Role of Translational Regulators Nanos and Dazl in Preserving Totipotency

描述(由申请人提供)：在后生动物中，在发育早期将生殖细胞排除在体细胞命运之外是确保物种延续的基本过程。原始生殖细胞(PGC)至少执行四种活动，以保护它们免受体细胞分化和启动自己独特的基因表达程序：1)激活隔离的母体生殖系mRNAs；2)抑制母体体细胞信息；3)瞬时全基因组转录抑制，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；以及4)PGC特异基因在母体体细胞mRNAs降解后的转录激活。所有这些活动都是在没有转录的情况下发生的，因此必须首先在翻译层面上进行监管。拟议项目的目标是确定这一复杂计划中的关键参与者，揭示他们活动的机械性细节，并为保护和指定生殖系的网络构建一个可行的工作模式。RNA结合蛋白Nanos和DAZL是包括青蛙和人类在内的不同物种胚系中的翻译调节因子。两者在PGCs中都被一种未知的机制翻译激活。Nanos和DAZL都与另一种(序列特异的)RNA结合蛋白Pumilio(Pum)相互作用，调节翻译，但具有相反的作用：Nanos抑制目标RNA的翻译，而DAZL促进翻译。我们在非洲爪哇的工作表明，缺乏Nanos的PGCs过早地启动Pol II转录，不适当地表达体细胞基因，从而无法存活。缺乏DAZL的非洲爪哇原生殖细胞不能迁移到原始性腺，并从生殖系中消失。关键问题是什么会激活Nanos和DAZL，以及它们的靶标mRNAs的身份。初步研究支持RNA结合蛋白Dead-End作为Nanos的翻译激活剂的新角色。我们的工作模型是，生殖系的保存是由DND启动的，DND激活了Nanos的翻译，可能还激活了包括DAZL在内的其他生殖系mRNAs的翻译。然后，Nanos抑制对体细胞命运至关重要的母体mRNAs的翻译，而DAZL促进激活PGC特异性特征的RNAs的翻译。本项目的目的是：1)确定导致Nanos和其他潜在RNA靶标翻译激活的DND/RNA相互作用；最终的相关性测试将是在定义的体外系统中与任何所需的蛋白质伙伴和DND重建Nanos翻译；2)鉴定其被Nanos/Pum抑制以防止体细胞RNA异常表达的母体RNA；使用一种新的在PGCs中进行翻译调控的新方法，我们将在体内验证Nanos/Pum/RNA相互作用；3)鉴定其被DAZL激活的母体RNA；与DAZL共免疫沉淀的候选mRNAs将通过评估它们在DAZL耗尽胚胎中恢复PGC特征的能力在体内得到验证。有了这些信息，我们希望能够构建一个相对详细的工作模式，在发育的最早阶段调控胚系命运。

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.