Specialized translational machinery in the Drosophila male germline

我的研究项目的长期目标是了解男性生殖细胞发育的转录后调控。mRNA翻译的起始依赖于一种名为eIF4E的5'帽结合蛋白，该蛋白与其他翻译起始因子结合以招募核糖体。后生动物物种已经进化出多种eIF4E变体，其体内功能尚未明确。例如，三种小鼠eIF4E相似物中的两种突变与明显的神经缺陷有关，五种线虫eIF4E相似物中的三种具有与生育有关的独特作用。然而，目前尚不清楚这些eif4e的不同功能是如何被指定的。我们的数据表明，果蝇为解决这个问题提供了一个理想的系统。黑腹果蝇有8个eIF4E同源基因，其中5个在雄性种系中高度表达。我们之前的研究表明，eIF4E-3是成功完成减数分裂所必需的（Hernandez等，2012）。在NSERC资助的研究中，我们敲除了剩余的四个富含睾丸的eif4e中的三个，并发现eIF4E-5在精子发育结束时需要分离成熟精子（Shao等人，In prep.）。在男性生殖细胞中，eIF4E-3和eIF4E-5表达的时间和位置相似。因此，我们假设eIF4E-3和eIF4E-5的不同功能是由于它们的蛋白质序列和分子关联的差异。因此，我们有独特的定位来定义使两种eIF4E蛋白在同一细胞中发挥不同作用的特征。因此，我们的具体目标是：目标1。定义对eIF4E-3和eIF4E-5功能重要的蛋白序列。目标2。鉴定eIF4E-3和eIF4E-5功能所需的相互作用蛋白。为了实现这些目标，我们将进行结构域交换实验来确定eIF4E-3和-5中对其在精子发育中独特功能重要的序列，我们将使用亲和纯化结合质谱（AP-MS）来鉴定eIF4E-3和-5相关蛋白。我们将在生物化学上验证已确定的相互作用，并使用分子遗传学和表型分析的组合来测试这些相互作用在体内的重要性。我们的研究结果将确定这些果蝇eIF4Es变体促进可育精子形成的机制。更广泛地说，我们的研究将为理解翻译起始在发育过程中如何被控制以产生对执行独特的细胞和亚细胞功能重要的不同蛋白质亚群提供一个模型。

The long-term goal of my research program is to understand post-transcriptional regulation of male germ cell development. Initiation of mRNA translation relies on a 5' cap binding protein called eIF4E, which associates with other translation initiation factors to recruit the ribosome. Metazoan species have evolved multiple eIF4E variants whose in vivo functions are not well defined. For example, mutations in two of the three mouse eIF4E paralogs are associated with distinct neurological defects, and three of the five nematode eIF4E paralogs have unique roles related to fertility. However, it is unclear how the different functions of these eIF4Es are specified. Our data suggest that fruit flies provide an ideal system to address this question. Drosophila melanogaster has eight eIF4E paralogs, of which five are highly expressed in the male germline. We previously showed that eIF4E-3 is needed for successful completion of meiosis (Hernandez et al., 2012). In research funded by NSERC, we knocked out three of the four remaining testis-enriched eIF4Es and discovered that eIF4E-5 is needed at the end of sperm development to separate the mature sperm (Shao et al., in prep.). In male germ cells, the timing and location of eIF4E-3 and eIF4E-5 expression are similar. We therefore hypothesize that the distinct functions of eIF4E-3 and eIF4E-5 are due to differences in their protein sequences and molecular associations. We are therefore uniquely positioned to define the features that enable two eIF4E proteins to play different roles in the same cells. Thus, our specific aims are: Aim 1. Define protein sequences that are important for eIF4E-3 vs. eIF4E-5 function. Aim 2. Identify interacting proteins that are needed for eIF4E-3 vs. eIF4E-5 function. To address these aims, we will perform domain-swapping experiments to define the sequences in eIF4E-3 and -5 that are important for their unique functions in sperm development, and we will use affinity-purification coupled with mass spectrometry (AP-MS) to identify eIF4E-3 and -5 associated proteins. We will validate identified interactions biochemically and use a combination of molecular genetics and phenotypic analysis to test the importance of these interactions in vivo. Our results will define the mechanisms by which these variant Drosophila eIF4Es promote formation of fertile sperm. More broadly, our studies will provide a model for understanding how translation initiation is controlled during development to produce distinct subsets of proteins important for carrying out unique cellular and subcellular functions.