ADAD1 and the post-meiotic male germ cell ribosome

项目总结/摘要 翻译产生细胞功能所必需的蛋白质，并依赖于核糖体，一种高度依赖于核糖体的蛋白质。 保守的蛋白质和RNA复合物。减数分裂后的雄性生殖细胞特别依赖于翻译 调节，以及随后的核糖体功能。该项目的长期目标是确定 ADAD 1是核减数分裂后雄性生殖细胞特异性RNA结合的分子机制， 蛋白质，影响翻译。我们实验室以前的工作表明，Adad 1是男性所必需的。 作为突变的生育力导致减数分裂后生殖细胞分化的缺陷。虽然以前 作为一种RNA编辑酶，我们的证据表明ADAD 1的缺失不会影响mRNA 编辑，使得ADAD 1的分子功能未知。 在我们实验室的初步数据中，我们证明ADAD 1缺失导致核糖体异常， 对减数分裂后生殖细胞分化重要的转录物的关联，减少的核糖体RNA 修饰和核糖体蛋白和核糖体生物发生因子的异常表达。在一起， 这些观察结果导致ADAD 1影响减数分裂后的翻译的假设， 通过调节核核糖体生物合成来转录。该提案的目标是产生 遗传工具和数据集，通过两个目标来有力地检验这一假设。目标1将定义 通过产生标记的表位在减数分裂后生殖细胞中与ADAD 1相互作用的蛋白质和RNA Adad 1等位基因，并结合免疫沉淀质谱和RNA测序。这些 这些努力将有助于阐明ADAD 1的分子作用机制。目标2将量化核糖体 Adad 1突变体和生殖细胞发育过程中的蛋白质组成 在一个实施方案中，将细胞特异性表位标记的核糖体蛋白的免疫沉淀与质谱联用。 总之，这些分析将量化ADAD 1对核糖体蛋白质组成的影响， 首次定义了减数分裂和减数分裂后生殖细胞核糖体蛋白质组。 历史上，核糖体被认为是在组织和细胞中不变的， 实验证据表明，核糖体蛋白或RNA组成可以变化，导致 具有独特翻译动力学的特殊核糖体。生殖细胞是否利用核糖体变异 控制翻译是一个尚未探索的问题，它可能会对我们的理解产生巨大的影响。 生殖细胞生物学本文提出的研究将为定义ADAD 1的作用奠定基础， 以及鉴定雄性生殖细胞作为核糖体水平翻译控制的模型。

PROJECT SUMMARY / ABSTRACT Translation produces the proteins necessary for a cell’s function and relies on the ribosome, a highly conserved protein and RNA complex. Post-meiotic male germ cells are especially reliant on translation regulation, and consequently ribosome function. The long-term goal of this project is to determine the molecular mechanism by which ADAD1, a nuclear post-meiotic male germ cell-specific RNA binding protein, impacts translation. Previous work from our laboratory demonstrated Adad1 is required for male fertility as mutation leads to defects in post-meiotic germ cell differentiation. Although previously proposed as an RNA editing enzyme, our evidence demonstrates ADAD1 loss does not influence mRNA editing, making the molecular function of ADAD1 unknown. In preliminary data from our laboratory, we demonstrate ADAD1 loss leads to abnormal ribosome association of transcripts important for post-meiotic germ cell differentiation, reduced ribosomal RNA modification, and aberrant expression of ribosomal proteins and ribosome biogenesis factors. Together, these observations lead to the hypothesis that ADAD1 influences translation of post-meiotic transcripts by modulating nuclear ribosome biogenesis. The goal of this proposal is to generate the genetic tools and datasets necessary to robustly test this hypothesis through two aims. Aim 1 will define ADAD1 interacting proteins and RNAs in post-meiotic germ cells by generating an epitope tagged Adad1 allele and combining it with immunoprecipitation mass spectrometry and RNA sequencing. These efforts will shed light on ADAD1’s molecular mechanism of action. Aim 2 will quantify ribosome protein composition in Adad1 mutants and during germ cell development using quantitative mass spectrometry coupled to immunoprecipitation of a cell-specific epitope-tagged ribosome protein. Together, these analyses will quantify ADAD1’s impact on ribosome protein composition as well as define, for the first time, the meiotic and post-meiotic germ cell ribosome proteome. Historically, the ribosome was considered invariant across tissues and cells, however recent experimental evidence indicates ribosomal protein or RNA composition can vary, resulting in a specialized ribosome with unique translation dynamics. Whether germ cells leverage ribosome variation to control translation is an unexplored question that could have a dramatic impact on our understanding of germ cell biology. The studies proposed here will lay the foundations for defining the role of ADAD1 as well as identifying the male germ cell as a model for ribosome-level translation control.