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

Erk 介导的线虫 Dicer 和 Drosha 功能调节

• 批准号: 8147376
• 负责人: Swathi Arur
• 金额: $ 29.7万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8147376
• 关键词: Antibodies; Area; Biogenesis; Biological; Biological Models; Caenorhabditis elegans; Cell Nucleus; Cell physiology; Cellular biology; Complex; Cytoplasm; Data; Development; Developmental Process; Dicer Pathway; Down-Regulation; Embryo; Embryonic Development; Enzymes; Event; Extracellular Signal Regulated Kinases; Fertilization; Fibroblasts; Generations; Genetic; Genetic screening method; Genomics; Goals; Homebound Persons; Human; Individual; Lead; Link; Malignant Neoplasms; Mediating; Mediator of activation protein; Meiosis; Methods; MicroRNAs; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Oncogenic; Oocytes; Oogenesis; Pathway interactions; Pattern; Pfeiffer Syndrome; Phosphorylation; Phosphotransferases; Production; Proteins; Proteomics; Regulation; Relative (related person); Research; Role; Signal Pathway; Signal Transduction; Small RNA; Sterility; Syndrome; System; Testing; Tumor Suppressor Proteins; Work; base; extracellular; fly; functional genomics; human DICER1 protein; in vivo; interest; neoplastic cell; novel; oocyte maturation; programs; protein complex; trophoblast; tumorigenesis

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. ! PUBLIC HEALTH RELEVANCE: This proposed project aims to investigate the role of the oncogenic RAS/Extracellular-signal regulated kinase (ERK) in regulating the tumor suppressors Dicer and Drosha, two key small RNA biogenesis enzymes. The study integrates multiple approaches ranging from classical genetics and cell biology to genomic and proteomic methods to assess the impact of ERK mediated phosphorylation on Dicer and Drosha function in vivo during Caenorhabitis elegans germline development. !

描述（由申请人提供）：卵子发生和胚胎发生需要多个信号网络和分子途径的精确协调。任何一个事件的干扰都可能导致一系列发育异常，例如阿佩尔综合征和普菲弗综合征以及癌症。其中关键成分包括 RAS 细胞外调节激酶 (ERK) 信号通路以及小 RNA 生物发生的重要介质 Dicer 和 Drosha。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信号传导的丧失会导致卵母细胞成熟缺陷和滋养层发育受到干扰，而在秀丽隐杆线虫中，ERK功能的丧失会破坏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，Dicer 或 Drosha 的缺失分别会破坏卵母细胞减数分裂成熟和胚胎发育。然而，RAS/ERK通路与小RNA生物发生之间的功能交叉仍不清楚，并且没有研究表明RAS/ERK通路调节Dicer或Drosha的活性。我们对 ERK 控制秀丽隐杆线虫种系发育的底物的鉴定工作最近发现了 RAS/ERK 通路与 Dicer 和 Drosha 之间的直接功能联系。在功能基因组学筛选中，我们将 Dicer 和 Drosha 鉴定为 ERK 的新型进化保守底物，它们各自以与 ERK 相互拮抗的方式发挥作用。通过生成针对 Dicer 磷酸化形式的特异性抗体，我们还观察到 ERK 介导的 Dicer 磷酸化诱导其从线虫种系中的细胞质易位到细胞核；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：i）阐明 ERK 依赖性磷酸化对 Drosha 定位的影响； ii) 测试 ERK 介导的磷酸化对 Dicer 和 Drosha 功能以及小 RNA 产生的影响； iii) 通过鉴定修饰蛋白的功能伙伴来确定磷酸 Dicer 功能的机制基础。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多个系统提供信息，包括人类卵子发生和肿瘤发生。 ！ 公共健康相关性：本拟议项目旨在研究致癌 RAS/细胞外信号调节激酶 (ERK) 在调节肿瘤抑制因子 Dicer 和 Drosha（两种关键的小 RNA 生物发生酶）中的作用。该研究整合了从经典遗传学和细胞生物学到基因组和蛋白质组学方法的多种方法，以评估 ERK 介导的磷酸化对秀丽隐杆线虫种系发育过程中体内 Dicer 和 Drosha 功能的影响。 ！