Role of Translational Regulators Nanos and Dazl in Preserving Totipotency

翻译调节因子 Nanos 和 Dazl 在保留全能性中的作用

• 批准号: 8506391
• 负责人: MARY LOU KING
• 金额: $ 36.82万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8506391
• 关键词: ATP phosphohydrolase; Address; Apoptosis; Binding; Binding Sites; Bioinformatics; Biological Assay; Biological Preservation; Cardiovascular Diseases; Cessation of life; Characteristics; Code; Complex; DNA Polymerase II; Data; Degenerative Disorder; Development; Diabetes Mellitus; Dorsal; Embryo; Endoderm; Ensure; Exclusion; Exhibits; Future; Gene Expression; Genes; Genetic; Genetic Transcription; Germ Cells; Germ Lines; Goals; Gonadal structure; Human; Immunofluorescence Immunologic; In Vitro; Infertility; Laboratories; Lead; Maternal Messenger RNA; Messenger RNA; MicroRNAs; Microarray Analysis; Modeling; Mus; Nature; Parkinson Disease; Pattern; Pluripotent Stem Cells; Process; Proteins; RNA; RNA Binding; RNA Recognition Motif; RNA-Binding Proteins; Rana; Recruitment Activity; Regulation; Repression; Rest; Role; Somatic Cell; Specific qualifier value; Staging; Sterility; Structure; Structure of primordial sex cell; System; Testing; Transcriptional Activation; Translational Activation; Translational Regulation; Translations; Untranslated Regions; Vertebrates; Work; Xenopus; falls; genome-wide; helicase; in vivo; migration; network models; novel; pluripotency; prevent; programs; public health relevance; reconstitution; stem cell biology; trait; transcription factor

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.

描述（由申请人提供）：在早期发育过程中将生殖细胞排除在体细胞命运之外是后生动物中确保物种延续的一个重要过程。原始生殖细胞 (PGC) 至少执行四种活动，这些活动是保护它们免于体细胞分化并启动其自身独特的基因表达程序所需的：1) 激活隔离的母体生殖系 mRNA； 2）母体躯体信息的压抑； 3）瞬时全基因组转录抑制，以确保当合子转录在胚胎其余部分启动时体细胞程序不会被激活； 4) 母体体细胞 mRNA 降解后 PGC 特异性基因的转录激活。所有这些活动都在没有转录的情况下发生，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计划中的关键参与者，揭示他们活动的机制细节，并为保护和指定种系的网络构建可行的工作模型。 RNA 结合蛋白 Nanos 和 Dazl 是不同物种（包括青蛙和人类）种系中的翻译调节因子。两者均通过未知机制在 PGC 中翻译激活。 Nanos 和 Dazl 均与另一种（序列特异性）RNA 结合蛋白 Pumilio (Pum) 相互作用来调节翻译，但效果相反：Nanos 抑制目标 RNA 的翻译，而 Dazl 则促进其翻译。我们在非洲爪蟾中的研究表明，缺乏 Nanos 的 PGC 会过早启动 Pol II 转录，不适当地表达体细胞基因，并且无法存活。缺乏dazl的非洲爪蟾PGC无法迁移至原始性腺并从种系中丢失。接下来的关键问题是什么激活 Nanos 和 dazl，以及它们的目标 mRNA 的身份。初步研究支持 RNA 结合蛋白 Dead-end 作为 Nanos 翻译激活剂的新作用。我们的工作模型是，种系的保存是由 Dnd 启动的，它激活 Nanos 以及可能包括 dazl 在内的其他种系 mRNA 的翻译。 Nanos 然后抑制对体细胞命运至关重要的母体 mRNA 的翻译，而 Dazl 则促进激活 PGC 特异性特征的 RNA 的翻译。该项目的目标是：1) 定义导致 Nanos 和其他潜在 RNA 靶标翻译激活的 Dnd/RNA 相互作用；相关性的最终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) 鉴定需要被 Nanos/Pum 抑制的母体 RNA，以防止体细胞 RNA 的异常表达；使用一种新的 PGC 翻译调控分析方法，我们将验证 Nanos/Pum/RNA 体内相互作用； 3) 鉴定PGC身份所需的母体RNA，其被Dazl激活；与 Dazl 共免疫沉淀的候选 mRNA 将通过评估它们在 Dazl 耗尽的胚胎中恢复 PGC 性状的能力进行体内验证。有了这些信息，我们期望能够构建一个相对详细的工作模型，用于在发育的最早阶段调控种系命运。