A molecular examination of mRNA localization and cell polarization

描述(由申请人提供)：导致人类多种疾病的一个因素是细胞极性缺陷。许多细胞类型使用信使核糖核酸定位作为建立极性的一种手段。目前，我们对mRNAs在高等真核生物中定位的理解存在着根本的差距。这项提议的目标是通过确定Oskar和FMR1 mRNAs的本地化机制来解决这一知识差距。Oskar基因定位于果蝇卵母细胞，FMR1基因定位于神经元。这些转录本是理想的研究对象，因为尽管它们定位在不同的组织中，但它们的定位利用了许多相同的因素。因此，一种共同的机制可能会对不同的mRNAs进行定位。因此，从这些研究中获得的见解有望普遍适用于我们对信使核糖核酸定位的理解。我们建议使用黑腹果蝇模型系统进行我们的研究。这个模型系统将使我们能够在整个生物体的背景下研究信使核糖核酸的定位过程。这项研究有三个主要目标。目标1的推动力是我们最近发现，剪接体的核心蛋白，即Sm蛋白，除了剪接外，还在mRNA定位中发挥作用。在本研究中，我们建议确定Sm蛋白在定位Oskar mRNA方面的作用机制。在目标2中，我们建议确定Oskar和FMR1 mRNA与马达蛋白偶联进行运输的机制。运动蛋白重链(KHC)是参与这两种mRNAs运输的主要马达。然而，KHC不使用其规范的接头与Oskar和FMR1 mRNA结合。因此，目前尚不清楚KHC如何与这些转录本结合。我们假设KHC与Oskar和FMR1mRNA之间存在一个未知的接头。目标2的目标是识别这个未知的适配器。最后，目标3集中在我们意想不到的发现上，一个独立的运动，细胞质动力蛋白，也在Oskar mRNA定位中发挥作用。已经证实了Dynein在FMR1mRNA定位中的作用。在这一目标中，我们将准确地确定Dynein在Oskar mRNA运输中的作用。此外，我们将检验这一假设，即一旦Dynein将Oskar mRNA传递到后极，它在维持卵母细胞内的不对称内吞活动方面发挥了额外的功能。这些目标的完成将极大地促进我们对细胞如何构建具有定位能力的mRNP，以及该mRNP与运动蛋白连接以进行运输的机制的理解。最终，从这些研究中获得的知识将使我们能够更有效地治疗由mRNA定位和细胞极性缺陷引起的疾病。)

DESCRIPTION (provided by applicant): A contributing factor to a wide spectrum of human diseases is defective cell polarity. Many cell types use mRNA localization as a means to establish polarity. At present, there is a fundamental gap in our understanding of how mRNAs are localized in higher eukaryotes. The goal of this proposal is to address this gap in knowledge by determining the mechanism by which oskar and FMR1 mRNAs are localized. oskar mRNA is localized in Drosophila oocytes and FMR1 mRNA is localized in neurons. These transcripts are ideal candidates for study because although they are localized in different tissues, their localization utilizes many of the same factors. Thus, a common mechanism might operate to localize diverse mRNAs. Insights gained from these studies are therefore expected to be generally applicable to our understanding of mRNA localization. We propose to perform our studies using the Drosophila melanogaster model system. This model system will enable us to study the process of mRNA localization in the context of the whole organism. This study has three main objectives. The impetus for Aim 1 was our recent discovery that core proteins of the spliceosome known as Sm proteins have a role, outside of splicing, in mRNA localization. In the present study, we propose to determine the mechanism by which Sm proteins function in localizing oskar mRNA. In Aim 2, we propose to determine the mechanism by which oskar and FMR1 mRNA are coupled to motor proteins for transport. The primary motor implicated in transporting both mRNAs is Kinesin heavy chain (Khc). However, Khc does not use its canonical adaptor to bind to oskar and FMR1 mRNA. Thus, it is currently unknown how Khc binds to these transcripts. We hypothesize that an unknown adaptor links Khc to oskar and FMR1 mRNA. The goal of Aim 2 is to identify this unknown adaptor. Finally, Aim 3 centers on our unexpected finding that a separate motor, cytoplasmic Dynein, also functions in oskar mRNA localization. A function for Dynein in the localization of FMR1 mRNA has already been demonstrated. In this aim, we will precisely define the role of Dynein in transporting oskar mRNA. Furthermore, we will test the hypothesis that once Dynein delivers oskar mRNA to the posterior pole, it performs an additional function in maintaining asymmetric endocytic activity within the oocyte. Completion of these aims will significantly advance our understanding of how the cell builds a localization competent mRNP, and the mechanism by which that mRNP is coupled to motor proteins for transport. Ultimately, the knowledge gained from these studies will enable us to more effectively treat diseases that result from defective mRNA localization and cell polarity. )