Characterization of how mRNA translation influences reproductive aging

总结 衰老是一个主要的危险因素，为广泛的疾病和下降的组织稳态 和功能这在女性生殖系统中尤其如此， 卵母细胞与流产和出生缺陷发生率的增加直接相关。我们 长期目标是确定和描述导致生殖老化的因素。在 哺乳动物的卵子可以储存数月、数年或数十年，使得分析生殖老化 缓慢且实验困难。在这里，我们试图建立在以前的努力，以建立果蝇 卵巢是研究生殖衰老的强大系统。有趣的是，鸡蛋产量的下降 质量与从苍蝇到人类的物种间较低水平的mRNA翻译相关。 尽管有这种常见的缺陷，我们对造成这种情况的机制却知之甚少， 降低储存卵子中mRNA的翻译能力。在这里，我们建议使用最先进的技术 基因操作和生化分析，系统地描述机器如何 所需的mRNA翻译的变化与母亲的年龄和在果蝇卵储存。 此外，我们试图从遗传学上测试是否操纵核糖体水平和翻译， 起始/延伸速率影响储存蛋的质量。我们已经建立了一个 进行本提案概述的所有实验的管道，并寻求利用 我们小组采用了一些创新的工具和技术。根据目标1，我们 将使用互补的分子和生物化学方法来全面表征 蛋白质合成和核糖体水平的变化在多大程度上在老龄女性的卵巢和 鸡蛋存放两周以上我们还将使用生物化学和创新的基于报告基因的检测方法， 评估翻译的忠实度是否随着年龄的增长而下降。在目标2中，我们将测试 增加或降低核糖体水平和翻译起始和延伸速率改善了细胞的功能。 储存鸡蛋的质量。在目标3下，我们将描述核糖体加氧酶NO 66如何影响 鸡蛋质量我们相信，这种对体内卵母细胞老化过程的综合分析将有助于 提供了关键的见解，为什么卵子的质量随着年龄的增长而下降，并将揭示新的分子靶点 用于开发旨在改善和延长生殖的疗法。考虑到我们的重点是 核糖体在这一过程中发挥的作用，我们相信我们的工作将广泛影响组织的研究。 衰老生物体内的稳态和再生。

Summary Aging represents a major risk factor for a broad range of diseases and declines in tissue homeostasis and function. This is particularly true in the female reproductive system where the aging of stored oocytes has been directly linked with an increased incidence of miscarriages and birth defects. Our long-term goal is to identify and characterize the factors that contribute to reproductive aging. In mammals, eggs can be stored months, years, or decades, making the analysis of reproductive aging slow and experimentally difficult. Here, we seek to build upon previous efforts to establish the Drosophila ovary as a powerful system with which to study reproductive aging. Interestingly, the decline in egg quality has been correlated with lower levels of mRNA translation across species, from flies to humans. Despite this common defect, we know surprisingly little about the mechanisms responsible for this reduction of mRNA translation capacity within stored eggs. Here, we propose to use state of the art genetic manipulation and biochemical analysis to systematically characterize how the machinery required for mRNA translation changes with maternal age and during egg storage in Drosophila. Moreover, we seek to genetically test whether manipulating ribosome levels and translation initiation/elongation rates prolongs the quality of stored eggs. We have established an operational pipeline for conducting all the experiments outlined under this proposal and seek to take advantage of a number of innovative tools and techniques that have been adopted by our group. Under Aim 1, we will use complementary molecular and biochemical approaches to comprehensively characterize the extent to which protein synthesis and ribosome levels changes in the ovaries of aging females and in eggs stored over two weeks. We will also use biochemical and innovative reporter based assays to evaluate whether translation fidelity declines with age. In aim 2, we will test the extent to which increasing or decreasing ribosome levels and translation initiation and elongation rates improves the quality of stored eggs. Under aim 3, we will characterize how the ribosome oxygenase NO66 influences egg quality. We believe this comprehensive analysis of in vivo oocytes during the course of aging will provide key insights into why the quality of eggs declines with age and will reveal new molecular targets for the development of therapies designed to improve and extend reproduction. Given our focus on the role ribosomes play in this process, we believe our work will broadly impact the study of tissue homeostasis and regeneration in aging organisms.