The Role of SSD1 in Handling of Oxidized RNAs in Saccharomyces Cerevisiae

描述（由申请人提供）： 项目摘要：活性氧（ROS）是呼吸作用的天然副产物，在细胞防御系统减弱或暴露于环境压力的细胞中具有破坏性。虽然DNA、蛋白质和脂质氧化的有害作用是众所周知的，但氧化损伤RNA的影响尚未研究。为了解决这个问题，酿酒酵母将被用作模型系统来研究氧化RNA在真核细胞中的处理机制。初步研究表明，S.缺乏SSD1的酿酒酵母比野生型细胞对氧化剂二酰胺更敏感。SSD1编码一种与核糖核酸外切酶同源的蛋白质（Ssd1），调节寿命，并且可能在人类中是潜在保守的。用二酰胺处理酵母细胞后，表位标记的Ssd1定位于P体，P体是被认为是经历衰变的抑制性mRNA的细胞质结构。这些结果表明，SSD1可能参与氧化剂损伤RNA的降解或修复。本研究的具体目的是研究SSD1在处理氧化损伤的RNA中的功能，阐明SSD1如何介导氧化应激抗性，并确定参与氧化RNA处理的其他组分。由于对细胞中氧化RNA的命运知之甚少，因此将使用免疫荧光和共聚焦显微镜分析P体以确定它们是否是氧化损伤RNA的定位位点。还将检查SSD1以确定其是否在抑制体内氧化损伤RNA的积累、P体形成、体内和体外氧化RNA的结合、翻译抑制、脱帽和氧化剂损伤RNA的降解中起作用。这项研究的结果可能阐明氧化损伤的RNA如何促进真核细胞的衰老，并可能深入了解与氧化RNA的细胞积累相关的神经退行性疾病如何发展。相关性：这项研究的目的是更好地了解我们的细胞如何检测和修复被氧化剂破坏的RNA，当食物转化为我们身体使用的能量时，所有人都会接触到氧化剂。这项研究的发现将大大提高我们对RNA损伤如何促进细胞衰老的认识，并可能为老年引起的神经退行性疾病的医学治疗提供建议。

DESCRIPTION (provided by applicant): Project Summary: Reactive oxygen species (ROS) are natural byproducts of respiration and are damaging in cells that have weakened cellular defense systems or are exposed to environmental stress. While the deleterious effects of DNA,protein, and lipid oxidation are well-known, the impact of oxidatively damaged RNAs has not been investigated. To address this issue, Saccharomyces cerevisiae will be used as a model system to study the mechanisms by which oxidized RNAs are handled in eukaryotic cells. Preliminary studies showed that S. cerevisiae lacking SSD1 was more sensitive to the oxidant diamide than wild-type cells. SSD1 encodes for a protein (Ssd1) that is homologous to exoribonucleases, regulates longevity, and may be potentially conserved in humans. Following treatment of yeast cells with diamide, epitope-tagged Ssd1 localizes to P-bodies, which are cytoplasmic structures that are thought to be translationally repressed mRNAs undergoing decay. These results suggest that SSD1may be involved in the decay or repair of RNAs damaged by oxidants. The specific aims of this research proposal are to examine if SSD1functions in the handling of oxidatively damaged RNAs, to elucidate how SSD1mediates oxidative stress resistance, and to identify other components involved in handling of oxidized RNAs. Since little is known about the fate of oxidized RNAs in cells, P-bodies will be analyzed to determine if they are the sites of localization for oxidatively damaged RNAs using immunofluorescence and confocal microscopy. SSD1 will also be examined to determineif it functions in repressing accumulation of oxidatively damaged RNAs in vivo, P- body formation, binding of oxidized RNAs in vivo and in vitro, translational repression, decapping, and degradation of RNAs damaged by oxidants. Results of this study may elucidate how oxidatively damaged RNAs contribute to aging in eukaryotic cells and may give insight into how neurodegenerative diseases that are associated with the cellular accumulation of oxidized RNAs develop. Relevance: The goal of this research is to better understand how our cells detect and repair RNAs damaged by oxidants, which all humans are exposed to when food is converted into energy that our bodies use. Findings from this study will greatly enhance our knowledge of how RNA damage contributes to cellular aging and may suggest ways of medically treating neurodegenerative diseases brought on by old age.