果蠅降解體成員與Dmoesin交互作用之分析

The analysis of interactions between Dorsophila Processing-body components and Dmoesin

呂智傑(Chih-Chieh Lu)

指導教授 ： 周子賓

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10404

摘要

在果蠅卵母細胞發育時，oskar (osk) 訊息核醣核酸(mRNA)決定極細胞(pole cells)與胚胎腹部的生成。oskar mRNA由護理細胞(nurse cells)產生並藉由微管蛋白(microtubule)及運輸蛋白Kinesin送至卵母細胞後端形成新月狀(crescent)的堆積。表現的Oskar 蛋白質會促進微絲延長(F-actin projection)以此固定oskar訊息核醣核酸複合體(mRNP)；在osk mRNP定位之前，它的轉譯(translation)和降解(degradation)是被抑制的。
裂解體(Processing body)主要功能為在細胞質中進行mRNA 降解、儲存以及抑制mRNA的轉譯。去蓋頭蛋白質1(Dcp1)、去蓋頭蛋白質2 (decapping protein2, dDcp2) 和人類促進去蓋頭大分子 (Human enhancer of decapping large subunit, Hedls又稱Ge-1)為裂解體的主要成員。果蠅去蓋頭蛋白質1 (Drosophila decapping protein 1, dDcp1)已知是osk mRNP的成員，並參與oskar mRNP在卵母細胞後端定位。
在卵母細胞中， dDcp1 調控osk mRNP的座落，它的功能與意義仍是我們研究的目標。已知在人類細胞及阿拉伯芥中Dcp1會與Dcp2 和Ge-1形成蛋白質複合體，這顯示其他降解體的成員亦可能是osk mRNP座落所需。在果蠅卵母細胞中，dDcp1與osk mRNP表現的新月形堆積訊號是貼附於表現在卵母細胞內緣的dGe-1或dDcp2A；而dDcp2A和dGe-1兩者突變時皆造成osk mRNP座落異常及產生胚胎腹部發育缺失的性狀；且在dDcp2A 發生突變的卵母細胞中dDcp1和dGe-1會產生不正常的座落；這皆顯示三者具有遺傳上的交互作用(genetic interaction)，而且dDcp1和dGe-1的正常分布需要dDcp2A的作用。另，藉由共同免疫沉澱法證實在果蠅卵母細胞中dDcp2A、dDcp1和dGe-1可以形成複合體，這顯示dDcp2A、dDcp1和dGe-1三者可能以複合體的形式調控osk mRNP的座落。
dGe-1已知參與裂解體形成與osk mRNP座落，然而現有的dGe-1突變株為N端缺失的突變(N-terminal deletion)，利用RT-PCR分析仍可以偵測到一段截斷的(truncated) dGe-1 transcript。由於抗體的限制，我們無法確定此段transcript能否表現出截斷蛋白(truncated protein)，故需重新篩選dGe-1的無效等位基因(null allele)。藉由P-element 不準確刪除法(P-element imprecise excision)篩選，並經過遺傳與分子生物分析得到dGe-172-8是一個無效等位基因突變株。此突變株可供進一步分析dGe-1參與在osk mRNP 坐落的角色。
　　我們推測當osk mRNP送至卵母細胞後端時，可以藉由分布在卵母細胞內緣的dDcp2-dGe-1 複合體與osk mRNP之中的dDcp1作用，將osk mRNP固定。為更進一步確認dDcp2A、dDcp1和dGe-1是否有直接的交互作用，我們利用體外GST-pull down 分析，證實dDcp2A的中間區域和dGe-1的C端可以直接與dDcp1作用。
　　另外，當dDcp2A突變時，卵母細胞中產生不正常的微絲結構，且後端微絲延長(F-actin projections)的現象會消失，顯示dDcp2A可調控微絲的結構。利用體外微絲結合實驗(F-actin association assay)發現dDcp2A的中間區域可與微絲交互作用。果蠅Dmoesin 突變時亦產生不正常的微絲結構，而Dmoesin可藉由穩固細胞膜與微絲的結構來幫助卵母細胞中osk mRNP的座落，我們推測Dmoesin很可能參與dDcp2-dGe-1複合體，以協助dDcp1-osk mRNP的固著。此項假說已被證實Dmoesin和dDcp2A具有物理性的交互作用(physical interaction)並存在一個相互調控(mutual dependent)的關係，本論文更證實dDcp2A的N端(dDcp2NA)和中間區域(dDcp2M)均與Dmoesin具有交互作用。已知Dmoesin N端的FERM功能區域(FERM domain)可結合細胞膜以及C端的微絲結合區域(actin binding domain, AB domain)可與微絲細胞骨架結合。本論文進一步將Dmoesin分成FERM區域、AB區域以及將兩者連接並帶有α-螺旋結構(α-helix)的α/tail 區域，將dDcp2M和Dmoesin三個區域以GST pull down分析，發現dDcp2M可與FERM區域具有直接的交互作用。但完整的dDcp2A卻無法與FERM作用，推測FERM與dDcp2A作用可能需要Dmoesin的其他區域，此推論仍需要進一步分析。另一方面，Dmoesin被磷酸化時會改變其在卵母細胞的分布與功能。原本分布在細胞質及細胞膜上的Dmoesin，在磷酸化後會集中至卵母細胞的內緣。故想詢問磷酸化是否會改變dDcp2A與Dmoesin的交互作用，然而磷酸化和去磷酸化的Dmoesin以GST pull down分析均可以和dDcp2A作用，顯示磷酸化不會改變兩者的交互作用。
　　綜合以上，本論文證實果蠅裂解體成員dDcp1、dDcp2A和dGe-1三者具有直接的交互作用，且dDcp2A也與Dmoesin作用而彼此之間作用的關係在osk mRNP座落的角色仍然需要進一步的實驗與分析。

