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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

基本信息

批准号：
9198248
负责人：
Kelly Ann Limoncelli
金额：
$ 2.8万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
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/9198248
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 Income Incubated Lead Lesion Lipids Malignant Neoplasms Messenger RNA Metabolic Modification Monitor Mutation Nerve Degeneration Normal Cell Oligonucleotides Oxides Pathway interactions Phenotype Plasmids Play Proteins Quality Control RNA RNA I 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 experimental study fitness in vivo macromolecule mutant nucleoside analog public health relevance repaired ultraviolet irradiation

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请方提供）：损伤剂，如紫外线照射和过氧化氢，可通过损伤细胞内大分子损害功能完整性。细胞具有处理破坏性修饰的质量控制系统，这些途径的缺陷可能导致慢性疾病，如癌症和神经变性3 -6。大量的工作集中在细胞如何处理受损的脂质，蛋白质和DNA;然而，对细胞如何处理受损的RNA知之甚少。RNA对破坏剂高度敏感，并且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）靶向具有可诱导核糖体停滞的序列特征的mRNA。NGD由失速位点的内切核酸裂解触发，随后是核糖体亚基的解离。负责初始切割的核酸内切酶目前尚不清楚17 -21。有趣的是，先前的体外实验已经显示小核糖体亚基S3（RPS 3）切割受损的dsDNA底物22 -27。然而，大多数RPS 3与核糖体相关，直接与传入的mRNA 28 -30相互作用。因此，我建议RPS 3的功能是识别和切割受损的RNA分子，作为针对受损RNA底物的质量控制系统的一部分。为了测试这一点，我将首先通过使用纯化的RPS 3和各种受损的RNA底物进行生物化学测定来确定RPS 3是否含有核酸内切酶活性。我还将评估RPS 3是否含有活性，同时与纯化的核糖体。接下来，我将确定RPS 3是否在体内识别和切割受损的RNA底物。我将尝试通过产生酵母rps 3变体并采用深度测序技术来监测存在或不存在破坏剂的情况下的适应性水平，来分离在处理受损RNA方面有缺陷的酵母rps 3突变体。为了排除影响正常核糖体功能的突变，我打算选择在正常条件下表现出野生型生长速率，但在损伤剂存在下敏感性增加的突变体。最后，由于最初提出RPS 3作为DNA损伤修复蛋白，我将确定RPS 3是否在体内DNA修复中发挥作用。我会测试酵母rps 3突变体是否与其他DNA损伤修复蛋白发生遗传相互作用。此外，我将进行ChIP检测，以确定RPS 3是否与DNA在细胞暴露于破坏剂后物理缔合。总的来说，这项研究旨在阐明有助于维持健康细胞功能的质量控制机制。