周子賓臺灣大學：分子與細胞生物學研究所林明德Lin, Ming-DerMing-DerLin2007-11-252018-07-062007-11-252018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/49903在果蠅卵發育的過程(oogenesis)中，母系訊息核醣核酸(maternal mRNA)在卵母細胞(oocyte)內特定位置的分佈與定位對於胚胎時期的發育扮演非常重要的角色。例如oskar (osk) 訊息核醣核酸在卵母細胞後端的定位決定極細胞(pole cells)與胚胎腹部腹節的形成。 除了輸送osk 訊息核醣核酸到後端的特定蛋白質外，參與訊息核醣核酸剪接(splicing)機制的成員也與osk 訊息核醣核酸的運送過程有關；例如一些Exon junction complex的蛋白質成員被破壞時，osk無法被運送到卵母細胞後端。此外，與蛋白質轉譯起始過程有關的蛋白質亦參與調控osk的運送；例如 eIF4E以及其他相關的轉譯起始調控因子都與osk 在卵母細胞後端的定位有關。 在特定之輸送機制、剪接機制與轉譯機制外，訊息核醣核酸的裂解(degradation)機制是否也參與osk 訊息核醣核酸在卵母細胞後端的定位並未被報導。本論文主要在說明果蠅的訊息核醣核酸裂解系統中的去頭蓋蛋白質1 (Drosophila decapping protein 1, dDcp1) 不但參與母系訊息核醣核酸在胚胎時期的裂解並且也是osk訊息核醣核酸輸送複合體的成員。本論文證實果蠅去頭蓋蛋白質1為輸送 osk往卵母細胞後端運送的過程所需，並揭示其參與並調控此運送過程的機制與其在卵發育過程中所扮演的其他可能角色。 在酵母菌與人類細胞中，訊息核醣核酸的裂解被發現坐落於細胞質中稱為Processing bodies (P-bodies)的特殊構造。P-bodies 的構造中包含了去頭蓋蛋白質1(Dcp1)、去頭蓋蛋白質2 (Dcp2)與5’ 往 3’ 外切核醣核酸酵素(5’ to 3’ exoribonuclease；Xrn1) 以及其它參與5’ 往3’ 之訊息核醣核酸裂解所需的相關蛋白質。在卵母細胞中去頭蓋蛋白質1坐落於卵母細胞的後端，其在卵母細胞後端的分佈狀態乃由osk 訊息核醣核酸所在之位置以及含量所決定。在護理細胞(nurse cells)中，去頭蓋蛋白質1分佈於細胞質並與去頭蓋蛋白質2、果蠅5’ 往 3’ 外切核醣核酸酵素(Pacman) 以及 Me31B 等蛋白質形成點狀的構造。本論文證實這些點狀構造就是果蠅護理細胞中參與訊息核醣核酸裂解的P-bodies。 人類去頭蓋蛋白質1的同源蛋白質，hDcp1a，也被發現為參與傳遞TGF-beta訊息路徑的轉錄激活因子(transactivation factor)並且被命名為SMIF (Smad4-Interacting Factor)。果蠅去頭蓋蛋白質1除了參與osk在卵母細胞後端的運送外，本論文並證實去頭蓋蛋白質1的N端會與Medea蛋白質(Drosophila Smad4)的MH1區域進行交互作用，而且其蛋白質的C端在酵母菌裡有激活基因轉錄活性的能力。唯，果蠅去頭蓋蛋白質1參與TGF-beta訊息傳遞路徑在卵的發育過程中所扮演的角色仍然需要進一步的實驗與分析。Specific subcellular localization of maternal mRNAs in the oocyte during oogenesis is very important for proper embryonic patterning in Drosophila. For example, the deposition of oskar (osk) mRNA at the posterior pole of the oocyte is critical for both pole cells and abdomen formation. Many mechanisms are involved in the posterior deposition of osk mRNA. In addition to those directly involved in the osk mRNA transportation, proteins participate in pre-mRNA splicing, such as Exon junction complex components, are implicated in osk mRNA transportation. Moreover, proteins that participate in mRNA translation, such as eIF4E and others related to translational initiation, can direct the posterior transportation of osk mRNA. Except for the transportation, splicing, and translation machineries, the mRNA degradation machinery has not been reported to participate in the regulation of osk mRNA transport. Evidence presented here indicates that Drosophila decapping protein 1 (dDcp1) is required for not only the maternal mRNA degradation during early embryogenesis but also the posterior transportation of osk mRNA. As a component of osk mRNP complex, the mechanism of dDcp1-directed osk mRNA transportation and the possible roles of dDcp1 during oogenesis are discussed. In both yeast and human cells, the sites of 5’ to 3’ mRNA degradation have been found to reside in specific cytoplasmic foci, named Processing bodies (P-bodies). P-bodies contain Decapping protein 1 (Dcp1), Decapping protein 2 (Dcp2), 5’ to 3’ exoribonuclease (Xrn1), and other proteins required for 5’ to 3’ mRNA degradation. In the oocyte, dDcp1 is localized at the posterior end and its localization is osk mRNA dosage- and position-dependent. In nurse cell cytoplasm, dDcp1 is localized in the cytosol and is colocalized with dDcp2, Drosophila 5’ to 3’ exoribonuclease (Pacman), and Me31B in discrete cytoplasmic foci. Evidence provided here indicates that these cytoplasmic dDcp1 bodies in nurse cells are Drosophila P-bodies. The human homolog of Dcp1, hDcp1a, has been found to involve in the TGF-beta signaling pathway and is also named as SMIF (Smad4-interacting factor). In addition to the requirement