Investigating the functional roles of RPS3 in RNA and DNA damage

摘要 紫外线辐射和过氧化氢等破坏性物质会通过以下方式损害功能完整性 破坏细胞内的大分子。细胞有质量控制系统来处理损害 这些通路的修饰和缺陷可能导致慢性疾病，如癌症和 神经退行性变3-6。相当多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含量很高 易受损害剂影响，这种损伤可能导致翻译错误和核糖体停滞10-12。 在翻译层面上出现了几个RNA质量控制系统。不可用衰变(NGD)靶向mRNA 可导致核糖体停滞的序列特征。NGD是由位于 失速部位，随后核糖体亚基解离。负责初始的核酸内切酶 卵裂目前尚不清楚，17-21。耐人寻味的是，之前的体外实验表明， 核糖体S3亚基(RPS3)裂解受损的dsDNA底物22-27。然而，RPS3的大多数是 与核糖体相关，直接与传入的mRNA28-30相互作用。因此，我建议RPS3 作为质量控制系统的一部分，识别和切割受损的RNA分子的功能 损坏的RNA底物。为了测试这一点，我将首先确定RPS3是否包含核酸内切酶活性，方法是 使用纯化的RPS3和各种受损的RNA底物进行生化分析。我也会评估 RPS3在与纯化的核糖体结合时是否具有活性。接下来，我将确定RPS3是否 在体内识别和切割受损的RNA底物。我将尝试分离酵母RPS3突变体 通过产生酵母RPS3变异体和采用深度测序来处理受损的RNA方面的缺陷 在存在或不存在破坏性因素的情况下监控健康水平的技术。排除突变 影响正常的核糖体功能，我打算选择在正常情况下表现出野生型生长速度的突变体。 但在有破坏性物质存在的情况下会增加敏感性。最后，因为它原来是 提出RPS3作为DNA损伤修复蛋白，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突变体是否在遗传上与其他DNA损伤修复相互作用 蛋白质。此外，我将进行芯片分析，以确定RPS3是否与DNA物理关联 当细胞暴露在破坏性物质中时。总体而言，这项研究试图阐明质量控制 维持细胞健康功能的机制。

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.