Mechanisms of mRNA localization and translational control in Drosophila development

项目总结 我们的长期研究目标是了解控制基因活性的转录后机制 早期的动物发育。我们关注的是细胞内信使核糖核酸的定位和翻译控制。 在调节母体提供的转录本中产生蛋白质方面起着至关重要的作用。因为这些 转录本控制着几乎所有动物的初始发育程序，它预先加载在卵子中，即 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。在动物身上 就像苍蝇和青蛙一样，mrna的定位和翻译的局部控制会产生不对称蛋白。 轴形成、图案化和生殖系发育所需的分布。通常有许多不同的成绩单 必须同时定位到不同的亚蜂窝位置。此外，平移控制必须是 叠加以抑制非本地化转录并激活适当本地化的转录。有多具体？ 对这些过程进行协商，以便将每一份文字记录都指向其正确的目的地并进行翻译 恰如其分地，人们对此知之甚少。我们的研究利用了果蝇卵，它严重依赖于 在母体转录本上，研究mRNA定位的机制及其与翻译的偶联 控制力。我们早期对Nanos信使核糖核酸的研究导致发现了一种扩散和捕获 被许多转录本用来定位到特定种质的机制 卵母细胞。由卵巢哺育细胞产生，然后转移到卵母细胞，这些转录本是共同的 在后端包装成核糖核蛋白复合体(RNP)，称为胚芽颗粒。晚些时候 胚胎发生，生殖粒mRNAs作为一个队列被分离到原始生殖细胞，它们在那里 是生殖系发育所必需的。尽管他们都依赖于细菌颗粒定位因子 翻译激活，不同的转录本有不同的时间需求。我们最近的研究导致了一个 细菌颗粒组装的逐步模型，该模型提供了理解组成的框架， RNPs的结构、翻译特性及其功能。确定共享和共享的特定角色 控制RNP组装和翻译的RNA特异性蛋白质反过来将是更深层次的 理解信使核糖核酸的定位是一种产生蛋白质的机制--进而是细胞-- 不对称性。为了阐明复杂RNA颗粒的局部组装和功能是如何受到控制的，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 识别顺式作用调控元件和相互作用蛋白的策略 并协调RNA的行为。核糖体足迹，一种基因组水平的翻译监测方法，将 被用来调查对非本地化转录实施翻译阻止的机制。最后，我们 将利用体内蛋白质合成的高分辨率成像来破译细菌颗粒之间的关系 联想和翻译活动。

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.