Mechanisms of mRNA localization and translational control in Drosophila development

项目摘要 我们的长期研究目标是了解转录后机制，控制基因活动， 动物早期发育我们专注于细胞内mRNA的定位和翻译控制， 在调节母体提供的转录本产生蛋白质方面起着关键作用。因为这些 控制几乎所有动物的初始发育程序的转录本预先装载在卵子中， 它们编码的蛋白质的空间和时间表达必须在转录后进行。动物中 像苍蝇和青蛙一样，mRNA的定位和翻译的局部控制产生了不对称蛋白质， 轴的形成，图案化和种系发育所需的分布。通常有许多不同的抄本 必须同时定位于不同的亚细胞位置。此外，翻译控制必须 叠加以抑制未定位的转录物并激活适当定位的转录物。如何具体化 赋予这些过程，使每个成绩单的目标是正确的目的地和翻译 正确地说，是很难理解的。我们的研究利用了果蝇卵， 在母体转录本上，研究mRNA定位及其与翻译的偶联机制， 控制我们早期的研究集中在纳米mRNA上，发现了一种扩散和捕获的机制， 许多转录本用于定位于细胞后部的特化种质的机制。 卵母细胞这些转录物由卵巢滋养细胞产生，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。晚些时候 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，在那里它们被 是生殖细胞发育所必需的。尽管它们都依赖于胚粒定位， 翻译激活，不同的转录物具有不同的时间需求。我们最近的研究导致了 逐步模型的胚芽颗粒组装，提供了一个框架，了解组成， RNP的结构和翻译性质及其功能。确定共享和 控制RNP组装和翻译的RNA特异性蛋白质反过来将是更深入研究RNP的基础。 理解mRNA定位作为产生蛋白质的机制-从而产生细胞- 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标记和新的生物化学技术， 策略，以确定顺式作用的调控元件和相互作用的蛋白质，介导的个人主义 并协调RNA的行为。核糖体足迹法是一种基因组水平的翻译监测方法， 可以用来调查机制，施加翻译逮捕非本地化的成绩单。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 结合和翻译活性。

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.