Investigating the functional roles of RPS3 in RNA and DNA damage

研究 RPS3 在 RNA 和 DNA 损伤中的功能作用

• 批准号: 8791845
• 负责人: Kelly Ann Limoncelli
• 金额: $ 2.97万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-06 至 2018-01-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8791845
• 关键词: Affect; Base Excision Repairs; Biochemical; Biological Assay; Cell physiology; Cells; ChIP-seq; Chemicals; Chronic Disease; Cleaved cell; Codon Nucleotides; Cytoplasm; DNA; DNA Damage; DNA Repair; Defect; Dissociation; Environmental Risk Factor; Exhibits; Exposure to; Frequencies; Genetic; Genome; Goals; Growth; Hydrogen Peroxide; In Vitro; Incubated; Lead; Lesion; Lipids; Malignant Neoplasms; Messenger RNA; Metabolic; Modification; Monitor; Mutation; Nerve Degeneration; Normal Cell; Oligonucleotides; Pathway interactions; Phenotype; Plasmids; Play; Proteins; Quality Control; RNA; RPS3 gene; Reaction; Recombinants; Research; Ribosomes; Role; Site; Site-Directed Mutagenesis; Structure; System; Techniques; Technology; Testing; Translations; Variant; Work; Yeasts; base; chromatin immunoprecipitation; combat; deep sequencing; ds-DNA; effective therapy; endonuclease; enzyme activity; fitness; in vivo; macromolecule; mutant; nucleoside analog; public health relevance; repaired; research study; ultraviolet irradiation

Abstract Damaging agents, such as UV irradiation and hydrogen peroxide, can compromise functional integrity by damaging intracellular macromolecules. Cells have quality control systems that deal with damaging modifications, and defects in these pathways could lead to chronic diseases such as cancer and neurodegeneration3-6. A considerable amount of work has focused on how cells deal with damaged lipids, proteins, and DNA; however, little is known about how cells deal with damaged RNA. RNA is highly susceptible to damaging agents and such lesions could lead to translation errors and ribosome stalling10-12. Several RNA quality control systems occur at the translational level. No-Go Decay (NGD) targets mRNAs with sequence features that can induce ribosome stalling. NGD is triggered by an endonucleolytic cleavage at the stall site, followed by the dissociation of the ribosomal subunits. The endonuclease responsible for the initial cleavages is currently unknown17-21. Intriguingly, previous in vitro experiments have shown that the small ribosomal subunit S3 (RPS3) cleaves damaged dsDNA substrates22-27. However, the majority of RPS3 is associated with ribosomes, directly interacting with incoming mRNA28-30. Therefore, I propose that RPS3 functions to recognize and cleave damaged RNA molecules as part of a quality control system that targets damaged RNA substrates. To test this, I will first determine whether RPS3 contains endonuclease activity by performing biochemical assays using purified RPS3 and various damaged RNA substrates. I will also assess whether RPS3 contains activity while associated with purified ribosomes. Next, I will determine if RPS3 recognizes and cleaves damaged RNA substrates in vivo. I will attempt to isolate yeast rps3 mutants that are defective at dealing with damaged RNA by generating yeast rps3 variants and employing deep sequencing technologies to monitor fitness levels in the presence or absence of damaging agents. To rule out mutations affecting normal ribosome function, I intend to select mutants that exhibit wild-type growth rates under normal conditions, but increased sensitivity in the presence of damaging agents. Finally, since it was originally proposed that RPS3 functions as a DNA damage repair protein, I will determine if RPS3 plays a role in DNA repair in vivo. I will test whether yeast rps3 mutants genetically interact with other DNA damage repair proteins. In addition, I will perform ChIP assays to determine whether RPS3 physically associates with DNA upon cellular exposure to damaging agents. Overall, this research seeks to elucidate quality control mechanisms that serve to maintain healthy cellular function.

抽象的 损伤剂，例如紫外线照射和过氧化氢，可能会通过以下方式损害功能完整性： 破坏细胞内大分子。细胞具有处理破坏性物质的质量控制系统 这些途径的改变和缺陷可能导致癌症等慢性疾病 神经变性3-6。大量的工作集中在细胞如何处理受损的脂质上， 蛋白质和DNA；然而，人们对细胞如何处理受损的 RNA 知之甚少。 RNA 高度 容易受到破坏性物质的影响，这种损伤可能导致翻译错误和核糖体停滞10-12。 一些 RNA 质量控制系统发生在翻译水平。 No-Go Decay (NGD) 靶向 mRNA 可以诱导核糖体停滞的序列特征。 NGD 由核酸内切酶引发 失速位点，然后是核糖体亚基的解离。负责初始的核酸内切酶 裂解目前未知17-21。有趣的是，之前的体外实验表明，小 核糖体亚基 S3 (RPS3) 切割受损的 dsDNA 底物22-27。然而，大多数 RPS3 是 与核糖体相关，直接与传入的 mRNA28-30 相互作用。因此，我建议RPS3 识别和切割受损 RNA 分子的功能，作为目标质量控制系统的一部分 RNA 底物受损。为了测试这一点，我将首先确定 RPS3 是否含有核酸内切酶活性 使用纯化的 RPS3 和各种受损的 RNA 底物进行生化测定。我也会评估 RPS3 与纯化核糖体相关时是否具有活性。接下来，我将确定是否 RPS3 识别并切割体内受损的 RNA 底物。我将尝试分离酵母 rps3 突变体 通过生成酵母 rps3 变体并采用深度测序来处理受损 RNA 方面存在缺陷 在存在或不存在破坏性物质的情况下监测健康水平的技术。排除突变 影响正常核糖体功能，我打算选择在正常情况下表现出野生型生长速率的突变体 条件下，但在存在破坏剂的情况下会增加敏感性。最后，既然是原来的 提出RPS3作为DNA损伤修复蛋白发挥作用，我将确定RPS3是否在DNA中发挥作用 体内修复。我将测试酵母rps3突变体是否在基因上与其他DNA损伤修复相互作用 蛋白质。此外，我将进行 ChIP 检测以确定 RPS3 是否与 DNA 物理结合 当细胞暴露于损伤剂时。总体而言，本研究旨在阐明质量控制 用于维持健康细胞功能的机制。