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

mRNA在细胞内的定位以及与mRNA定位相关联的翻译调节对于细胞极性的建立是必不可少的。果蝇卵母细胞是研究mRNA定位和翻译调控机制的理想模型。最近的研究表明，RNA在细胞核中的剪接对于胞浆mRNA的定位是重要的。我们已经确定了一个果蝇核质穿梭蛋白，Hrp 48，这是必不可少的本地化oskar mRNA的卵母细胞的后部，并为oskar RNA的翻译抑制。hrp 48是哺乳动物A/B型hnRNP的同源物，并且被证明对于抑制p元件RNA的剪接是必需的。本研究的目的是通过对Hrp 48蛋白复合物的分析，阐明细胞核剪接对细胞质mRNA定位和翻译调控的影响。体内Hrp 48的结构域分析2. Hrp 483中的核定位信号。通过分析1，我们表明Hrp 48的富含甘氨酸的结构域（其没有RNA结合结构域）对于复合物的形成是重要的，而复合物的形成对于oskar mRNNA的翻译抑制是必需的。此外，我们提出该结构域与对gurken mRNA在细胞的前背部分的细胞质定位至关重要的因子相互作用。通过分析2，我们鉴定了Hrp 48中的核定位信号，其与哺乳动物中作为hnRNPAl中的核定位信号的M9序列部分同源。我们确定了在细胞核中与Hrp 48形成复合物的因子。这些因子不能与突变的Hrp 48形成不可能进入细胞核的复合物。

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.