周子賓臺灣大學：分子與細胞生物學研究所吳美齡Wu, Mei-LingMei-LingWu2007-11-252018-07-062007-11-252018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/49889在5端到3端訊息核糖核酸(mRNA)降解路徑中，先去除訊息核糖核酸3端的多個腺嘌呤核糖核酸(polyA)，然後去頭蓋蛋白質Dcp1及Dcp2 複合體除掉5端的頭蓋結構，最後5端到3端外切脢降解訊息核糖核酸本體。去除多個腺嘌呤核糖核酸及蓋頭結構決定降解速度。在酵母菌及人類去頭蓋蛋白質複合體，Dcp2同源蛋白的N端具有主要降解頭蓋的MutT活性區域(MutT motif)。透過同源比對,果蠅的CG6169是Dcp2的唯一同源蛋白，故命名果蠅去頭蓋蛋白質2，dDcp2。 在酵母菌及人類細胞中，Dcp1和Dcp2一起坐落在細胞質的不同點上。在酵母菌中，Dcp1和Dcp2可以直接互相作用; 在人類細胞中，兩者則經由Hedls間接作用。在早期胚胎形成(embryogenesis)過程中，dDcp1為母源訊息核糖核酸降解所需，並是oskar訊息核糖核酸輸送複合體的成員。oskar訊息核糖核酸定位在卵母細胞(oocyte)後端，決定胚胎腹部腹節與極細胞(pole cells)的發育。在護理細胞(nurse cells)中，dDcp1在細胞質和dDcp2有同位分佈(colocalization)。dDcp2突變可以產生胚胎腹節缺失的後端突變性狀，因此，dDcp2可以被視為果蠅體軸後端發育可能所需的基因。我們推測dDcp2可能涉及在oskar訊息核糖核酸輸送複合體中。 我們構築五個基因片段進行互補實驗，以釐清兩個dDcp2突變的基因形式(allele) dDcp2de21和BG1766對基因影響的範圍。再者，我們研究dDcp2de21和BG1766對oskar訊息核糖核酸輸送複合體定位的影響。dDcp2de21是dDcp2 的無效基因形式，且可能些微影響下游基因diablo。但是，在BG1766，跳躍元素(P element)插入同時影響dDcp2 和diablo。 在dDcp2de21生殖細胞突變株(germline clone)，oskar訊息核糖核酸輸送複合體成員Exu、Me31B和Orb在卵母細胞後端的分佈受阻撓。然而卵母細胞中的微管組織 (microtubule organization)似乎正常。此外，Exu和Me31B在護理細胞的細胞質中累積。這暗示著在dDcp2de21生殖細胞突變株中，母源物質由護理細胞到卵母細胞的運輸受阻撓。綜合這些實驗數據，dDcp2可能參與調控母源物質由護理細胞到卵母細胞的運輸。 藉由CytoTrap酵母菌雙雜合系統實驗，在活體外，單獨的dDcp1不能和dDcp2-RB結合。dDcp1和dDcp2之間的關聯尚不明確，仍需要進一步實驗分析。After deadenylation, the Dcp1-Dcp2 decapping complex hydrolyses the 5’ capped structure of mRNA in 5’ to 3’ mRNA degradation pathway. Finally, the transcript is digested by a 5’ to 3’ exonuclease. This degradation pathway is rate-determined by a combination of deadenylation and decapping. In yeast and human, Dcp2 contains a MutT motif at its N-terminus, which is necessary for mRNA decapping. Through the blast search, CG6169 is the only Drosophila homologue of Dcp2, and therefore is named as Drosophila decapping protein 2 (dDcp2). In yeast and human cells, Dcp1 co-localizes with Dcp2 in distinct cytoplasmic foci. In yeast, yDcp1p can physically interact with yDcp2p while in human their interaction depends on the existence of Hedls. Drosophila decapping protein 1 (dDcp1) is required for degradation of maternal mRNAs in early embryogenesis. dDcp1 is one component of the oskar (osk) mRNP complex. The localization of osk at the posterior of the oocyte is critical for both the development of abdomen and pole cells. dDcp1 co-localizes with dDcp2 in nurse cell cytoplasm. Since dDcp2 mutant embryos exhibit the abdomen deletion phenotypes, dDcp2 is proposed as a posterior group gene. These data suggest that dDcp2 may also have a role in the localization pathway of the osk mRNP complex. To clarify the specificity of two dDcp2 mutant alleles, dDcp2de21 and BG1766, we constructed five genomic fragments for complementation tests. Next, we investigated the effects of dDcp2de21 and BG1766 on the localization of the osk mRNP complex. According to our data, dDcp2de21 is confirmed as a null allele specific to dDcp2, and might mildly affect the downstream gene, diablo. However, in BG1766, the P element insertion affects both dDcp2 and diablo. In dDcp2de21 GLC egg chambers, the localization of components of the osk mRNP complex is disrupted at the posterior end of the oocyte, such as Exu, Me31B and Orb. However, microtubule organization seems to be normal in the oocyte. In addition, Exu and Me31B accumulate in nurse cell cytoplasm. It implies that the transport of maternal materials from nurse cells to oocytes is affected in dDcp2de21 GLC egg chambers. Taking these data together, dDcp2 may participate in regulating the transport of maternal materials from nurse cells to oocytes. Through the CytoTrap yeast two-hybrid system, dDcp1 alone does not interact with dDcp2-RB form in vitro. Therefore, the relationship between dDcp1 and dDcp2 still remains elusive.Table of Content 中文摘要…………………………………………………………………………….1 Abstract……………………………………………………………………………...3 Table of Content.