of dDcp1 for the posterior transport of osk mRNA, the N-terminal of dDcp1 can interact with the MH1 domain of Medea (Drosophila Smad4 homolog). Besides, dDcp1 contains intrinsic transactivation activity in its C-terminal region and is able to translocate into the nucleus in response to the Dpp signaling. However, the possible roles of dDcp1 in the TGF-beta signaling during oogenesis remain to be clarified.Acceptance of the dissertation 論文授權書 致謝 中文摘要 Abstract Table of contents List of table and figures Chapter 1: The discovery of Drosophila decapping protein 1, dDcp1 Summary 2 Introduction 3 Using FLP-DFS technique to study the specific maternal effect of an essential gene 3 The cFRT2L2R chromosome 5 The discovery of b53 mutation 7 Identification and analysis of b53 mutant 7 Results Molecular characterization of P-element insertion site in b53 mutant 9 Imprecise P-element excision of b53 mutant 9 Genomic rescue of b53 mutation 11 Bioinformatic analysis of CG11183 13 Phenotypes of b53 escapers 17 Maternal effects of b53 mutant 18 dDcp1442P is a null allele 19 Phenotypes of dDcp1442P GLC egg chambers 20 Eggshell defects in egg produced by dDcp1442P GLC females 22 Discussion dDcp1 mutant has pleiotropic phenotypes 25 Possible post-translational modification of dDcp1 26 Chapter 2: dDcp1 resides in Drosophila Processing bodies in nurse cell cytoplasm and is required for maternal mRNA degradation Summary 29 Introduction The 5’ to 3’ mRNA degradation and decapping proteins 30 Decapping protein 1 (Dcp1) in yeast and human 31 Decapping protein 2 (Dcp2) in yeast and human 32 Processing bodies are sites of 5’ to 3’ mRNA degradation and contain factors required for NMD, miRNA, and siRNA pathways in human and yeast 34 P-body components identified by auto-antibodies 35 P-bodies can function as sites of mRNA storage in yeast 36 Characterization of P-bodies in nurse cells 37 Results dDcp1 is required for maternal mRNAs degradation including oskar, bicoid and twine 38 dDcp1 can not stimulate the decapping activity of dDcp2 in an in vitro decapping assay 41 The production of dDcp1 antibody 44 The expression pattern of dDcp1 in oogenesis 44 Oogenesis in germarium region 45 Distribution pattern of dDcp1 in germarium region 46 Distribution pattern of dDcp1 in egg chambers 47 From syncytial blastoderm to cellular blastoderm in early embryogenesis 48 Maternal expressed dDcp1 is able to colocalize with dDcp2 in early cellular blastoderm embryos 49 dDcp2 is localized in discrete cytoplasmic foci in nurse cells 50 dDcp1 is colocalized with dDcp2 and Me31B in nurse cell cytoplasm 52 Pacman is localized in discrete foci in nurse cell cytoplasm 53 dDcp1 forms a complex with dDcp2/Me31B/Pacman but not Tazman in nurse cell cytoplasm 54 dDcp1 is not colocalized with Ccr4 in either nurse cells or gastrulating embryos 55 Both dDcp1 and dDcp2 bodies are increased in pacman mutants 57 YFP-dDcp1 cytoplasmic foci are reduced both in number and size after cycloheximide treatment 58 YFP-dDcp1 cytoplasmic foci are vanished after RNase A treatment 59 YFP-dDcp1 bodies are dramatically increased under heat stress 60 Discussion dDcp1 can not enhance the decapping activity of dDcp2 63 The dynamic distribution of P-bodies during oogenesis 64 The dDcp1 bodies at the posterior pole of stage 2-6 oocyte may not function as P-bodies 65 Sites of deadenylation may different from sites of degradation in Drosophila 65 Chapter 3: dDcp1 is a component of the oskar mRNP complex and directs its posterior transportation in the oocyte Summary 68 Introduction The deposition of maternal