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

项目总结： 了解如何实现和保持多能性是使研究人员能够 在需要的时候诱导多能性，或者在对癌症有害的时候阻止它。一条潜在的途径 研究在于对细菌颗粒的研究。细菌颗粒是异质核糖核蛋白(RNP)复合体。 发现于从蠕虫到人类的生殖细胞细胞质中。在线虫中，被称为P颗粒的细菌颗粒是 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颗粒会引发不孕症，并导致精子、肌肉和PAN的错误表达。 生殖细胞中的神经元标记。这可能是由于P颗粒拮抗了 错误表达的转录本或选择性地抑制其翻译。区分这些可能性的原因是 对阐明P颗粒在多能性中的作用至关重要。然而，到目前为止，研究一直受到阻碍。 受P-颗粒RNAi技术限制。P颗粒是母系遗传的，因此必须启动RNAi 在分析之前的那一代。因此，目前很难阐明短期和长期影响。 诱发P颗粒丢失。这项申请中提出的工作利用了一种新技术来耗尽 P颗粒被称为生长素诱导降解(AID)系统。磷颗粒有三种关键蛋白质 成分-PGL-1、PGL-2和PGL-3。为了使这些目标降级，CRISPR被用来设计降级标签 直到线虫中所有三个内源基因的末端。然后，在生长素暴露后，这些蠕虫(参考 AS PgAID)会降解PGL-1、PGL-2和PGL-3，从而消除这些P颗粒的核因子 磷颗粒的组装和分散。 这些PgAID蠕虫将允许识别P-AID的直接、中期和长期影响 颗粒在成虫生殖系中分散。这将通过比较PgAID蠕虫和对照来识别 生殖细胞的增殖、不育、孵化大小和形态因P颗粒的分散而改变。mrna 还将检测精选精子和神经元基因的报告表达，并与以前的 用P-颗粒RNAi方法产生的数据。PgAID蠕虫还将用于确定序列 P颗粒耗竭后mRNA表达的变化。这将通过执行mrna-seq on 在P颗粒扩散后立即解剖的种系，以及在体细胞开始之前和开始的时候 记者用错了词。总体而言，这项提案将为阐明所用机制提供关键的下一步 在生殖系中保持细胞的多能性，这将有助于解锁成功使用这些 治疗效益的机制。

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.