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

 描述(由申请人提供)：该研究项目的目标是定义卵子是如何产生、供应和保护的，以及卵子是如何为从卵母细胞向胚胎的转变而戏剧性地重组的。对这一关键发育窗口的分析在果蝇中是可以获得的，由于发育中调节因子的保守，对果蝇的研究将确定调节机制和成分，这些调节机制和成分将阐明早期发育的基本特征，并在理解发育缺陷和不孕不育方面对人类健康产生影响。此外，分析果蝇卵是如何产生的，为控制后生动物DNA复制提供了模型，从而深入了解了癌细胞中的基因拷贝数是如何改变的。这项研究将研究DNA复制、mRNA翻译和蛋白质分解的关键过程，所有这些过程在从卵母细胞到胚胎的转变过程中都受到发育控制。在后生动物中，复制起始点的调控仍然难以捉摸，因为还不可能确定什么定义起始点，定位基本起始点识别复合体(ORC)，或捕获起始点激发及其控制。果蝇具有发育调控起源活动的巨大优势和在分化组织中定位ORC以定位起源的能力。分化和细胞周期修饰对ORC定位的影响将被确定，ORC结合、转录和复制抑制发育之间的关系将被确定。通过广泛的区域结合ORC并对分化信号进行重复的原点激发的复制起始点将被用来破译复制起始是如何被控制的。从分化的卵母细胞到全能胚胎的转变是在没有转录的情况下发生的，包括渗透压的快速变化和蛋白质组通过mRNA翻译和蛋白分解的急剧变化而发生的广泛的重塑。通过基因和蛋白质组筛选确定的调节器表明氧化还原状态和甘氨酸转运体在卵子激活中的关键作用，这将被描述。在这一发育过程中，PNG激酶被证明是mRNA翻译的主要调节者，在卵子中提供了完成减数分裂和控制mRNA翻译之间的联系。PNG激活翻译的机制(S)将通过检验以下假设来探索：它使翻译抑制物磷酸化和失活，并影响鸡蛋中mRNAs到RNP复合体的定位。后期促进复合体(APC)的一种独特形式，APCCort，专门针对蛋白质的降解，这些蛋白质的去除是从减数分裂到有丝分裂的变化所必需的。将评估对最近发现的在卵母细胞到胚胎转变中至关重要的底物进行APCCort靶向降解的要求，并将确定在这一发育时期特定表达的泛素途径的其他成分的功能。

 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.