Producing, provisioning, and protecting the egg: Regulation of DNA replication, mRNA translation, and proteolysis for the transition from oocyte to embryo

卵子的生产、供应和保护：从卵母细胞到胚胎过渡的 DNA 复制、mRNA 翻译和蛋白水解的调节

• 批准号: 9071147
• 负责人: Terry L. ORR-WEAVER
• 金额: $ 61.98万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-04 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9071147
• 关键词: Affect; Binding; Cell Cycle; Complex; Congenital Abnormality; DNA biosynthesis; Defect; Development; Drosophila genus; Embryo; Excision; Fertility; Gene Dosage; Genetic; Genetic Transcription; Genetic Translation; Goals; Health; Human; Infertility; Light; Link; Maps; Meiosis; Messenger RNA; Mitosis; Modeling; Modification; Oocytes; Osmolalities; Osmolar Concentration; Oxidation-Reduction; Pathway interactions; Phosphotransferases; Process; Proteins; Proteolysis; Proteome; Proteomics; Regulation; Replication Initiation; Replication Origin; Repression; Research; Role; Signal Transduction; Testing; Tissues; Totipotent; Translations; Ubiquitin; Work; anaphase-promoting complex; cancer cell; egg; glycine transporter; insight; origin recognition complex; prevent; programs; protein degradation; public health relevance; response

 DESCRIPTION (provided by applicant): The goal of this research program is to define how an egg is produced, provisioned, and protected and how the egg is dramatically reorganized for the transition from oocyte to embryo. Analysis of this key window in development is accessible in Drosophila, and due to the conservation of regulators in development, studies in Drosophila will identify regulatory mechanisms and components that will shed light on fundamental features of early development, with implications for human health in understanding developmental defects and infertility. In addition, analysis of how a Drosophila egg is produced has provided models for the control of metazoan DNA replication, generating insights into how gene copy number can become altered in cancer cells. The proposed research will study the crucial processes of DNA replication, mRNA translation, and proteolysis, all of which are subjected to developmental control in the transition from oocyte to embryo. The regulation of replication origins has remained elusive in metazoans because it has not been possible to determine what defines an origin, to localize the essential Origin Recognition Complex (ORC), or to capture origin firing and its control. Drosophila offers the tremendous advantages of developmental control of origin activity and the ability to localize ORC in differentiated tissues to map origins The effect of differentiation and cell cycle modification on ORC localization will be determined, and the relationship between ORC binding, transcription and developmental repression of replication defined. Replication origins that bind ORC across a broad zone and undergo repeated rounds of origin firing in response to differentiation signals will be exploited to deciphr how replication initiation is controlled. The transition from the differentiated oocyte to the totipotent embryo occurs in the absence of transcription, involving rapid changes in osmolarity and extensive remodeling of the proteome by dramatic changes in mRNA translation and proteolysis. Regulators identified by genetic and proteomic screens indicate a crucial role for redox state and a glycine transporter in egg activation that will be delineated. The PNG kinase was shown to be a master regulator of mRNA translation specifically at this developmental transition, providing the link between completion of meiosis and control of mRNA translation in the egg. The mechanism(s) through which PNG activates translation will be explored by testing the hypotheses that it phosphorylates and inactivates translational repressors and affects the localization of mRNAs to RNP complexes in the egg. A unique form of the Anaphase Promoting Complex (APC), APCCort, specifically targets proteins for degradation whose removal is required for the change from meiosis to mitosis. The requirement for APCCort-targeted degradation of recently identified substrates that are critical in the oocyte-to-embryo transition will be evaluated, and the function of other components of the ubiquitin pathway expressed specifically in this developmental period will be defined.

 描述（由申请人提供）：本研究计划的目标是确定卵子是如何产生、供应和保护的，以及卵子是如何戏剧性地重组以从卵母细胞过渡到胚胎的。在果蝇中可以分析发育中的这一关键窗口，并且由于发育中的调节因子的保守性，在果蝇中的研究将确定调节机制和组分，这些调节机制和组分将揭示早期发育的基本特征，并对人类健康理解发育缺陷和不育产生影响。此外，对果蝇卵如何产生的分析为控制后生动物DNA复制提供了模型，使人们了解癌细胞中基因拷贝数如何改变。拟议的研究将研究DNA复制，mRNA翻译和蛋白质水解的关键过程，所有这些都受到从卵母细胞到胚胎过渡的发育控制。 在后生动物中，复制起点的调节仍然是难以捉摸的，因为它还不可能确定什么定义了一个起点，定位基本的起点识别复合物（ORC），或捕获起点发射及其控制。果蝇提供了巨大的优势，发育控制的起源活动和定位ORC在分化的组织中的能力，以映射起源。分化和细胞周期修饰对ORC定位的影响将被确定，并确定ORC结合，转录和发育抑制复制之间的关系。将利用跨越宽区域结合ORC并响应于分化信号而经历多轮原点激发的复制起点来解密如何控制复制起始。 从分化的卵母细胞到全能胚胎的转变发生在转录的情况下，涉及渗透压的快速变化和蛋白质组的广泛重塑，mRNA翻译和蛋白质水解的急剧变化。通过遗传和蛋白质组学筛选确定的调节剂表明氧化还原状态和甘氨酸转运蛋白在卵活化中的关键作用，将被描绘出来。PNG激酶被证明是mRNA翻译的主要调节因子，特别是在这个发育过渡期，提供了减数分裂完成和卵中mRNA翻译控制之间的联系。PNG激活翻译的机制将通过测试其磷酸化和失活翻译阻遏物并影响mRNA在卵中定位于RNP复合物的假设来探索。后期促进复合物（APC）的一种独特形式，APCCort，特异性靶向蛋白质降解，其去除是从减数分裂到有丝分裂的变化所必需的。将评价最近鉴定的在卵母细胞至胚胎转变中至关重要的底物的APCCort靶向降解的要求，并将定义在该发育期特异性表达的泛素途径的其他组分的功能。