Mechanisms of mRNA localization to Drosophila germ granules

项目摘要/摘要 任何物种的繁殖成功都关键依赖于生殖细胞的发育。生殖细胞是全能细胞 将他们的基因组从一代转移到下一代。一旦受精，它们就会分化为 体细胞谱系，同时留出一部分细胞，以产生未来的生殖细胞。他们意识到了这一壮举 通过转录抑制和染色质建立生殖系特有的基因组图谱 重塑伴随着涉及mRNA定位的扩张性转录后调控。生殖细胞 形成生殖细胞颗粒，所有的生物都依赖这些颗粒来实现生殖细胞的功能。MRNAs在 它们的翻译和降解受到调控的颗粒。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，如信使核糖核酸的定位。这一过程是实现 果蝇生殖细胞分化过程中mRNA翻译的时空调控和稳定性 而且在哺乳动物精子发生过程中也是如此。因此，要了解生殖细胞的发育和生殖 任何物种的成功，最重要的是要确定控制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单个RNA，特别是在 经过发育的完整生物体中的生殖系已经深刻地阻碍了我们充分理解 基因定位在生殖细胞发育中的作用机制及影响。使用一种基因上可服从的 像果蝇这样的模型生物，再加上生物化学和单个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 单链mRNA荧光原位杂交(SmFISH)与结构照明结合的方法 显微镜(SIM)和显示多个mRNA形成不对称的同型簇- 呈颗粒状分布的。相反，蛋白质在颗粒中分布均匀。我的假设是 信使核糖核酸本身在信使核糖核酸定位和胚芽颗粒组装中起着指导作用。在这里，我向大家介绍一位 有重点的策略来解决我的假设，并描述同型集群和胚胎的形成 体外和体内颗粒剂。与SIM相比，分辨率大大提高，我将实现随机光学 重建显微镜(STORM)超分辨率和全内反射荧光(TIRF) 显微镜下解剖在胚胎和重建的胚胎颗粒中如何形成信使核糖核酸簇 果蝇S2细胞，确定驱动聚集的序列，并确定聚集的生物学相关性。我 然后将识别促进高阶RNA聚集的蛋白质。最后，我将研究mRNAs和 颗粒蛋白质促进颗粒组装。在这项研究中获得的知识将有助于理解 基因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是 在物种之间保存，识别的任何基因、网络和机制以及开发的技术 可用于研究生殖细胞在其他生物体中的发育和繁殖。

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.