ORB GENE REGULATION OF TRANSLATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在果蝇卵和胚胎中，mRNA的定位在极性的建立中起着关键作用。它通常始于在哺育细胞核的RNP复合体中转录和包装定位的mRNAs。这些复合体“屏蔽”mRNAs，防止它们在运输过程中被翻译，并“标记”它们进行本地化。被遮盖的mRNAs通过哺育细胞运输，并沉积在卵母细胞中。根据其顺式作用的定位元件，mRNAs随后被定位于卵母细胞中的特定位置，在那里它们被锚定到细胞酮网络。局部化消息的翻译激活提供了蛋白质产品的空间受限来源。或者，可以将mRNAs以屏蔽的形式存储，直到适当的信号，例如卵子沉积，激活翻译。这为协调蛋白质的局部合成和其他发育事件提供了一种机制。 虽然首先在果蝇卵巢和早期胚胎中发现了mRNA定位的重要性(加上“就地”翻译调控)，但现在清楚的是，这种调控机制在许多不同的环境中都有应用，几乎在每一个真核生物中都存在。例如，在酵母中，ASH-1mRNA对子细胞的不对称分离提供了一种机制，确保该细胞不会发生交配类型转换。体细胞中的mRNA定位也发生在运动、分化或生长过程中。在神经细胞中，编码MAP2和aCaMKII的mRNAs聚集在树突中，而不是轴突中。局部的信使核糖核酸翻译也与突触的可塑性和学习记忆有关。在所有这些情况下，涉及mrna定位和翻译调控的步骤很可能与上述在果蝇卵巢中描述的步骤大致相似。 建议的研究集中在果蝇orb基因，它是苍蝇卵巢mRNA定位机制的关键组成部分之一。ORB是高度保守的RRM RNA结合蛋白CPEB家族的创始成员。在以前的研究中，我们已经证明了orb在mRNA本地化过程的最后一步发挥作用，一旦它在现场就调节了mRNA的翻译。球体绑定到3？Oskar(OSK)、Bicudal-D(Bic-D)和K(10)体内许多定位的mRNAs的UTR，并需要促进这些mRNAs的翻译。此外，ORB蛋白通过与3？定位的orb基因的非翻译区和激活翻译。这个正反馈环对于ORB在发育中的卵母细胞中的正确表达至关重要。 我们研究的目的是更好地了解ORB如何自动调节自己的表达，以及它如何控制mRNAs的现场翻译，编码因子是建立卵母细胞/哺育细胞身份和确定卵室和胚胎的极性的关键。这些问题的部分答案将来自于识别和表征其他对ORB自动调节和/或功能重要的基因。为此，我们建议在体内分离与ORB相关的蛋白质，并通过质谱分析鉴定它们。为了验证这些生化研究，我们设计了一个敏化的球体突变背景。敏化的背景将使我们能够测试候选基因中的突变，这些候选基因编码与体内ORB相关的蛋白质，用于与ORB的遗传相互作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. mRNA localization plays a pivotal role in the establishment of polarity in the Drosophila egg and embryo. It typically begins with the transcription and packaging of the localized mRNAs in RNP complexes in nurse cell nuclei. These complexes 'mask' the mRNAs, preventing them from being translated while in transit, and 'mark' them for localization. The masked mRNAs are transported through the nurse cells and deposited in the oocyte. Depending upon their cis-acting localization elements, the mRNAs are then targeted to specific locations in the oocyte where they are anchored to the cytosketal network. Translational activation of the localized message provides a spatially restricted source of the protein product. Alternatively the mRNAs can be stored in a masked form until an appropriate signal, such as egg deposition, activates translation. This provides a mechanism for coordinating the localized synthesis of the protein with other developmental events. While the importance of mRNA localization (coupled with 'on site' translational regulation) was first documented in fly ovaries and early embryos, it is now clear that this regulatory mechanism is employed in many different contexts and occurs in virtually every eukaryote. For example, in yeast, the asymmetric segregation of ash-1 mRNA to the daughter cell provides a mechanism for ensuring that mating type switching does not occur in that cell. mRNA localization in somatic cells also occurs during the processes of movement, differentiation or growth. In neuronal cells, mRNAs encoding MAP2, a dendrite-specific microtubule-associated protein, and aCaMKII accumulate in the dendrites, but not in the axons. Localized mRNA translation has also been implicated in synaptic plasticity and learning and memory. In all of these cases, it is likely that the steps involved in mRNA localization and translational regulation are similar in broad outline to those described above in fly ovaries The proposed studies focus on the Drosophila orb gene which is one of the key components of the fly ovarian mRNA localization machinery. Orb is a founding member of the highly conserved CPEB family of RRM RNA binding proteins. In previous studies, we have shown that orb functions at the last step in the mRNA localization process, regulating the translation of the mRNA once it is on site. Orb binds to the 3? UTR of many localized mRNAs in vivo oskar (osk) Bicudal-D (Bic-D), and K(10) and is required to promote the translation of these mRNAs. In addition, Orb protein autoregulates its own expression by binding to the 3? UTR of localized orb mRNA and activating translation. This positive feedback loop is critical for the proper expression of Orb in the developing oocyte. The goal of our studies is to better understand how Orb autoregulates its own expression and how it controls the on site translation of mRNAs encoding factors critical for establishing oocyte/nurse cell identity and defining the polarity of the egg chamber and embryo. Part of the answer to these questions will come from identifying and characterizing other genes that are important for Orb autoregulation and/or function. For this purpose we propose to isolate proteins that are associated with Orb in vivo and identify them by Mass Spec analysis. To validate these biochemical studies we have devised a sensitized orb mutant background. The sensitized background will permit us to test mutations in candidate genes which encode proteins that are associated Orb in vivo for genetic interactions with Orb.