周子賓臺灣大學：分子與細胞生物學研究所李沛珍Lee, Pei-ChenPei-ChenLee2007-11-252018-07-062007-11-252018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/49947根據蛋白質序列的比對分析，果蠅CG11183 基因的人類同源物SMIF是一個轉錄共同調控蛋白，透過N端的EVH1/WH1 domain與Smad4作用，參與變形生長因子貝它的信息傳遞路徑。CG11183因此最先被命為dSMIF，但是CG11183參與果蠅變形生長因子貝它信息傳遞路徑的證據並不確實。人類SMIF之後亦被證實是一個去頭蓋酵素而被命名為hDcp1a. CG11183突變所產生的胚胎腹節缺失的後端突變性狀，並經證實其具有去頭蓋酵素活性並參與osk mRNA的定位及裂解機制的調控。我們因此確定CG11183為果蠅的去頭蓋酵，dDcp1。 dDcp1 具有兩種不同的分子特性，dDcp1的N端具有一個EVH1/WH1 區塊可以進行蛋白質間的相互作用，而dDcp1的去頭蓋酵素活性亦經試管實驗的證實。在本論文中,為了要進一步了解去頭蓋酵素的作用以及EVH1/WH1 domain 所負責的功能,我們針對dDcp1蛋白質中負責去頭蓋作用以及EVH1/WH1 domain中負責作用的胺基酸進行點突變,並利用轉殖基因表現於dDcp1完全喪失的突變果蠅中，以分析這些點突變所造成的性狀以及其本身的後端定位的機制。為了更詳細了解dDcp1蛋白在卵母細胞後端的定位，一系列不同長短蛋白質片段亦用於解析其所參與的機制。 負責dDcp1的去頭蓋作用或是EVH1/WH1 domain作用的胺基酸都是果蠅生存所需要的。負責去頭蓋作用的點突變對卵巢發育並沒有顯著的影響,但會造成早期胚胎的死亡；但是dDcp1蛋白質在卵母細胞後端的分部型態並未受到影響。 dDcp1的EVH1/WH1domain 中負責與其他蛋白作用的三個氨基酸發生點突變時,卵母細胞的決定與極性都受到影響。但這些突變並不改變dDcp1在卵母細胞的分布位置。由不同蛋白片段的缺失突變分析顯示，dDcp1N-端300個胺基酸在卵巢中的行為與正常dDcp1蛋白相似,也未明顯影響卵巢的發育。而dDcp1N-端183個胺基酸本身分布雖然不正常,無法集中於卵母細胞的後端，但不影響Osk 蛋白在卵母細胞的後端的表現。 綜合以上結果，我們推測在dDcp1中，EVH1/WH1 domain與去頭蓋作用分別具有不同生物功能但並不負責其蛋白的定位。Based on the analysis of protein sequence, the human homologue of Drosophila CG11183, SMIF, a transcriptional co-activator, interacts with Smad4 by the EVH1/WH1 domain in N-terminal region of dDcp1 involving in transforming growth factorβ(TGFβ) signaling pathway. Thus, CG11183 was named dSMIF(Drosophila homologue of SMad Interacting Factor)originally. However, up to now, the evidences that CG11183 participate in TGFβ signaling pathway in vivo are not manifested certainly. Human SMIF was also identified as a decapping enzyme subsequently, named hDcp1a. CG11183 mutant results in the embryonic abdomen deletion phenotypes. It was proven that CG11183 possesses decapping activity involved in posterior localization and degradation regulation of osk mRNA. Therefore, we confirmed that CG11183 is a Drosophila decapping enzyme, dDcp1. dDcp1 possesses two different molecular properties. The N-terminus of dDcp1 belongs to an EVH1/WH1 domain which is responsible for protein-protein interaction and the decapping activity of dDcp1 has been corroborated in vitro. In this thesis, to better understand the function of dDcp1 and the capability of EVH1/WH1 domain, we mutated the residues which are critical for decapping activity and ligand binding of EVH1/WH1 domain and expressed the mutant proteins in dDcp1 null background by transgenic flies to analyze the phenotype caused by these mutant proteins and the mechanism of dDcp1 posterior localization. To gain insight the mechanism of Dcp1 posterior localization, a series of dDcp1 deleted fragments are also used to analyze in the same experiment. The residues responsible for decapping activity and protein-protein interaction in EVH1/WH1 domain are both required for Drosophila survival. The development of ovary is not affected substantially when the residues of dDcp1 which critical for decapping activity are mutated, but the embryos die in early embryogenesis. However, the posterior localization of dDcp1 is not affected. On the other hand, oocyte determination and polarity of oocyte are disrupted as the residues important to ligand binding are mutated but the posterior localization of dDcp1 is not affected. The results of deletion analysis display that dDcp1N300owns most of the biological activity sufficienlyt for oogenesis. And although the frangment, dDcp1N183, do not affect the expression of Osk , dDcp1N183 can not concentrate at the posterior pole of oocyte.Table of Content 中文摘要………………………………………………………………………………1 Abstract………………………………………………………………………………..3 Table of content ……………………………………………………………………….5 List of tables...