…………………………………………………………………..5 List of Tables……………………………………………………………………….9 List of Figures.……………………………………………………………………10 Abbreviations……………………………………………………………………..11 Introduction……………………………………………………………………….12 Drosophila oogenesis…………………………………………………………..12 Anterior-posterior axis determinants in the oocyte……………………………..14 Dorsal-ventral axis determination of the oocyte………………………………..17 The oskar mRNP complex and its posterior localization……………………….18 Two major RNA degradation pathways………………………………………...20 Deadenylation……………………………………………………………...21 5’ to 3’ decay and 3’ to 5’ decay…………………………………………..21 Overview of decapping proteins………………………………………………...22 The Dcp1/Dcp2 decapping complex………………………………………22 Substrate recognization of Dcp1/Dcp2……………………………………22 Effectors associated with Dcp1/Dcp2……………………………………..23 DcpS……………………………………………………………………….24 Drosophila decapping protein 1 (dDcp1), is a component of the oskar mRNP complex…………………………………………………………………………24 Drosophila decapping protein 2 (dDcp2) is a putative posterior group gene…..25 The themes………………………………………………………………………...26 Materials and methods…………………………………………………………27 Fly stocks and maintenance……………………………………………………..27 The autosomal FLP-DFS technique…………………………………………….27 Cuticle preparation……………………………………………………………...27 Western analysis………………………………………………………………...28 Molecular cloning……………………………………………………………….28 Constructs for complementation analyses of putative dDcp2 mutant alleles……..28 Constructs for cytotrap two hybrid test……………………………………………29 Cytotrap…………………………………………………………………………30 Preparation of cdc25H yeast competent cells……………………………..30 Transforming yeast…………………………………………….…………..31 To detect protein-protein interactions……………………………………..31 Immunoflourescence staining for ovaries………………………………………31 Results ……………………………………………………………………………..33 CG6169 encodes the Drosophila decapping protein 2 (dDcp2)………………..35 The dDcp2 locus and mutant alleles…………………………………………….35 Constructs of transgenic genes: complementation analyses and genomic rescue of dDcp2 mutant alleles………………………………………………………....37 The genomic fragments containing dDcp2 locus can rescue the female sterility of homozygous dDcp2de21, but not BG1766……………………………………….38 The genomic fragment containing the truncated C-terminal of dDcp2 can prolong the lethal phase of homozygous dDpc2de21..……………………………………39 W4 has adverse effects on BG1766 GLC females, but not dDcp2de21 GLC females………………………………………………………………………….40 dDcp2 mutant affects the distribution pattern of Orb, Exu, and Me31B……….42 dDcp2 is required for the posterior localization of Orb…………………...43 dDcp2 is required for the posterior localization of Exu…………………...44 dDcp2 is required for the posterior localization of Me31B………………..45 dDcp2 mutants do not affect microtubule organization in the oocyte………….46 Microtubule plus end………………………………………………………47 Microtubule minus end…………………………………………………….47 dDcp2-RB does not interact with dDcp1 in yeast system………………………48 Discussion …………………………………………………………………………51 In addition to dDcp2, the P element insertion in BG1766 has the effect on another gene…………………………………………………………………….51 The C-terminal region of dDcp2 is required for proper pupation and contains unknown function……………………………………………………………….53 The organization of microtubule is not affected in dDcp2 mutant oocyte……...54 dDcp2 may participate in the transportation of the posterior-localized complex…………………………………………………………………………54 Putative P-bodies increase in dDcp2 mutant ovaries…………………………...56 dDcp2-RB does not interact with dDcp1 directly………………………………58 Acknowledgement……………………………………………………………….60 References…………………………………………………………………………61 Tables and Figures………………………………………………………………721928371 bytesapplication/pdfen-US果蠅去頭蓋蛋白質2decapping protein 2otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49889/1/ntu-95-R93b43021-1.pdf