核糖核酸降解複合體成員及膜突蛋白在果蠅卵細胞中調控oskar核糖核酸的運送與定錨

Abstract

在果蠅(Drosophila. melanogaster)卵發育(oogenesis)過程中，oskar 訊息核醣核酸(oskar mRNA)被運送、坐落於卵母細胞後端，決定了胚胎後端體節的發育與生殖細胞的前驅細胞(極細胞pole cell)的產生。核糖核酸代謝裂解體(mRNA processing bodies, P-bodies)主要功能負責5’到3’核糖核酸裂解，也參與核糖核酸運送、剪接、儲存或轉譯抑制等相關機制。之前的研究顯示去蓋頭蛋白質1 (dDcp1)為oskar mRNA蛋白質複合體的成員；但對於dDcp1為何參與在oskar mRNA座落於卵後端調控機制中扮演的角色尚不清楚。本論文研究動機為找到P-bodies在oskar mRNA後端坐落調控機制扮演的功能探討。在此論文中，我提出P-bodies成員dDcp1， 去蓋頭蛋白質2 (dDcp2)，促進去蓋頭大蛋白(dGe-1)與果蠅膜突蛋白(Drosophila moesin, Dmoesin)共同作用於oskar mRNA在卵母細胞的運送與正確定錨。 首先、我發現dDcp1、dDcp2和dGe-1在卵母細胞中以去蓋頭複合體(decapping complex)的方式坐落於細胞皮層(Cortex)，而且dDcp2決定dDcp1和dGe-1正確坐落在細胞皮層，比較之前單個基因對於oskar mRNA在卵母細胞中扮演角色分析，我推測整個複合體必須先形成才進而調控oskar mRNA在卵母細胞後端的定位。再者、文獻指出微管骨架 (microtubule cytoskeleton)和纖維絲肌動蛋白(Filamentous-actin, F-actin)分別參與oskar mRNA的運送和定錨。我也發現dDcp2對微管生長和纖維性肌動蛋白的建構皆有正面調控的功能，綜合以上推測decapping complex可能藉由對微管骨架和纖維絲肌動蛋白的正面調控來幫助oskar mRNA的運送和定錨。 其三、Dmoesin負責連結細胞膜和纖維絲肌動蛋白，以維持細胞形狀，在卵母細胞的皮層上，可以明顯地測到果蠅膜突蛋白。我發現dDcp2與Dmoesin互相依附地(mutually-dependent)座落於皮層上；當一方突變缺失時，另一方伴隨纖維絲肌動蛋白同時脫落於卵母細胞質中。先前研究指出，在卵發育的滋養細胞(nurse cells)與卵母細胞質中，oskar mRNA蛋白質複合體與P-bodies處於動態地進行成員互換；意即oskar mRNA處在隨時可被裂解的P-bodie中，然而Dmoesin抑制dDcp2的mRNA去蓋頭酵素活性，可避免oskar mRNA在卵發育過程被降解掉。此外，Dmoesin的磷酸化狀態可決定dDcp2在卵母細胞中的分布位置，過量表現磷酸化的Dmoesin可累積較多的dDcp2坐落在細胞皮層，反之表現非磷酸化Dmoesin使dDcp2散布在細胞質。 我綜合上述結果與實驗室前人研究成果，提出:一、果蠅膜突蛋白和去蓋頭蛋白質2在卵母細胞皮層形成oskar訊息核醣核酸的定錨者(anchor)；二、去蓋頭蛋白2和果蠅膜突蛋白組成oskar訊息核醣核酸的輸送複合體(the transporting complex)和定錨複合體(the anchoring complex)，藉由調控果蠅膜突蛋白的磷酸化狀態，兩複合體在卵母細胞後端進行動態轉換，最終完成oskar訊息核醣核酸在卵母細胞後端的定錨。

During oogenesis in the fruit fly (Drosophila melanogaster), oskar mRNA is delivered and localized to the posterior, and thereby also promotes the assembly of germ plasm, which is a specialized cytoplasm for germ-cell formation. Processing bodies (P-bodies) of mRNA are sites that 5’ to 3’ mRNA degradation happens, including the removal of 5’ cap on mRNA and mRNA exonuclease activity. In a previous study, the Drosophila decapping protein 1 (dDcp1) has been found as a component of the oskar messenger ribonucleoprotein (mRNP), directing its posterior localization. In this study, I aimed to the three components of a decapping complex (dDcp1, Drosophila decapping protein 2 (dDcp2) and dGe-1) cooperating with Drosophila moesin (Dmoesin) participates in the transport and anchoring of oskar mRNP in the oocyte. First, I found that the trimeric complex forming by dDcp1, dDcp2, and dGe-1 stands along the oocyte cortex and is required for the posterior localization of oskar mRNA. Moreover, the presence of dDcp2 plays a critical role in sustaining the cortical localizations of dDcp1 and dGe-1 in oocytes. Second, previous studies point out that microtubule cytoskeleton is responsible for the oskar mRNA transport and F-actin microfilaments facilitate the oskar mRNA anchorage. I found that dDcp2, except the mRNA decapping activity, shows a positive regulation both on the microtubule growth and F-actin proper formation in oocytes. Third, Dmoesin is a crosslinker connecting the membrane and F-actin along the cortex. dDcp2 and Dmoesin show a mutually-dependent adherecne along the oocyte cortex. Lastly, the phosphorylation status of Dmoeisn determines the allocation of dDcp2 in the oocyte. Overexpression of phospho-Dmoesin accumulates the cortical dDcp2, whereas overexpression of nonphospho-Dmoeisn draws dDcp2 into the ooplasm. Hence, we propose that from stage 6 dDcp2 and phospho-Dmoesin form a pre-localized anchor for locate oskar mRNA along the cortex. And Dmoesin-dDcp2 recruit oskar mRNA-dDcp1-dGe-1 to be the anchoring complex. nonphospho-Dmoe and dDcp2 organize a transporting complex to move oskar mRNA-dDcp1 from the cortex. At the posterior, phosphorylation status exchange the components of the two complex and oskar mRNA can be anchored properly.