Taking advantage of this unique tool, I will determine how human cells cope with defective ribosomes harboring mutant 18S rRNA, the rRNA component of the small ribosomal subunit. I will first characterize how mutations in the 18S rRNAs interfere with translation. Second, I will determine the fate of defective 18S rRNA in mammalian cells. Lastly, I will identify the mammalian factors coping with defective rRNA. Together, the proposed studies will for the first time delineate mammalian rRNA quality control mechanisms, and provide the foundation for understanding how dysregulation of this process leads to human disease.

项目总结 蛋白质的产生是绝对必要的，错误的翻译与一系列疾病有关 包括癌症、神经退行性变和一类以发育障碍为主的疾病，称为 核糖体疾病。为了确保遗传密码的忠实翻译，细胞需要监测 翻译装置的所有组件，包括mRNAs、tRNAs和核糖体。虽然质量不高 监控mRNAs和tRNAs的控制机制相对较好地描述了，对此知之甚少 核糖体本身的质量控制。真核细胞核糖体是由4个核糖体组成的复杂分子机器 核糖体RNA(RRNA)和80种蛋白质，所有这些成分都受到突变、氧化应激、 和紫外线辐射。值得注意的是，rRNA在翻译过程中执行核糖体的核心酶功能。 尽管rRNA对核糖体功能和人类健康很重要，但质量控制机制 应对存在缺陷的rRNA在哺乳动物细胞中还没有被研究过。以前在酵母方面的工作 已经发现含有突变的rRNA优先被降解，证实了质量的存在 针对错误rRNA的控制机制。然而，检测和降解过程中涉及的成分 其中，rRNA的特征仍然很差。这种知识差距主要是由于技术限制， 包括有限的rRNA检测方法和适用于全基因组筛选的适当报告系统。 最近的技术进步为不偏不倚和机械论的研究奠定了基础。由于危急状况 核糖体质量控制的本质对人类健康，我寻求定义的质量控制机制 哺乳动物细胞通过rRNA传递。我已经设计了一种哺乳动物荧光rRNA报告程序，它可以 与无偏见的基因筛查和假设驱动的方法相结合。利用这一独特的 工具，我将确定人类细胞如何应对含有突变的18S rRNA的有缺陷的核糖体， 小核糖体亚基的组成部分。我将首先描述18S rRNA的突变是如何干扰 翻译。其次，我将确定有缺陷的18S rRNA在哺乳动物细胞中的命运。最后，我将确定 哺乳动物应对缺陷rRNA的因素。总之，拟议的研究将首次勾勒出 哺乳动物rRNA的质量控制机制，并为理解失调是如何 这一过程会导致人类疾病。