Mechanisms of mRNA localization and translational control in Drosophila development

果蝇发育中 mRNA 定位和翻译控制的机制

• 批准号: 10377348
• 负责人: ELIZABETH R GAVIS
• 金额: $ 62.04万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377348
• 关键词: Animals; Behavior; Biochemical; Cells; Code; Complex; Coupling; Cytoplasmic Granules; Dependence; Destinations; Development; Diffusion; Drosophila genus; Embryonic Development; Ensure; Event; Fertility; Genes; Genetic Transcription; Genome; Genomic approach; Germ; Germ Cells; Goals; Impairment; Infertility; Label; Location; Mediating; Messenger RNA; Methods; Microscopy; Modeling; Monitor; Movement; Neoplasm Metastasis; Neurodegenerative Disorders; Nurses; Oocytes; Ovarian; Pattern; Play; Process; Production; Property; Protein Biosynthesis; Proteins; RNA; Rana; Regulatory Element; Research; Ribonucleoproteins; Ribosomes; Role; Site; Specificity; Structure; Structure of primordial sex cell; Transcript; Translating; Translational Activation; Translations; cohort; egg; fly; high resolution imaging; in vivo; in vivo imaging; innovation; nano; programs; protein distribution; protein expression

PROJECT SUMMARY Our long-term research goal is to understand post-transcriptional mechanisms that control gene activity during early animal development. We focus on intracellular mRNA localization and translational control, which play crucial roles in regulating the production of proteins from maternally supplied transcripts. Because these transcripts, which control the initial developmental program of nearly all animals, are pre-loaded in the egg, the spatial and temporal expression of the proteins they encode must be exerted post-transcriptionally. In animals as diverse as flies and frogs, mRNA localization and local control of translation produce asymmetric protein distributions required for axis formation, patterning, and germline development. Often many different transcripts must be localized concurrently to various subcellular locations. Additionally, translational control must be superimposed to repress unlocalized transcripts and activate properly localized transcripts. How specificity is conferred on these processes, so that each transcript is targeted to its correct destination and translated appropriately, is poorly understood. Our research has capitalized on the Drosophila egg, which relies heavily on maternal transcripts, to investigate mechanisms of mRNA localization and its coupling to translational control. Our early studies focusing on nanos mRNA led to the discovery of a diffusion-and-entrapment mechanism used by numerous transcripts for localization to the specialized germ plasm at the posterior of the oocyte. Produced by the ovarian nurse cells and then transferred to the oocyte, these transcripts are co- packaged at the posterior end into ribonucleoprotein complexes (RNPs) called germ granules. Later during embryogenesis, germ granule mRNAs are segregated as a cohort to the primordial germ cells, where they are required for germline development. Despite their shared dependence on germ granule localization tor translational activation, different transcripts have distinct temporal demands. Our recent studies have led to a stepwise model for germ granule assembly that provides a framework for understanding the composition, structure, and translational properties of RNPs and their functions. Determining the specific roles of shared and RNA-specific proteins in controlling RNP assembly and translation will, in turn, be fundamental to a deeper understanding of mRNA localization as a mechanism for generating protein – and consequently cellular – asymmetries. To elucidate how localized assembly and function of complex RNA granules is controlled, we will take advantage of quantitative high resolution imaging, in vivo fluorescent RNA labeling, and new biochemical strategies to identify cis-acting regulatory elements and interacting proteins that mediate both individualistic and coordinate RNA behaviors. Ribosome footprinting, a genome-level approach for monitoring translation, will be employed to investigate mechanisms that impose translational arrest on unlocalized transcripts. Finally, we will use high resolution imaging of protein synthesis in vivo to decipher the relationship between germ granule association and translational activity.

项目概要 我们的长期研究目标是了解在转录过程中控制基因活性的转录后机制。 早期动物发育。我们专注于细胞内 mRNA 定位和翻译控制，这在 在调节母体提供的转录物产生蛋白质方面发挥着至关重要的作用。因为这些 控制几乎所有动物初始发育程序的转录本被预先加载到鸡蛋中， 它们编码的蛋白质的空间和时间表达必须在转录后发挥作用。在动物中 与苍蝇和青蛙一样多样化，mRNA 定位和翻译的局部控制产生不对称蛋白质 轴形成、图案化和种系发育所需的分布。通常有许多不同的转录本 必须同时定位到不同的亚细胞位置。此外，翻译控制必须是 叠加以抑制未定位的转录本并激活正确定位的转录本。特异性如何 赋予这些过程，以便每个转录本都被定位到正确的目的地并被翻译 适当地，理解甚少。我们的研究利用了果蝇卵，它在很大程度上依赖于 母体转录本，研究 mRNA 定位及其与翻译的耦合机制 控制。我们早期针对纳米 mRNA 的研究发现了扩散和截留 许多转录本使用的机制来定位到位于后部的专门种质 卵母细胞。这些转录本由卵巢护理细胞产生，然后转移到卵母细胞， 在后端包装成核糖核蛋白复合物（RNP），称为胚芽颗粒。后来期间 在胚胎发生过程中，生殖颗粒 mRNA 作为一个群体被分离到原始生殖细胞中，它们在那里 种系发育所需。尽管它们共同依赖于细菌颗粒定位 翻译激活时，不同的转录本具有不同的时间需求。我们最近的研究导致了 胚芽颗粒组装的逐步模型为理解其成分提供了框架， RNP 的结构、翻译特性及其功能。确定共享和共享的具体角色 反过来，控制 RNP 组装和翻译的 RNA 特异性蛋白将成为更深层次研究的基础。 将 mRNA 定位理解为生成蛋白质（进而生成细胞）的机制 不对称。为了阐明复杂 RNA 颗粒的局部组装和功能是如何控制的，我们将 利用定量高分辨率成像、体内荧光 RNA 标记和新的生化技术 识别顺式作用调节元件和介导个体主义的相互作用蛋白质的策略 并协调 RNA 行为。核糖体足迹是一种用于监测翻译的基因组水平方法，将 用于研究对非本地化转录本施加翻译抑制的机制。最后，我们 将利用体内蛋白质合成的高分辨率成像来破译胚芽颗粒之间的关系 关联和翻译活动。