RNA binding protein networks and translational control in mammalian oocytes

在细胞的整个生命周期中，转录和转录后调控的组合控制着 基因表达，特别是在关键的发育决定和细胞周期中。这是 尤其是配子的发育。事实上，差异化需要许多关键的转变 这些细胞更多地依赖于mRNA的翻译调控，而不是转录。要克服技术上的 在生殖细胞实验模型中固有的障碍，我们开发了新的策略来研究如何 翻译在小鼠卵母细胞中是受调控的。监测母体rRNA与核糖体的关联 与单卵母细胞翻译试验一起，我们可以看到卵母细胞的翻译图景 在成长和成熟的过程中，以以前不可能实现的方式。通过在全基因组水平上监控翻译， 我们已经确定了在卵母细胞重新进入减数分裂细胞时发生的翻译上的整体切换。 周而复始。卵母细胞生长所需的mRNAs的翻译停止，而编码细胞周期的mRNAs的翻译停止 与胚胎发育所必需的成分的翻译一起被激活。 使用关键细胞周期成分Cyclin B1和Cyclin B2的翻译作为范例，我们有 证明了翻译定义了减数分裂细胞周期的一些独特属性。在此，我们建议 通过进一步探索母体mRNAs的差异翻译是如何在 卵母细胞的生长和成熟。这些实验是按照三个具体目标组织的。与第一个具体的 目的：探讨高水平翻译的3‘非编码区在卵母细胞中的内在特征。 生长和静止。我们将使用新的策略来确定一个信使核糖核酸3‘非编码区的功能元件 包括那些存在于Cyclin B2 mRNA中的基因，并了解它们如何在 体外和体内。带着第二个特定的目标，我们将探索蛋白质复合体的生化性质。 这是翻译的抑制因子，包括那些参与抑制细胞周期蛋白B1翻译的因子。在 最后一个特定的目标，我们将调查在卵母细胞重新进入 细胞周期。我们将在翻译抑制和细胞周期成分调控之间建立联系。 此外，我们还将探讨定义适时抑制翻译和不稳定的3‘UTR码。这个 这些研究的主要成果将是更好地理解抑制和激活的机制 配子发育过程中的母体基因。这些研究意义重大，因为它们将阐明 信使核糖核酸的翻译与细胞周期的调节和翻译程序如何引导密切相关。 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.