關鍵字

果蠅裂解體 ； 去頭蓋蛋白質 ； 卵發育

並列摘要

In Drosophila oogenesis, oskar (osk) mRNA at posterior end of oocyte determines both pole cells and abdomen formation of the embryo. Osk mRNA are generated in nurse cell and transported to the posterior end of the oocyte by microtubules and the motor protein, Kinesin. Osk mRNP complex is located and accumulated to form a crescent at posterior. Before osk mRNP localization, its translation and degradation are repressed. Osk protein promotes F-actin projections to anchor more osk mRNP in the posterior end of the oocyte.
Decapping protein 1(Dcp1), decapping protein 2 (Dcp2) and Human enhancer of decapping large subunit (Hedls also called Ge-1) are components of Processing bodies (P-bodies) are involved in mRNA degradation, storage and translation repression in cytoplasm foci. Drosophila decapping protein 1 (dDcp1) is not only a component of osk mRNP but also required for its posterior localization in Drosophila oocyte.
We attempt to figure out the function and meaning which dDcp1 is involved in the osk mRNP localization. In human cell lines and Arabidopsis, Dcp1, Dcp2, and Ge-1 can form a complex; thus, it indicated whether dDcp2A and dGe-1 are involved in dDcp1-osk mRNP localization. In the oocyte, we observed dDcp1-oskar mRNP present a closely abutting localization pattern with dDcp2 and dGe-1. Both dDcp2 and dGe-1 causes the mislocalized osk mRNP and posterior group embryonic phenotype in its mutant phenotype individual. In dDcp2 null mutant, dDcp1 and dGe-1 were mislocalized in the oocyte. It indicated they have genetic interactions in the oocyte, and the dDcp2 is required for proper distribution of dDcp1 and dGe-1. Besides, the formation of dDcp2A/dDcp1/dGe-1 complex in Drosophila oocyte has been proved by coimmunoprecipitation. It indicated that dDcp2A, dDcp1, and dGe-1 might form a complex to regulate osk mRNP localization.
dGe-1 has been proved promote P body formation and osk mRNP localization. However, the dGe-1 mutant allele we obtained is an N-terminal deletion mutant. It was detected a truncated dGe-1 transcript by RT-PCR. Due to our antibody restriction, we cannot detect whether this transcript is translated to produce a truncated protein. Thus, we preformed P-element imprecise excision to screen a null allele of dGe-1. After genetic and molecular analysis, dGe-172-8 is a null mutant for analyze the role of dGe-1 in osk mRNP localization.
We suggested the osk mRNP was anchored by the interaction between dDcp2-dGe-1 complex along the cortex and dDcp1 in osk mRNP complex, as osk mRNP was transported to the posterior end. In the oocyte, the complex formed by dDcp2A, dDcp1 and dGe-1 has been proved by co-immunoprecipitation. In order to figure out whether this interaction among dDcp2, dDcp1 and dGe-1 is direct or indirect, we used in vitro GST-pull down assay as approach. We found the middle of dDcp2A and the C terminal of dGe-1 can interact with dDcp1 directly.
　　On the other hand, abnormal actin structure is presented, and the posterior actin projection is absent in dDcp2 null mutant oocyte. It indicated dDcp2 regulates the actin in the oocyte. Using F-actin association assay, it revealed the middle of dDcp2A can associate with F-actin. Besides, abnormal actin structure is also observed in Dmoesin mutant oocyte. Dmoesin is a well-known cross-linker connected the plasma membrane and F-actin, and has been proved to anchor the osk mRNP in the oocyte. Thus we suggested Dmoesin is involved in the osk mRNP localization which dDcp2-dGe-1-dDcp1 complex assisted. Dmoesin has been proved in a mutual- dependent relationship and interacts with dDcp2A physically. Here, we identified both the N-terminal (dDcp2NA) and middle region (dDcp2M) of dDcp2A can associate with Dmoesin directly. Dmoesin contains 2 functional domains: one is FERM domain which can associate with cell membrane; the other is actin binding domain, AB domain, which can bind to actin cytoskeleton. We divide Dmoesin into FERM domain, AB domain, and the α/tail domain which has α-helix structure and connects the FERM and AB domains. The GST pull down assay was preformed and revealed the FERM domain can interact with dDcp2M. However, the full length dDcp2A cannot interact with FERM domain. This result suggested that the other region of Dmoesin is required for the interaction between FERM and dDcp2A. It needs to be clarified. In addition, the Threonine 559 phosphorylation of Dmoesin affects the localization and function of Dmoesin. Phosphorylated Dmoesin is expressed on the cortex in the oocyte; whereas dephosphorylated Dmoesin is expressed in cytosol and plasma membrane. However, both phospho/dephospho form Dmoesin can interact with dDcp2A, it revealed the interaction between dDcp2A and Dmoesin is independent on phosphorylation.
　　To sum up, this thesis proved Drosophila P body components, dDcp1, dDcp2A and dGe-1 contain direct interactions; dDcp2A interact with Dmoesin directly. However, the possible roles of these interactions in the osk mRNP localization during oogenesis remain to be clarified.

並列關鍵字

processing body ； decapping protein ； oogenesis

參考文獻

References

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