Role of Translational Regulators Nanos and Dazl in Preserving Totipotency

描述（由申请方提供）：在早期发育中，体细胞命运中排除生殖细胞是后生动物确保物种延续的重要过程。原始生殖细胞（PGCs）至少执行四种必需的活动以保护它们免于体细胞分化并启动它们自己独特的基因表达程序：1）激活隔离的母体生殖系mRNA; 2）抑制母体体细胞信息; 3）瞬时基因组-广泛抑制转录，以确保当合子转录在胚胎的其余部分启动时，体细胞程序不被激活;和4）在母体体细胞mRNA降解后PGC特异性基因的转录激活。所有这些活动都是在没有转录的情况下发生的，因此必须主要在翻译水平上进行调节。拟议项目的目标是定义这个复杂程序中的关键参与者，揭示其活动的机械细节，并为保护和指定生殖系的网络构建一个可行的工作模型。 RNA结合蛋白Nanos和Dazl是不同物种（包括青蛙和人类）生殖系中的翻译调节因子。两者在PGCs中通过未知的机制被激活。Nanos和Dazl都与另一种（序列特异性）RNA结合蛋白Pumilio（Pum）相互作用，以调节翻译，但具有相反的效果：Nanos抑制靶RNA的翻译，而Dazl促进它。我们在非洲爪蟾中的工作表明，缺乏Nanos的PGCs过早启动Pol II转录，不适当地表达体细胞基因，并且不能存活。缺乏dazl的爪蟾PGCs不能迁移到原始性腺，并从种系中丢失。关键问题是什么激活了Nanos和dazl，以及它们的靶mRNA的身份。初步研究支持RNA结合蛋白Dead-end作为Nanos的翻译激活剂的新作用。我们的工作模型是，生殖系的保存是由Dnd启动的，它激活了Nanos和可能包括dazl在内的其他生殖系mRNA的翻译。然后，Nanos抑制对体细胞命运至关重要的母体mRNA的翻译，而Dazl促进激活PGC特异性性状的RNA的翻译。该项目的目的是：1）确定导致Nanos和其他潜在RNA靶点翻译激活的Dnd/RNA相互作用;相关性的最终测试将是在具有任何所需蛋白质伴侣和Dnd的定义的体外系统中重建Nanos翻译; 2）鉴定需要Nanos/Pum抑制以防止体细胞RNA异常表达的母体RNA;使用一种新的PGC翻译调节测定法，我们将在体内鉴定Nanos/Pum/RNA相互作用; 3）鉴定其被Dazl激活是PGC身份所需的母体RNA;通过评估与Dazl共免疫沉淀的候选mRNA在Dazl耗尽的胚胎中恢复PGC性状的能力，将在体内验证它们。有了这些信息，我们希望能够构建一个相对详细的工作模型，在发育的最早阶段调控生殖系的命运。

DESCRIPTION (provided by applicant): The exclusion of germ cells from somatic cell fates in early development is an essential process in metazoans that ensures continuation of the species. Primordial germ cells (PGCs) execute at least four activities that are required to both protect them from somatic differentiation and to initiate their own unique gene expression programs: 1) activation of sequestered maternal germline mRNAs; 2) repression of maternal somatic messages; 3) transient genome-wide suppression of transcription to ensure that somatic programs are not activated when zygotic transcription initiates in the rest of the embryo; and 4) transcriptional activation of PGC-specific genes after degradation of maternal somatic mRNAs. All of these activities occur in the absence of transcription and therefore must be regulated primarily at the level of translation. The goal of the proposed project is to define key players in this intricate program, to uncover mechanistic details of their activities, and to construct a viable working model for the network that protects and specifies the germline. The RNA-binding proteins Nanos and Dazl are translational regulators in the germlines of diverse species, including frogs and humans. Both are translationally activated in PGCs by an unknown mechanism. Nanos and Dazl both interact with another (sequence-specific) RNA-binding protein, Pumilio (Pum), to regulate translation, but with opposite effects: Nanos represses translation of target RNAs, while Dazl promotes it. Our work in Xenopus has shown that PGCs lacking Nanos prematurely initiate Pol II transcription, inappropriately express somatic genes, and do not survive. Xenopus PGCs deficient in dazl fail to migrate to the primordial gonads and are lost from the germline. Key questions then are what activates Nanos and dazl, and the identities of their target mRNAs. Preliminary studies support a new role for the RNA-binding protein, Dead-end, as a translational activator of Nanos. Our working model is that preservation of the germline is initiated by Dnd, which activates the translation of Nanos and probably other germline mRNAs including dazl. Nanos then represses translation of maternal mRNAs essential for somatic fates, while Dazl promotes translation of RNAs that activate PGC-specific traits. The aims of this project are: 1) to define the Dnd/RNA interactions that result in translational activation of Nanos and other potential RNA targets; the definitive test for relevance will be to reconstitute Nanos translation in a defined in vitro system with any required protein partners and Dnd; 2) to identify the maternal RNAs whose repression by Nanos/Pum is required to prevent aberrant expression of somatic RNAs; using a novel assay for translational regulation in PGCs, we will authenticate Nanos/Pum/RNA interactions in vivo; 3) to identify the maternal RNAs whose activation by Dazl is required for PGC identity; candidate mRNAs that co-immunoprecipitate with Dazl will be validated in vivo by assessing their abilities to restore PGC traits in Dazl-depleted embryos. With this information, we expect to be able to construct a relatively detailed working model for the regulation of germ-line fate in the earliest stages of development.