The function of germ granules in maintaining pluripotency in the C. elegans germline

胚芽颗粒在维持线虫种系多能性中的功能

• 批准号: 9023577
• 负责人: Dustin Lynn Updike
• 金额: $ 33.3万
• 依托单位: MOUNT DESERT ISLAND BIOLOGICAL LAB
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9023577
• 关键词: Adult; Affinity; Animal Model; Binding; Caenorhabditis elegans; Cell Nucleus; Cells; Complex; Cytoplasm; Cytoplasmic Granules; Data; Disease; Event; Exhibits; Genetic; Genetic Models; Genetic Transcription; Germ; Germ Cells; Goals; Health; Human; Meiosis; Messenger RNA; Methods; Modeling; Molecular; Mutation; Pathway interactions; Patients; Phenocopy; Post-Transcriptional Regulation; Process; Property; Proteins; RNA; RNA-Binding Proteins; Regenerative Medicine; Repression; Research; Ribonucleoproteins; Role; Small Interfering RNA; Somatic Cell; Spermatogenesis; Stem cells; Structure; Surveys; Techniques; Testing; Time; Transcript; Translating; Translations; cell type; cofactor; epigenetic memory; improved; innovation; mRNA Expression; mutant; neuronal cell body; novel strategies; patient safety; pluripotency; protein expression; self-renewal; sex determination; sperm cell; stemness; translation factor

 DESCRIPTION (provided by applicant): Stem cells are defined by their pluripotent and self-renewing properties. A significant amount of research is focused on unlocking these properties in differentiated cells in order to create patient-derived stem cells that can be used to treat disease. However, the process of creating patient-derived stem cells relies heavily on expressing pluripotency factors within the nucleus; using techniques that often render stem cells too dangerous for regenerative medicine. This proposal takes a unique approach to understand how cytoplasmic components called germ granules regulate cellular "stemness". The ultimate goal is to be able to manipulate germ-granule pluripotency factors within the cytoplasm to improve the quality and safety of patient-derived stem cells. Germ granules are a heterogeneous mix of RNA and RNA-binding proteins, and they are conserved in the germline from worms to humans. The transparency and sophisticated genetics of C. elegans offers an opportunity to study germ-granule function in a way that is not feasible in more complex model organisms. Our recent studies have shown that germ-granules create a cytoplasmic microenvironment that facilitates post- transcriptional regulation events that are central to the pluripotent and self-renewing properties of the germline. We have also shown that depleting germ granules causes germ cells to lose pluripotency, initiate somatic differentiation, and express transcripts associated with spermatogenesis. Our preliminary data suggests that germ granules maintain pluripotency by 1) silencing somatic mRNAs that are stochastically expressed in the germline, and 2) repressing the function of associated proteins required for sperm-specific mRNA expression. We will test these ideas by inducing the expression of mRNAs that exhibit high germ-granule affinity, and then we will compare changes in their distribution, accumulation, and translation in the presence and absence of germ granules (Aim 1). We will also determine how and which sperm-promoting pathways are negatively regulated by germ granules (Aim 2). And finally, we examine a specific germ-granule protein, CSR-1, that has the capacity to recognize and promote the expression of germline mRNAs, and will determine how it and its cofactors function in germ granules to promote germ-cell pluripotency (Aim 3). These findings will provide new approaches to safely induce cellular pluripotency when it is needed and shut off pluripotency when it is not.

 描述（由申请人提供）：干细胞的定义是其多能和自我更新的特性，大量的研究集中在解锁分化细胞的这些特性，以创建可用于治疗疾病的患者来源的干细胞。然而，创建患者来源的干细胞的过程在很大程度上依赖于在细胞核内表达多能性因子；使用的技术通常会使干细胞对于再生医学来说过于危险。生殖颗粒调节细胞“干性”，最终目标是能够操纵细胞质内的生殖颗粒多能性因子，以提高患者来源的干细胞的质量和安全性。线虫的透明性和复杂的遗传学提供了以在更复杂的模型中不可行的方式研究生殖颗粒功能的机会。我们最近的研究表明，生殖颗粒创造了一个细胞质微环境，促进转录后调控事件，这对于生殖系的多能和自我更新特性至关重要。我们还表明，耗尽生殖颗粒会导致生殖细胞丢失。我们的初步数据表明，胚芽颗粒通过 1) 沉默体细胞 mRNA 来维持多能性。 2) 抑制精子特异性 mRNA 表达所需的相关蛋白的功能，我们将通过诱导表现出高胚芽颗粒亲和力的 mRNA 的表达来测试这些想法，然后我们将比较它们的变化。在存在和不存在胚芽颗粒的情况下的分布、积累和翻译（目标 1）我们还将确定胚芽颗粒如何以及哪些促进精子的途径受到负调控（目标 2）。特定的生殖颗粒蛋白 CSR-1 具有识别和促进生殖系 mRNA 表达的能力，并将决定其及其辅助因子如何在生殖颗粒中发挥作用以促进生殖细胞多能性（目标 3）。提供新的方法来在需要时安全地诱导细胞多能性，并在不需要时关闭多能性。