Understanding germ cell pluripotency through an inducible P-granule degradation system

通过诱导性 P 颗粒降解系统了解生殖细胞多能性

• 批准号: 9788750
• 负责人: Elisabeth Adkins Marnik
• 金额: $ 6.43万
• 依托单位: MOUNT DESERT ISLAND BIOLOGICAL LAB
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-09 至 2020-09-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788750
• 关键词: Adult; Animals; Auxins; Biological Assay; CRISPR/Cas technology; Caenorhabditis elegans; Cell Nucleus; Cell Proliferation; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoplasm; Cytoplasmic Granules; Data; Development; Engineering; Exposure to; Fertility; Generations; Genes; Germ; Germ Cells; Goals; Human; Imagery; Inherited; Long-Term Effects; Malignant Neoplasms; Messenger RNA; Morphology; Muscle; Neurites; Neurons; Oocytes; Organism; Postdoctoral Fellow; Process; Proteins; RNA Interference; Regulation; Reporter; Research; Research Personnel; Ribonucleoproteins; Stem cells; Sterility; System; Techniques; Testing; Therapeutic; Time; Transcript; Translations; Work; base; cell type; in vivo; innovation; insight; mRNA Expression; mRNA sequencing; neuronal cell body; pluripotency; premature; prevent; protein degradation; rapid technique; regenerative; safety net; sperm cell; young adult

Project Summary: Understanding how pluripotency is achieved and maintained is a critical first step in enabling researchers to induce pluripotency when it is needed or prevent it when it is harmful in cancer. One potential avenue of research lies in studying germ granules. Germ granules are heterogeneous ribonucleoprotein (RNP) complexes found in germ cell cytoplasm from worms to humans. In C.elegans, germ granules, known as P granules, are critical for maintaining fertility and germ cell pluripotency. Research done in the Updike lab has shown that depletion of P granules through RNAi triggers sterility and causes misexpression of sperm, muscle and pan- neuronal markers in the germ cells. This could be due to P granules antagonizing the accumulation of misexpressed transcripts or selectively repressing their translation. Distinguishing between these possibilities is critical to elucidating the function of P granules in pluripotency. However, research so far has been hampered by the technical limitations of P-granule RNAi. P granules are maternally inherited, thus RNAi must be initiated in the generation before analysis. Therefore, it is currently difficult to elucidate immediate and long term effects of induced P-granule loss. The work proposed in this application takes advantage of a new technique to deplete P granules called the auxin-inducible degradation (AID) system. P granules have three critical protein components – PGL-1, PGL-2 and PGL-3. To degrade these targets CRISPR was used to engineer a degron tag to the end of all three endogenous genes in C. elegans. Then, upon auxin exposure, these worms (referenced as PgAID) will degrade PGL-1, PGL-2 and PGL-3 resulting in elimination of these nucleators of P-granule assembly and dispersal of P granules. These PgAID worms will allow for the identification of the immediate, mid-, and long-term effects of P- granule dispersal in the adult germline. This will be done by comparing PgAID worms to controls to identify alterations in germ-cell proliferation, sterility, brood size, and morphology due to P-granule dispersal. mRNA and reporter expression of select sperm and neuronal genes will also be examined and compared to previous data generated with the P-granule RNAi approach. PgAID worms will also be used to determine the sequential changes in mRNA expression following P-granule depletion. This will be done by performing mRNA-seq on dissected germlines immediately after P-granule dispersal, and at times before and at the onset of somatic reporter misexpression. Overall, this proposal will provide a critical next step in elucidating the mechanisms used in the germline to maintain cellular pluripotency, which will help unlock the possibility of successfully using these mechanisms for therapeutic benefit.

项目概要： 了解多能性是如何实现和维持的，是使研究人员 在需要时诱导多能性，或在癌症中有害时预防多能性。一个潜在的途径， 研究在于研究胚芽颗粒。胚粒是一种异质性核糖核蛋白复合体 在从蠕虫到人类的生殖细胞质中发现。在秀丽隐杆线虫中，称为P颗粒的胚芽颗粒， 对维持生育能力和生殖细胞多能性至关重要。厄普代克实验室的研究表明， 通过RNAi耗尽P颗粒触发不育，并导致精子、肌肉和泛精子表达错误。 生殖细胞中的神经元标记这可能是由于P颗粒拮抗 错误表达的转录本或选择性抑制其翻译。区分这些可能性是 这对阐明P颗粒在多能性中的功能至关重要。然而，迄今为止的研究受到了阻碍， 受到P-颗粒RNAi技术的限制。P颗粒是母系遗传的，因此必须启动RNAi 在分析之前的一代。因此，目前很难阐明即时和长期影响 诱导性P颗粒丢失。本申请中提出的工作利用了一种新技术来消耗 P颗粒称为生长素诱导降解（AID）系统。P颗粒有三个关键蛋白 PGL-1、PGL-2和PGL-3。为了降解这些靶标，CRISPR被用于设计降解决定子标签 在C.优美的然后，在生长素暴露后，这些蠕虫（参考文献 如PgAID）将降解PGL-1、PGL-2和PGL-3，从而消除P-颗粒的这些成核剂 P颗粒的组装和分散。 这些PgAID蠕虫将允许鉴定P- 在成体生殖系中的颗粒扩散。这将通过比较PgAID蠕虫和对照来完成，以识别 生殖细胞增殖，不育，育雏大小和形态由于P-颗粒分散的改变。mRNA 选择精子和神经元基因的报告基因表达也将被检查，并与先前的研究进行比较。 用P-颗粒RNAi方法产生的数据。PgAID蠕虫还将用于确定 P颗粒耗竭后mRNA表达的变化。这将通过对以下进行mRNA-seq来完成： 在P颗粒分散后立即解剖生殖系，并在体细胞分裂前和体细胞分裂开始时解剖生殖系。 记者误传。总的来说，这一建议将为阐明所使用的机制提供关键的下一步 保持细胞的多能性，这将有助于解锁成功使用这些细胞的可能性。 治疗益处的机制。