components during Drosophila oogenesis 69 Microtubules are essential for mRNA localization 69 The determination of dorsal-ventral (DV) body axis of Drosophila embryo 70 The determination of anterior-posterior (AP) body axis of Drosophila embryo 72 Targeting osk mRNA to the posterior pole is microtubule dependent 73 Translational control of osk mRNA 73 Actin is involved in the anchorage of osk mRNA and Osk proteins at the posterior pole 74 Factors required for oskar mRNA transportation 76 The involvement of dDcp1 in osk mRNA transport 78 Results dDcp1 is a novel posterior group gene 79 dDcp1 specifically affects the posterior deposition of osk mRNA 80 The localization of dDcp1 is dependent on osk mRNA as well as on microtubule organization 82 dDcp1 is required for the posterior localization of Exu, Yps, and Orb 85 Live-imaging of the osk mRNP particles 86 dDcp2 mutants can display a posterior group embryonic phenotype 89 Discussion The possible mechanisms that dDcp1 can stably associated with osk mRNA during transportation 93 dDcp1 dose not cause premature translation of osk mRNA 94 The conversion between polar granules and P-bodies may exist during development 95 A dynamic switch of mRNAs among translational activation, translational repression, and degradation 95 Using CytoTrap yeast two-hybrid system to screen the possible interaction partners of dDcp1 97 dDcp2 mutants present posterior group embryonic phenotypes 98 Chapter 4: dDcp1 is a putative transcriptional co-activator in TGF-b signaling pathway Summary 102 Introduction An overview of TGF-b superfamily 103 TGF-b family members in Drosophila 103 Intracellular signal transduction of TGF-b signaling 104 Type I and type II receptors in Drosophila 105 Smad proteins 106 Receptor-regulated Smad (R-Smad), common Smad (Co-Smad), and inhibitory Smad (I-Smad) in Drosophila 107 Dpp signaling in Drosophila oogenesis 108 SMIF, a human Smad4-interacting factor, carries mRNA decapping enzymatic activity 109 Dose dDcp1 involve in TGF-b signaling pathway in Drosophila? 110 Results Ectopically expressed HA-dDcp1 is able to response to the Dpp signaling 112 dDcp1 contains intrinsic transactivation activity in its C-terminal region 114 The N-terminal region of dDcp1 can interact with MH1 domain of Medea but not full length Medea 116 Defects in Dpp signaling dose not cause posterior group embryonic phenotype 118 Discussion dDcp1 has the ability to enter the nucleus 121 dDcp1 is a shuttle protein with triple functions 122 Possible role of dDcp1 in germline stem cell maintenance 123 Future Aspects Drosophila Hedls/Ge-1 may trigger the interaction between dDcp1 and dDcp2 126 How dDcp1 directs the posterior transportation of osk mRNA? 127 The implication of the involvement of dDcp1 in TGF-b signaling 127 Dynamic distribution of P-bodies from oogenesis to early embryogenesis 129 Material and Methods Plasmid constructions 135 Drosophila stocks 144 Cuticle preparation 145 Antibody generation 145 Western blot analysis 146 Whole-mount ovary antibody staining 146 Co-Immunoprecipitation 147 Northern blot analysis 147 Whole-mount Ovary in situ hybridization 148 RNase A treatment of hand-dissected egg chambers 148 Cycloheximide treatment of hand-dissected egg chambers 149 Heat-shock of YFP-dDcp1 flies 149 Yeast one-hybrid assay 149 Yeast two-hybrid assay 150 References 151 Table and Figures 171 Appendix I. Isogenization of cFRT2L2R chromosome3865821 bytesapplication/pdfen-US果蠅去頭蓋蛋白質1Drosophiladecapping protein 1dDcp1otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49903/1/ntu-95-D90225003-1.pdf