Mechanisms of mRNA localization to Drosophila germ granules

项目总结/摘要 任何物种的繁殖成功都依赖于生殖细胞的发育。生殖细胞是全能细胞 将它们的基因组从一代转移到下一代。受精后，它们分化成 体细胞谱系，同时留出一部分细胞，产生未来的生殖细胞。他们意识到这一壮举 通过转录抑制和染色质表达来建立种系特异性基因组景观， 重塑加上广泛的转录后调控涉及mRNA定位。生殖细胞 形成生殖颗粒，所有生物体都依赖这些颗粒来实现生殖细胞功能。mRNA富集在 颗粒，其中它们的翻译和降解受到调节。首先，生殖细胞发育的关键步骤依赖于 完全依赖于转录后事件，如mRNA定位。这一进程是实现 不仅在果蝇生殖细胞特化过程中， 而且在哺乳动物精子发生期间也是如此。因此，要了解生殖细胞的发育和生殖 任何物种的成功，它是至关重要的特点，管理mRNA定位到胚芽的机制 颗粒。缺乏足够的空间和时间敏感性来分辨特异性存在于细胞中的单个mRNA。 生殖细胞在完整生物体中的发育，深刻地阻碍了我们充分理解 mRNA定位对生殖细胞发育的影响。使用一种基因上可接受的 模式生物，如果蝇，加上生物化学和单mRNA成像，提供了一个理想的 补充哺乳动物研究的战略。在我的博士后研究期间，我开发了一种超分辨率 方法，其中我耦合单mRNA荧光原位杂交（smFISH）与结构照明 在SIM显微镜下，发现多个mRNA形成不对称的同型簇。 呈颗粒状分布。相反，蛋白质均匀分布在颗粒内。我的假设是 mRNA本身在mRNA定位和胚粒组装中起指导作用。在这里，我提出一个 一个集中的战略，以解决我的假设和特点的形成同型集群和细菌 颗粒在体外和体内。随着SIM的分辨率大大提高，我将实现随机光学 重建显微镜（STORM）超分辨和全内反射荧光（TIRF） 显微镜下解剖mRNA簇如何在胚胎中形成，并在重建的生殖颗粒中形成。 果蝇S2细胞，识别驱动聚类的序列并识别聚类的生物学相关性。我 然后将识别促进高阶RNA聚类的蛋白质。最后，我将研究mRNA和 颗粒蛋白促进颗粒组装。本研究中获得的知识将有助于理解 mRNA定位如何影响生殖细胞发育。鉴于生殖细胞发育的许多特征 物种之间的保守性，任何基因，网络和机制以及开发的技术 可以用来研究其他生物体的生殖细胞发育和繁殖。

PROJECT SUMMARY/ABSTRACT Reproductive success of any species relies critically on germ cell development. Germ cells are totipotent cells that transfer their genome form one generation into the next. Upon fertilization, they differentiate into all somatic lineages while setting aside a fraction of cells that give rise to future germ cells. They realize this feat by establishing a germline specific genomic landscape through transcriptional repression and chromatin remodeling coupled with an expansive post-transcriptional regulation involving mRNA localization. Germ cells form germ granules and all organisms depend on these granules for germ cell function. mRNAs enrich within granules where their translation and degradation is regulated. First, critical steps of germ cell development rely entirely on post-transcriptional events such as mRNA localization. This process is fundamental in achieving spatial and temporal control of mRNA translation and stability not only during Drosophila germ cell specification but also during mammalian spermatogenesis. Thus, to understand germ cell development and the reproductive success of any species it is paramount to characterize the mechanisms that govern mRNA localization to germ granules. Lack of adequate spatial and temporal sensitivity to resolve single mRNAs specifically in the germline in intact organism through development has profoundly hindered our ability to fully understand the mechanisms and the impact of mRNA localization on germ cell development. Using a genetically amenable model organism such as the fruit fly, coupled with biochemistry and single-mRNA imaging, provides an ideal strategy to complement mammalian research. During my postdoctoral studies, I developed a super-resolution approach where I coupled single-mRNA fluorescent in situ hybridization (smFISH) with structural illumination microscopy (SIM) and showed that multiple mRNAs form homotypic clusters that are asymmetrically- distributed in granules. In contrast, proteins were evenly distributed within granules. My hypothesis is that the mRNA itself plays an instructive role in mRNA localization and germ granule assembly. Here, I present a focused strategy to address my hypothesis and characterize the formation of homotypic clusters and germ granules in vitro and in vivo. With much improved resolution over SIM, I will implement stochastic optical reconstruction microscopy (STORM) super-resolution and total internal reflection fluorescence (TIRF) microscopy to dissect how mRNA clusters form in the embryo and in reconstructed germ granules in Drosophila S2 cells, identify sequences that drive clustering and identify the biological relevance of clustering. I will then identify proteins that promote higher-order RNA clustering. Finally I will examine how mRNAs and granule proteins promote granule assembly. The knowledge gained in this study will provide understanding of how mRNA localization shapes germ cell development. Given that many features of germ cell development are conserved among species, any genes, networks and mechanisms identified as well as technologies developed could be implemented to study germ cell development and reproduction in other organisms.