Erk-mediated regulation of Dicer and Drosha function in C. elegans

描述(申请人提供)：卵子发生和胚胎发生需要多个信号网络和分子通路的精确协调。任何一个事件的扰动都可能导致一系列发育异常，如Apert和Pfieffer综合征和癌症。其中关键的组成部分是RAS-细胞外调节激酶(ERK)信号通路和DICER和DROSHA，它们是小RNA生物发生的重要中介。它们控制着广泛的细胞和发育过程，包括卵子发生和胚胎发生的许多关键方面。例如，在小鼠中，ERK信号的缺失会导致卵母细胞成熟缺陷和滋养细胞发育受扰，而在秀丽线虫中，ERK功能的缺失会扰乱卵母细胞的分化和成熟，导致不育或早期胚胎死亡。同样，在线虫和小鼠中，Dester或DROSHA的缺失分别扰乱了卵母细胞的减数分裂成熟和胚胎发育。然而，Ras/ERK通路和小RNA生物发生之间的功能交集尚不清楚，也没有研究表明Ras/ERK通路调节DICER或DROSHA的活性。我们对ERK调控秀丽线虫生殖系发育的底物的鉴定工作最近发现了RAS/ERK途径与DICER和DROSHA之间的直接、功能联系。在功能基因组学筛选中，我们确定了DICER和DROSHA是ERK的新的、进化上保守的底物，每个底物都以相互拮抗的方式与ERK发挥作用。通过产生针对磷酸化形式的抗体，我们还观察到ERK介导的DICER的磷酸化诱导它在线虫种系中从胞浆移位到核；这种磷酸化事件及其对DICER定位的影响在小鼠胚胎成纤维细胞和人类肿瘤细胞中是保守的。因此，我们提出的研究目标有三个：(1)阐明ERK依赖的磷酸化对DROSHA定位的影响；(2)测试ERK介导的磷酸化对DICER和DROSHA功能以及小RNA生产的影响；(3)通过确定修饰蛋白的功能伙伴来确定磷酸化DICER功能的机制。鉴于RAS/ERK信号通路与DICER和DROSHA的普遍相关性，我们的工作结果可能会为包括人类卵子发生和肿瘤发生在内的多种系统提供信息。好了！

DESCRIPTION (provided by applicant): Oogenesis and embryogenesis require the precise orchestration of multiple signaling networks and molecular pathways. Perturbations in any one event can lead to a host of developmental abnormalities such as the Apert and Pfieffer syndromes and cancer. Among the crucial components are the RAS-extracellular regulated kinase (ERK) signaling pathway and Dicer and Drosha, essential mediators of small RNA biogenesis. They govern a wide array of cellular and developmental processes, including many critical aspects of oogenesis and embryogenesis. For example, in mice, loss of ERK signaling results in defective oocyte maturation and perturbed trophoblast development, while in Caenorhabditis elegans loss of ERK function disrupts oocyte differentiation and maturation, leading to sterility or early embryonic lethality. Similarly, in C. elegans and mice, loss of Dicer or Drosha disrupts oocyte meiotic maturation and embryonic development, respectively. However, the functional intersection between the RAS/ERK pathway and small RNA biogenesis remains unclear, and no studies have shown that the RAS/ERK pathway regulates the activity of Dicer or Drosha. Our work on the identification of substrates through which ERK governs germline development in Caenorhabditis elegans recently uncovered a direct, functional link between the RAS/ERK pathway and Dicer and Drosha. In a functional genomics screen, we identified Dicer and Drosha as novel, evolutionarily conserved substrates of ERK that each function in a reciprocally antagonistic manner with ERK. By generating antibodies specific to the phosphorylated form of Dicer, we also observed that ERK-mediated phosphorylation of Dicer induces it to translocate from the cytoplasm to the nucleus in the C. elegans germline; this phosphorylation event and its consequences on Dicer localization are conserved in mouse embryonic fibroblasts and human tumor cells. Therefore, the goals of our proposed study are three-fold: i) to elucidate the effect of ERK-dependent phosphorylation on the localization of Drosha; ii) to test the impact of ERK-mediated phosphorylation on Dicer and Drosha function and small RNA production; and, iii) to determine the mechanistic basis of phospho-Dicer function by identifying functional partners of the modified proteins. Given the general relevance of RAS/ERK signaling pathway and Dicer and Drosha, results from our work will likely inform multiple systems, including human oogenesis and oncogenesis. !