Novel mechanisms regulating translation elongation during male germ cell differentiation

项目摘要 雄性生殖力依赖于雄性生殖细胞发育过程中mRNA翻译的适当调节。这是 通过RNA结合蛋白（RBP）的协同作用实现。在减数分裂的雄性生殖细胞中，RBP通常是 定位于RNA致密的细胞质颗粒，称为生殖细胞RNA颗粒，尽管许多这些颗粒 功能不明。因此，这项工作的长期目标是确定RNA颗粒在男性中的作用。 生殖细胞分化，并在这样做，潜在地发现新的转录后调控模式 控制着生殖细胞的发育ADAD 2是一种新的RBP，其分子功能未知， RNA颗粒。ADAD 2的缺失，以及与之相关的颗粒的缺失，导致严重的减数分裂后生殖细胞缺失， 完全男性不育使用遗传模型和核糖体分离程序，我们已经表明， 减数分裂转录物子集具有增加的核糖体结合，同时具有减少的蛋白质丰度 独立于Adad2突变体中的总mRNA水平。这些看似不同的结果表明， 翻译延伸缺陷。支持这一观点的是，ADAD 2与多种蛋白质发生物理相互作用， 影响翻译，包括eEF1B延伸复合物的组分和翻译的调节物 阻遏蛋白PIWIL1。总之，这些观察结果导致了ADAD 2颗粒作为一种蛋白质的假设。 是减数分裂mRNA子集正确翻译的中心。利用遗传模型，蛋白质组学， 先进的RNA测序分析，该提案将定义Adad2中改变翻译的驱动因素 突变体，并揭示ADAD 2颗粒在精子发生中的作用。在目标1中，ADAD 2如何影响 翻译延伸和延伸复合体本身将被探索。这一目标将利用多个“经济学” 标度平移分析方法沿着定量质谱法，以确定伸长率的变化 和Adad2突变体中的延伸复合物。目的2将描述ADAD 2和 PIWIL1介导的翻译调控主要通过应用各种RNA测序范例。 最后，Aim 3将通过利用遗传学方法来定义ADAD 2颗粒的蛋白质和RNA组分。 影响ADAD2颗粒形成的模型。RNA颗粒的描述是不完全的， 组成和功能，尽管它们对男性生育力很重要。这项工作将连接男性不育症 在Adad2突变体中观察到的ADAD2颗粒的分子功能，将其定义为一个基本的 调节哺乳动物生育能力，并最终建立框架，以确定如何翻译 伸长在雄性生殖细胞发育中受到调节。

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.