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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760831
• 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.

从单细胞到多细胞动物的发育是一个高度复杂的过程，需要细胞的协调和沟通。 几个高度保守的信号转导通路被反复用于调节发育的许多方面。 线虫C. Ras/MAPK信号传导调节几种发育事件，包括生殖细胞的减数分裂进程、卵母细胞生长、细胞命运特化和细胞迁移。 对于Ras/MAPK信号传导引发不同的生物学事件，必须有独特的调节剂和下游靶点来定制信号传导，以达到适当的结果。 我们确定了几个积极的调节Ras/MAPK信号在指定的排泄管细胞（EDC）。 EDC是组成排泄系统的三个管状细胞之一。缺少EDC的动物积累废液并作为幼虫死亡。 大多数这些正调节剂促进Ras/MAPK信号传导，特别是在EDC中。然而，UBC-25似乎在促进卵母细胞生长中具有Ras/MAPK信号传导的第二作用。 我们NSERC研究计划的目标是了解这些细胞类型特异性调节剂促进Ras/MAPK信号传导的机制。 本提案的目的是1）表征UBC-25和Ras/MAPK信号传导在卵母细胞生长中的作用，2）表征EKL-7在EDC的特化中的作用，和3）确定未克隆调节物的分子身份。 UBC-25是一种保守的E2泛素结合酶。 除了指定EDC的命运，UBC-25的功能与Ras/MAPK信号调节卵母细胞的生长，但不减数分裂的进展提供了一个独特的机会，以确定Ras/MAPK促进卵母细胞生长的机制。 我们假设UBC-25拮抗Ras/MAPK信号的负调节剂来调节卵母细胞的生长和EDC的命运。 EKL-7缺乏已知的功能结构域，但具有一簇MAPK Erk磷酸化位点。 EKL-7与已知的Erk底物发生遗传相互作用。 我们假设EKL-7是Ras/MAPK信号传导的直接靶点，其与其他Erk底物合作以指定EDC命运。最后，我们将完成两个新的基因突变的遗传图谱，促进Ras/MAPK信号在EDC中，以确定其分子身份，以确定他们如何提供特异性信号。 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。 这项工作将培养HQP在批判性思维，模型构建，科学写作，分子，遗传和细胞生物学技能和知识的职业生涯在生物科学和工程。