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，对削弱细胞防御系统或暴露在环境压力下的细胞具有损害作用。虽然DNA、蛋白质和脂肪氧化的有害影响是众所周知的，但氧化损伤的RNA的影响还没有被调查。为了解决这个问题，酿酒酵母将被用作一个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，缺乏SSD1的酿酒酵母比野生型细胞对氧化剂联胺更敏感。SSD1编码一种蛋白质(SSD1)，它与外切核酸酶同源，调节寿命，并可能在人类中潜在地保守。在用联胺处理酵母细胞后，带有表位标记的SSD1定位于P小体，P小体是一种细胞质结构，被认为是正在腐烂的翻译抑制的mRNAs。这些结果表明，SSD1可能参与了氧化剂损伤的RNA的衰退或修复。这项研究计划的具体目的是研究SSD1是否在处理氧化损伤的RNA中发挥功能，阐明SSD1如何介导氧化应激抵抗，并确定参与处理氧化RNA的其他成分。由于对氧化RNA在细胞中的去向知之甚少，将使用免疫荧光和共聚焦显微镜对P小体进行分析，以确定它们是否是氧化损伤RNA的定位部位。SSD1还将被检测以确定它是否在抑制体内氧化损伤的RNA的积累、P-小体的形成、体内和体外氧化的RNA的结合、翻译抑制、去帽和被氧化剂损伤的RNA的降解中发挥作用。这项研究的结果可能会阐明氧化损伤的RNA如何导致真核细胞的衰老，并可能深入了解与氧化RNA细胞积累相关的神经退行性疾病是如何发展的。相关性：这项研究的目标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，当食物被转化为我们身体使用的能量时，所有人类都会接触到氧化剂。这项研究的发现将极大地提高我们对RNA损伤如何导致细胞衰老的了解，并可能提出从医学上治疗老年引起的神经退行性疾病的方法。

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.