RNA binding protein networks and translational control in mammalian oocytes

在细胞的整个生命周期中，转录和转录后调节的组合控制着细胞的生长。 基因表达，特别是在关键的发育决定和细胞周期期间。这是 尤其是配子的发育。事实上，差异化所需的许多关键转变 这些细胞更多地依赖于mRNA的翻译调节而不是转录。攻克技术 在生殖细胞实验模型中固有的障碍，我们已经开发了新的策略来研究如何 翻译在小鼠卵母细胞中受到调节。监测母体mRNA与核糖体的关联 结合单个卵母细胞翻译试验， 以以前不可能的方式生长和成熟。通过在全基因组水平上监测翻译， 我们已经确定了在卵母细胞重新进入减数分裂细胞时， 周期卵母细胞生长所需的mRNA的翻译停止，而细胞周期编码的mRNA的翻译 这些成分与胚胎发育所必需的成分的翻译一起被激活。 使用翻译的关键细胞周期组分细胞周期蛋白B1和细胞周期蛋白B2作为范例，我们有 证明了翻译定义了减数分裂细胞周期的一些独特特性。在此，我们建议 通过进一步探索母体mRNAs的差异翻译如何在哺乳动物中被调节来扩展这些观察结果。 卵母细胞生长和成熟。这些实验是按照沿着三个具体目标组织的。述第一特定 目的：研究卵母细胞高水平翻译的mRNA的3 '非翻译区的内在特征 生长与静止我们将使用新的策略来鉴定mRNA的3' UTR中的功能元件 包括存在于细胞周期蛋白B2 mRNA中的那些，并了解它们如何有助于细胞周期蛋白B2 mRNA的翻译控制。 体外和体内。在第二个特定目标中，我们将探索蛋白质复合物的生化特性 作为翻译抑制因子发挥作用，包括参与抑制CyclinB1翻译的那些。在 最后一个具体目标，我们将研究如何抑制翻译是触发时，卵母细胞重新进入 细胞周期我们将建立翻译抑制和细胞周期组分调控之间的联系。 此外，我们将探索定义翻译和不稳定的及时抑制的3' UTR代码。的 这些研究的主要成果将是更好地了解抑制和激活的机制， 配子发育过程中的母体mRNA。这些研究意义重大，因为它们将阐明 mRNA的翻译与细胞周期的调控以及翻译程序如何指导细胞周期的调控密切相关。 发展

Throughout the lifespan of a cell, a combination of transcriptional and post-transcriptional regulations control gene expression, particularly at the time of critical developmental decisions and during the cell cycle. This is especially true for the development of gametes. Indeed, many pivotal transitions required for the differentiation of these cells rely more on translational regulation of mRNA than on transcription. To overcome the technical hurdles inherent in the germ cell experimental model, we have developed novel strategies to investigate how translation is regulated in mouse oocytes. Monitoring the association of maternal mRNAs with ribosomes together with single oocyte translation assays has allowed us to view the translational landscape in oocytes during growth and maturation in ways previously not possible. By monitoring translation at a genome-wide level, we have identified a global switch in translation that occurs at the time of oocyte reentry into the meiotic cell cycle. Translation of mRNAs required for oocyte growth ceases, while translation of mRNA coding for cell cycle components is activated together with translation of components that will be necessary for embryo development. Using the translation of the critical cell cycle components Cyclin B1 and Cyclin B2 as a paradigm, we have demonstrated that translation defines some unique properties of the meiotic cell cycle. Here, we propose to extend these observations by exploring further how differential translation of maternal mRNAs is regulated during oocyte growth and maturation. The experiments are organized along three specific aims. With the first Specific Aim, we will investigate the intrinsic features of the 3'UTR of mRNAs translated at high levels during oocyte growth and quiescence. We will use novel strategies to identify functional elements in the 3' UTR of an mRNA including those present in the Cyclin B2 mRNA, and to understand how they contribute to translational control in vitro and in vivo. With the second Specific Aim, we will explore the biochemical properties of protein complexes that function as repressors of translation, including those involved in repression of CyclinB1 translation. In the last Specific Aim, we will investigate how repression of translation is triggered at the time of oocyte reentry into the cell cycle. We will establish links between translational repression and regulation of cell cycle components. In addition, we will explore the 3' UTR code that defines timely repression of translation and destabilization. The major outcome of these studies will be a better understanding of the mechanism of repression and activation of maternal mRNA during gamete development. These studies are significant because they will shed light on how mRNA translation is intimately involved in regulation of the cell cycle and how translational programs direct development.