Characterization of how mRNA translation influences reproductive aging

摘要 衰老是导致多种疾病和组织内稳态下降的主要风险因素 和功能。在女性生殖系统中尤其如此，在那里储存着老化的 卵母细胞与流产和出生缺陷发生率的增加直接相关。我们的 长期目标是确定和描述导致生殖衰老的因素。在……里面 哺乳动物的卵子可以储存几个月、几年或几十年，做出生殖老化的分析 速度慢，实验难度大。在这里，我们试图在以前的努力基础上建立果蝇 卵巢是研究生殖衰老的强大系统。有趣的是，鸡蛋的减少 质量与从苍蝇到人类的不同物种之间较低的mRNA翻译水平有关。 尽管有这种共同的缺陷，但我们对导致这种缺陷的机制知之甚少，这令人惊讶 储藏鸡蛋内的mRNA翻译能力降低。在这里，我们建议使用最先进的 基因操作和生化分析来系统地表征机器是如何 果蝇在卵子储存过程中，信使核糖核酸翻译所需的时间随母体年龄的变化而变化。 此外，我们试图从基因上测试是否操纵核糖体水平和翻译 起始/伸长速率延长了储藏鸡蛋的品质。我们已经建立了一个行动小组 进行本提案中概述的所有实验的渠道，并寻求利用 我们团队已经采用了一些创新的工具和技术。在目标1下，我们 将使用互补的分子和生化方法来全面表征 老年女性卵巢中蛋白质合成和核糖体水平变化的程度 鸡蛋储存超过两周。我们还将使用生化和创新的记者化验方法来 评估翻译保真度是否会随着年龄的增长而下降。在目标2中，我们将测试 增加或减少核糖体水平以及翻译起始和延伸率可以改善 储藏鸡蛋的质量。在目标3中，我们将描述核糖体加氧酶NO66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全面分析将 提供关于鸡蛋质量随年龄下降的关键见解，并将揭示新的分子靶点 用于开发旨在改善和延长生殖能力的治疗方法。鉴于我们的重点是 核糖体在这一过程中扮演的角色，我们相信我们的工作将广泛影响组织的研究 衰老生物体的动态平衡和再生。

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.