Dmoesin調控果蠅卵母細胞中奧斯卡核糖核酸複合體的定錨

Abstract

osk mRNA坐落在果蠅卵細胞後端決定胚胎未來的體軸，以及影響生殖細胞形成與腹部發育，然而目前對osk mRNA坐落機制仍不清楚。文獻指出在khc突變或osk-K10中，osk mRNA坐落於卵細胞周圍，這暗示在此存在負責定錨osk mRNA的物質。我們先前許多研究結果指出dDcp2與Dmoe皆為osk坐落所需，且兩者形成複合體。因此我們認為dDcp2-Dmoe複合體可能擔任此角色。 本論文第一部份透過免疫螢光染色證實p-Dmoe自stage7/8開始將dDcp2從細胞質帶至卵細胞周圍，此dDcp2-Dmoe複合體能做為osk mRNA定錨根基。先前的研究指出當dDcp2與p-Dmoe大量表現時，卵細胞後端Osk增加；當dDcp2與nonp-Dmoe大量表現時，卵細胞質中產生一個明顯的異位Osk訊號點，dDcp2、nonp-Dmoe、dDcp1、dGe-1及Kinesin皆存在於相同的異位點。我們根據以上結果提出假說：Dmoe磷酸化狀態改變時，dDcp2-Dmoe複合體會改變Osk的坐落位置。當Dmoe磷酸化時，osk mRNP會被定錨在卵細胞後端形成定錨複合體(anchoring complex)。成功坐落的osk mRNA會轉譯出Osk。當Dmoe去磷酸化時，Osk則被送往卵細胞內部形成運輸複合體(transporting complex)。 論文第二部分為檢驗假說。首先證實了大量表現osk mRNA亦產生運輸複合體。接著在此背景下調控dDcp2、p-Dmoe及nonp-Dmoe表現量，並檢驗Osk在運輸複合體及定錨複合體之間的平衡變動。結果發現單獨提升dDcp2或p-Dmoe表現量皆能促進定錨複合體形成，而提升nonp-Dmoe表現量則促進運輸複合體形成。此外，同時提升dDcp2及total Dmoe表現量能使所有卵細胞產生定錨複合體。這些結果證實了我們的假說。然而，同時提升dDcp2及p-Dmoe表現量時卻使運輸複合體再度形成，目前仍無法解釋此現象。 論文第三部分為透過共同免疫沉澱法證實Osk和dDcp2具有交互作用。 總結，本論文證實Dmoe磷酸化狀態調控osk mRNP的定錨，Osk透過與dDcp2交互作用維持在卵細胞後端的坐落。

The localization of osk mRNA at the posterior pole of Drosophila oocyte determines the posterior axis for embryo, and affects the germ cells formation and abdomen development; however, the mechanism remains unclear. Previous studies showed that in khc mutant or osk-K10 oocytes, osk mRNA is localized along oocyte cortex. It implies that there is a factor responsible for anchoring osk mRNP. Many of our previous studies showed that both dDcp2 and Dmoe are required for osk localization and they form a complex. Therefore, we assume that the dDcp2-Dmoe complex is a candidate of the cortical anchor for osk mRNP. In the first part of this thesis, immunofluorescent staining confirms that since stage7/8, p-Dmoe recruits dDcp2 from the ooplasm to the cortex. Thus, the cortical dDcp2-Dmoe complex stands by osk mRNP and acts an anchor. Our previous studies showed that the posterior Osk is elevated when both dDcp2 and p-Dmoe are upregulated. Overexpressing both dDcp2 and nonp-Dmoe generates a significantly ectopic Osk dot in the ooplasm. And nonp-Dmoe, dDcp2, dDcp1, dGe-1 and Kinesin are all localized in the dot. Based on the above, we propose a hypothesis that dDcp2-Dmoe complex regulates osk localization through the change of Dmoe phosphorylation status. When Dmoe is phosphorylated, osk mRNP is anchored at the posterior pole, resulting in the translation of Osk and forming the anchoring complex. When Dmoe is dephosphorylated, Osk is moved inwardly and forms the transporting complex. The second part of this thesis is to confirm our hypothesis. Firstly, we prove that overexpressing osk mRNA generates the transporting complex in the oocyte. Next, under this condition, we regulate the expression of dDcp2, p-Dmoe or nonp-Dmoe to examine the change of Osk in the balance between the transporting complex and the anchoring complex. As a result, increasing the expression of dDcp2 or p-Dmoe alone promotes the formation of the anchoring complex while upregulating of nonp-Dmoe enhances the formation of the transporting complex. Moreover, overexpressing dDcp2 and total Dmoe simultaneously leads all oocytes to generate the anchoring complex. These results verify our hypothesis. However, overexpressing both dDcp2 and p-Dmoe generates the transporting complex again. We cannot interpret the phenomena. In the third part of this thesis, the co-immunoprecipitation assay indicates that Osk interacts with dDcp2 in the ovary. In conclusion, this thesis reveals that the phosphorylation status of Dmoe regulates osk mRNP anchoring and Osk maintains its posterior localization through the interaction with dDcp2.