…………………………………………………………………………8 List of Figures ………………………………………………………………………...9 Introduction…………………………………………………..11 Overview of Drosophila oogenesis…………………………………………………..11 The axis formation during oogenesis………………………………………………...12 Localization and regulation of oskar RNP complex…………………………………14 The function and structure of EVH1/WH1 domain………………………………….15 RNA degradation…………………………………………………………………….16 The pathway of mRNA turnover………………………………………………..16 Decapping enzymes……………………………………………………………..17 Control of Decapping…………………………………………………………...18 Drosophila Decapping protein, dDcp1 ……………………………………………...20 dDcp1 is a novel posterior group gene …………………………………………20 dDcp1 contains EVH1/WH1 domain…………………………………………...21 dDcp1 has decapping activity in vitro and vivo...................................................21 Materials and methods………………………………………24 Drosophila maintenance…………………………………………………………….24 The autosomal FLP-DFS technique…………………………………………………24 Germ-line clone production…………………………………………………………25 Cuticle preparation…………………………………………………………………..25 Fluorescence ovary antibody staining……………………………………………….25 Transgene construction………………………………………………………………27 Imaging GFP-dDcp1…………………………………………………………………28 Results ……………………………………………………….29 The GFP-dDCP1 fusion has biological activity……………………………………..30 The dDcp1 mutant shortens the duration of egg production……………………31 Both EVH1/WH1 domain and decapping activity are required for survival…………………………………………………………………….32 The amino acid point mutations in EVH1/WH1 domain in dDcp1………………….34 The amino acid point mutations in EVH1/WH1 domain in dDcp1 disrupt the egg chamber formation and oocyte polarity…………………34 dDcp1 is required for development of follicle cells during oognensis………..35 The ligand binding site of EVH1/WH1 domain in dDcp1 is not required for posterior localization ……………………………………..36 The critical amino acid mutations for decapping activity of dDcp1…………………37 The dDcp1R57A/G172D mutant does not disrupt the development of egg chamber…………………………………………………………….37 The posterior localization of dDcp1 protein does not be affected when Arg57 and Gly 172 are mutated………………………………….38 The dDcp1R57A mutant does not affect the localization of Osk and Stau proteins………………………………………………………..39 The truncated mutations of dDcp1………………………………………………….39 The dDcp1N183 fails to localize at the posterior pole during oogenesis………..40 The dDcp1N183 is sufficient for posterior localization of Osk but not Orb proteins……………………………………………………..41 The dDcp1N300 acts as the full length protein during the oogenesis…………..42 Discussion ……………………………………………………44 There are two different functions in dDcp1…………………………………………44 The function of EVH1/WH1 domain of dDcp1……………………………………..45 EVH1/WH1 domain of dDcp1 is critical for Drosophila ……………...........45 The substrate binding site of EVH1/WH1 domain is crucial for oogenesis…………………………………………………….46 dDcp1 is required for proper development of follicle cells………………46 The role of Decapping activity of dDcp1……………………………………………47 Decapping activity is not required for oogenesis…………………………….47 dDcp1 may involve in mRNA degradation during…………………………. 48 early embryogenesis. dDcp1 may be required for germline stem cell maintenance………………………..48 The C-terminus of dDcp1 is required for posterior localization…………………….49 The dDcp1 possesses the ability to translocate to nucleus………………………….50 Acknowledgement……………………………………………52 References…………………………………………………….53829016 bytesapplication/pdfen-US去頭蓋酵素果蠅drosophiladecappingEVH1/WH1otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49947/1/ntu-93-R91225008-1.pdf