Analysis of genetic enhancers of Ras/MAPK signaling in C. elegans

线虫 Ras/MAPK 信号传导遗传增强子分析

• 批准号: RGPIN-2018-05673
• 负责人: Rocheleau, Christian
• 金额: $ 3.06万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=663536
• 关键词: Analysis; genetic; enhancers; Ras; MAPK

Development of a multicellular animal from a single cell is a highly complex process requiring cell coordination and communication. Several highly-conserved signal transduction pathways are repeatedly used to regulate many aspects of development. In the nematode C. elegans, Ras/MAPK signaling regulates several developmental events including meiotic progression of germ cells, oocyte growth, cell fate specification and cell migration. For Ras/MAPK signaling to elicit different biological events, there must be unique regulators and downstream targets that tailor signaling such that the appropriate outcomes are reached. ******We identified several positive regulators of Ras/MAPK signaling during the specification of the excretory duct cell (EDC). The EDC is one of three tubular cells that make up the excretory system. Animals missing the EDC accumulate waste fluid and die as larvae. Most of these positive regulators promote Ras/MAPK signaling specifically in the EDC. However, UBC-25 appears to have a second role in promoting oocyte growth with Ras/MAPK signaling. The objective of our NSERC research program is to understand the mechanisms by which these cell type specific regulators promote Ras/MAPK signaling. The objectives of this proposal are 1) characterize the role of UBC-25 and Ras/MAPK signaling in oocyte growth, 2) characterize the role of EKL-7 in specification of the EDC and 3) determine the molecular identities of uncloned regulators. ******UBC-25 is a conserved E2 ubiquitin-conjugating enzyme. In addition to specifying the EDC fate, UBC-25 functions with Ras/MAPK signaling to regulate oocyte growth, but not meiotic progression providing a unique opportunity to determine the mechanisms by which Ras/MAPK promotes oocyte growth. We hypothesize that UBC-25 antagonizes a negative regulator of Ras/MAPK signaling to regulate oocyte growth and the EDC fate. EKL-7 lacks known functional domains, but has a cluster of MAPK Erk phosphorylation sites. EKL-7 genetically interacts with known Erk substrates. We hypothesize that EKL-7 is a direct target of Ras/MAPK signaling that cooperates with other Erk substrates to specify the EDC fate. Finally, we will complete genetic mapping of two novel mutations in genes that promote Ras/MAPK signaling in the EDC to determine their molecular identities to determine how they provide specificity to signaling. This research will provide new insights into the molecular functions of UBC-25 and EKL-7 proteins, the mechanisms that regulate Ras/MAPK signaling, and how Ras/MAPK regulates oocyte growth. This work will train HQP in critical thinking, model building, scientific writing, and molecular, genetic, and cell biological skills and knowledge for careers in the biological sciences and engineering.

从单细胞发育成多细胞动物是一个高度复杂的过程，需要细胞协调和沟通。 一些高度保守的信号转导途径被反复用于调节发育的许多方面。 在线虫中，Ras/MAPK 信号传导调节多种发育事件，包括生殖细胞的减数分裂进程、卵母细胞生长、细胞命运规范和细胞迁移。 为了使 Ras/MAPK 信号传导引发不同的生物事件，必须有独特的调节因子和下游靶标来定制信号传导，从而达到适当的结果。 ******我们在排泄管细胞 (EDC) 的规范过程中发现了 Ras/MAPK 信号传导的几个正调节因子。 EDC 是构成排泄系统的三个管状细胞之一。缺少 EDC 的动物会积聚废液并以幼虫的形式死亡。 大多数这些正调节因子特别促进 EDC 中的 Ras/MAPK 信号传导。然而，UBC-25 似乎在通过 Ras/MAPK 信号传导促进卵母细胞生长方面发挥着第二个作用。 我们 NSERC 研究计划的目标是了解这些细胞类型特异性调节因子促进 Ras/MAPK 信号传导的机制。 该提案的目标是 1) 表征 UBC-25 和 Ras/MAPK 信号在卵母细胞生长中的作用，2) 表征 EKL-7 在 EDC 规范中的作用，3) 确定未克隆调节因子的分子身份。 ******UBC-25 是一种保守的 E2 泛素结合酶。 除了指定 EDC 命运外，UBC-25 还与 Ras/MAPK 信号传导一起调节卵母细胞生长，但不调节减数分裂进展，从而为确定 Ras/MAPK 促进卵母细胞生长的机制提供了独特的机会。 我们假设 UBC-25 拮抗 Ras/MAPK 信号传导的负调节因子来调节卵母细胞生长和 EDC 命运。 EKL-7 缺乏已知的功能域，但具有一组 MAPK Erk 磷酸化位点。 EKL-7 与已知的 Erk 底物存在遗传相互作用。 我们假设 EKL-7 是 Ras/MAPK 信号传导的直接靶标，与其他 Erk 底物合作指定 EDC 命运。最后，我们将完成 EDC 中促进 Ras/MAPK 信号传导的两个新基因突变的遗传图谱，以确定它们的分子身份，从而确定它们如何提供信号传导的特异性。 这项研究将为 UBC-25 和 EKL-7 蛋白的分子功能、调节 Ras/MAPK 信号传导的机制以及 Ras/MAPK 如何调节卵母细胞生长提供新的见解。 这项工作将培训 HQP 的批判性思维、模型构建、科学写作以及生物科学和工程职业所需的分子、遗传和细胞生物学技能和知识。