Analyses of nuclear-RNP complex essential for RNA localization and translation of Drosophila maternal RNA

mRNA在细胞内的定位和与mRNA定位相关的翻译调控是细胞极性形成的必要条件。果蝇卵母细胞是研究mRNA定位机制和翻译调控的理想模型。近年来的研究表明，细胞核内RNA的剪接对胞质内mRNA的定位起着重要的作用。我们已经确定了一种果蝇核细胞质穿梭蛋白Hrp48，它对于oskar mRNA定位到卵母细胞前部以及oskar RNA的翻译抑制至关重要。Hrp48是哺乳动物a /B型hnRNP的同源物，并且被证明是抑制p元素RNA剪接所必需的。本研究旨在通过分析含Hrp48的蛋白复合物，阐明细胞核剪接如何影响胞质mRNA的定位和翻译调控。我们分析如下：人体内Hrp48结构域分析2。Hrp483的核定位信号。鉴定在细胞核中与Hrp48形成复合物的因子通过分析1，我们发现Hrp48的glycin富结构域（没有RNA结合结构域）对于复合物的形成是重要的，这对于oskar mrna的翻译抑制至关重要。此外，我们认为该结构域与对细胞前背部分gurken mRNA的细胞质定位至关重要的因子相互作用。通过分析2，我们发现Hrp48中的核定位信号部分同源于哺乳动物hnRNP Al中的核定位信号M9序列。通过分析，我们确定了在细胞核中与Hrp48形成复合物的因子。这些因子不能与突变的Hrp48形成复合物，因此不可能进入细胞核。

mRNA localization within cells and translational regulation that couples to the mRNA localization is an essential for the establishing of cell polarity. Drosophila oocyte is the ideal model for studying the mechanism of the mRNA localization and the translational control. Recent studies have revealed that splicing of RNA in the nucleus is important for the cytosolic mRNA localization. We have identified a Drosophila nuclear-cytoplasm shuttling protein, Hrp48, that is essential for the localization of oskar mRNA to the poaterior of the oocyte, and for the translational repression of oskar RNA. Hrp48 is a homologue of mammalian A/B type hnRNP, and is shown to'be essential for the inhibition of the splicing of p-element RNA. The purpose of this research is to clarify how splicing in nucleus affects the cytoplasmic mRNA localization and translational regulation by analyzing Hrp48 containing protein complex.We have analyzed these below :1. Domain analysis of Hrp48 in vivo2. Nuculear localization signal in Hrp483. Identification of factor(s) that makes a complex with Hrp48 in the nucleusBy the analysis 1, we showed that glycin rich domainin of Hrp48 (which has no RNA binding domain) is important for the complex formation that is essential for the translational repression of oskar mRNNA. In addition, we suggested that this domain interacts with factor(s) that is critical for the cytoplasmic localization of gurken mRNA in anterior-dorsal part of the ocyte.By the analysis 2, we identified the nuclear localization signal in Hrp48, which is partly homologous to M9 sequence that act as a nuclear localization signal in hnRNP Al in mammals.By the analysis 3, we identified factors that make a complex with Hrp48 in the nucleus. These factors cannot make a complex with mutated Hrp48 that is impossible to go into the nucleus.