Novel mechanisms regulating translation elongation during male germ cell differentiation

项目总结 在男性生殖细胞发育过程中，男性的生育能力依赖于mRNA翻译的适当调控。这是 通过RNA结合蛋白(RBPs)的协同作用实现。在减数分裂的雄性生殖细胞中，限制性商业惯例通常是 定位于RNA的致密胞浆颗粒称为生殖细胞RNA颗粒，尽管其中许多颗粒 具有未知的功能。因此，这项工作的长期目标是确定RNA颗粒在男性体内的作用 生殖细胞分化，在这样做的过程中，可能会发现转录后调控的新模式 管理生殖细胞的发展。ADAD2是一种未知分子功能的新型RBP，它形成了一个独特的 核糖核酸颗粒。ADAD2的丢失，并通过结合颗粒，导致减数分裂后严重的生殖细胞丢失和 完全的男性不育症。使用遗传模型和核糖体分离程序，我们已经证明了 减数分裂转录子亚群在蛋白质丰度降低的同时增加了核糖体关联 与Adad2突变体中的总mRNA水平无关。这些看似不同的结果表明 平移伸长缺陷。支持这一观点的是，ADAD2在物理上与多种蛋白质相互作用 影响翻译，包括eEF1B伸长复合体的成分和翻译的调节因子 抑制因子PIWIL1。总之，这些观察结果导致了ADAD2颗粒作为一种 减数分裂mRNAs子集的正确翻译中心。使用遗传模型、蛋白质组学和 先进的RNA测序分析，该提案将定义Adad2中更改翻译的驱动因素 并揭示ADAD2颗粒在精子发生中的作用。在目标1中，ADAD2如何影响 翻译伸长和伸长复合体本身也将被探究。这一目标将充分利用多组学 标度平移分析方法与定量质谱仪一起确定延伸率的变化 Adad2突变体的伸长复合体。目标2将描述ADAD2和ADAD2之间的相互作用 PIWIL1介导的翻译调控主要通过应用各种RNA测序范例来实现。 最后，Aim 3将通过利用遗传基因来定义ADAD2颗粒的蛋白质和RNA成分 影响ADAD2颗粒形成的模型。RNA颗粒不完全用以下术语描述 组成和功能，尽管它们对男性生育能力很重要。这项工作将把男性不育联系起来 在Adad2突变体中观察到ADAD2颗粒的分子功能，将其定义为基本的 哺乳动物生育力的调节器，并最终建立定义如何翻译的框架 伸长在雄性生殖细胞发育过程中受到调节。

PROJECT SUMMARY Male fertility is reliant on the proper regulation of mRNA translation during male germ cell development. This is achieved by the concerted actions of RNA binding proteins (RBPs). In meiotic male germ cells, RBPs are often localized to RNA dense cytoplasmic granules termed germ cell RNA granules although many of these granules are of unknown function. Thus, the long-term goal of this work is to define the role of RNA granules in male germ cell differentiation and in doing so, potentially discover new modalities of post-transcriptional regulation governing germ cell development. ADAD2 is a novel RBP of unknown molecular function that forms a unique RNA granule. Loss of ADAD2, and by association the granule, results in severe post-meiotic germ cell loss and complete male infertility. Using genetic models and ribosome isolation procedures, we have shown that a subset of meiotic transcripts have increased ribosome association concurrent with reduced protein abundance independent of total mRNA levels in Adad2 mutants. These seemingly disparate outcomes are indicative of translation elongation defects. Supporting this notion, ADAD2 physically interacts with multiple proteins that influence translation including a component of the eEF1B elongation complex and a regulator of the translation repressor PIWIL1. Together, these observations lead to the hypothesis that the ADAD2 granule acts as a hub for the proper translation of a subset of meiotic mRNAs. Using genetic models, proteomics, and advanced RNA-sequencing analyses, this proposal will define the drivers of altered translation in Adad2 mutants and uncover the role of the ADAD2 granule in spermatogenesis. In Aim 1, how ADAD2 influences translation elongation and the elongation complex itself will be explored. This aim will leverage multiple ‘omics scale translation analysis methods along with quantitative mass spectrometry to define changes in elongation and the elongation complex in Adad2 mutants. Aim 2 will delineate the interaction between ADAD2 and PIWIL1-mediated translation regulation primarily by the application of various RNA-sequencing paradigms. And lastly, Aim 3 will define the protein and RNA components of the ADAD2 granule by leveraging genetic models that impinge on ADAD2 granule formation. RNA granules are incompletely described in terms of composition and function, in spite of their importance for male fertility. This work will connect the male infertility observed in Adad2 mutants to the molecular function of the ADAD2 granule, defining it as a fundamental regulator of mammalian fertility and ultimately building the framework in which to define how translation elongation is regulated in male germ cell development.