Mechanisms of mRNA localization to Drosophila germ granules

果蝇胚芽颗粒 mRNA 定位机制

• 批准号: 10011571
• 负责人: Tatjana Trcek
• 金额: $ 24.59万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011571
• 关键词: Address; Animal Model; Biochemistry; Biological; Biological Assay; Biophysics; Cell Fraction; Cell physiology; Cells; Code; Complement; Coupled; Cytoplasmic Granules; Data; Development; Drosophila genus; Embryo; Ensure; Event; Excision; Fertilization; Fluorescence Microscopy; Fluorescent in Situ Hybridization; Future; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Genetic Translation; Genome; Genomics; Germ; Germ Cells; Hydrogels; Image; In Situ; In Situ Hybridization; In Vitro; Instruction; Knowledge; Learning; Lighting; Liquid substance; Messenger RNA; Microscopy; Molecular; Mutagenesis; Oocytes; Optics; Organism; Play; Post-Transcriptional Regulation; Process; Property; Proteins; Publishing; RNA; RNA Sequences; RNA interference screen; Reproduction; Research; Resolution; Ribonucleoproteins; Role; Seeds; Shapes; Spermatogenesis; Structure; Technology; Testing; Totipotent cell; Transcript; Translations; Visualization; Work; biophysical properties; chromatin remodeling; fly; gene repression; genetic approach; improved; in vitro Assay; in vivo; insight; mRNA Decay; mRNA Stability; microscopic imaging; mutant; nano; nanocluster; particle; reconstitution; reconstruction; reproductive success; single molecule; tool

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.

项目摘要/摘要 任何物种的生殖成功都依赖生殖细胞的发育。生殖细胞是全能细胞 那将他们的基因组形成一代。受精后，它们分为所有 体细胞谱系搁置一小部分会导致未来生殖细胞的细胞。他们意识到这一壮举 通过转录抑制和染色质建立种系特定的基因组景观 重塑以及涉及mRNA定位的广泛的转录后调节。生殖细胞 形成胚芽颗粒，所有生物都取决于这些颗粒来获得生殖细胞功能。 mRNA富含在内 调节其翻译和降解的颗粒。首先，生殖细胞发育的关键步骤依赖 完全涉及转录后事件，例如mRNA定位。这个过程对于实现 mRNA翻译和稳定性的空间和时间控制不仅在果蝇生殖细胞规范期间 而且在哺乳动物的精子发生过程中。因此，了解生殖细胞发育和生殖 任何物种的成功，最重要的是表征控制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解决单一mRNA。 通过发育完整生物的种系极大地阻碍了我们完全理解的能力 机制和mRNA定位对生殖细胞发育的影响。使用遗传上的 诸如果蝇之类的模型有机体，再加上生物化学和单个MRNA成像，提供了理想 补充哺乳动物研究的策略。在博士后研究中，我开发了一个超分辨率 我将单个mRNA荧光原位杂交（Smfish）与结构照明耦合 显微镜（SIM），表明多个mRNA形成同型簇，不对称 分布在颗粒中。相反，蛋白质均匀分布在颗粒中。我的假设是 mRNA本身在mRNA定位和生殖颗粒组装中起启发性作用。在这里，我提出了 重点策略来解决我的假设并表征同型簇和细菌的形成 体外和体内颗粒。有了大大改进的分辨率，我将实施随机光学 重建显微镜（Storm）超分辨率和总内反射荧光（TIRF） 显微镜剖析mRNA簇如何在胚胎中形成和重建的细菌颗粒中的形成方式 果蝇S2细胞，识别驱动聚类的序列并确定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件。这项研究中获得的知识将提供对 mRNA定位如何塑造生殖细胞的发育。鉴于生殖细胞发育的许多特征是 在物种中保守，确定的任何基因，网络和机制以及开发的技术 可以实施以研究其他生物的生殖细胞发育和繁殖。