果蠅S2細胞內訊息核醣核酸裂解體(Processing-bodies)之定義與探討

Abstract

真核生物的訊息核醣核酸裂解在許多不同的生物機能層面扮演了很重要的角色，例如：基因表現的調控（何時啟動或停止）、訊息核醣核酸品質上面的控制（有無受損）、抵抗外來病毒的侵襲。在人類細胞與酵母菌方面，由於過去十幾年來的努力研究，發現訊息核醣核酸的裂解有主要兩條路徑：在去掉poly A之後，分別進行5’端往3’端方向的裂解以及3’端往5’端方向的裂解。 近期研究顯示，在人類細胞與酵母菌中，訊息核醣核酸裂解所需的蛋白質會在細胞質特定位置呈現一種聚集的現象，稱之為裂解體 (Processing bodies, P bodies)。而這些被稱為裂解體的地方，確實是訊息核醣核酸被裂解之處。基於這些參與訊息核醣核酸裂解之蛋白質有其同源物存在於不同的物種間以及整個訊息核醣核酸裂解途徑機制的相似性，我們因而推測在果蠅的細胞中理應存在著類似於人類與酵母菌裂解體之構造。 本篇論文著眼於定義與確認果蠅S2細胞中是否有相似於人類與酵母菌裂解體的存在。在觀察一些參與果蠅訊息核醣核酸裂解時所需的蛋白質時，如dDcp1, dDcp2, Me31B, Pacman, Xrn1…等，發現他們在S2細胞質中也有聚集的現象產生。然而，造成此一現象的原因不見得就是訊息核醣核酸裂解所造成，亦有可能其本身的存在只是這些蛋白質的一個儲存場所。我們將以一連串的實驗來釐清這些可能性，例如：對於這些不同的蛋白質加以定位觀察是否處於同一構造中、破壞參與訊息核醣核酸裂解的蛋白質並觀察對於這些假設性的裂解體的影響、觀察進行裂解中的訊息核醣核酸是否出現在這些構造中…等等。實驗結果證明，這些出現在S2細胞質中的構造絕非單純的只是這些蛋白質暫時存放的處所。相反的，我們認為這些假定的裂解體與在人類細胞或是酵母菌中發現的構造具有相似的功能：也就是訊息核醣核酸裂解發生的地方，尤其是對於進行5’端往3’端方向的訊息核醣核酸裂解而言。

The degradation of eukaryotic mRNAs plays important roles in the modulation of gene expression, quality control of mRNA biogenesis and antiviral defenses. Two general pathways of mRNA decay have been identified in eukaryotic cells. After shortening of the poly(A) tail at the 3′ end of the mRNA, mRNA may be degraded in a 5’ to 3’ direction or in a 3’ to 5’ direction. Recently, in yeast and human cells, many enzymes of the basic decay machinery are found in the cytoplasmic foci, called processing bodies (P bodies). Further experiments have proved that P-bodies are actual sites of mRNA decay, especially for 5’ to 3’ decay pathway. Based on the conserved mRNA decay factors and the similar mRNA decay mechanisms between species, we suggest that the processing body related structure probably also exists in Drosophila. Here, we observed that the mRNA decay factors, such as Ccr4, dDcp1, dDcp2, Me31B, and Pacman, are concentrated in the cytoplasmic foci of Drosophila S2 cells. Whether these proteins are in a unique set of structures? We construct a series of proteins which involve in the different mRNA decay pathways to transfect the cells then co-immunostain the transfected protein and endogenous dDcp1 (as a marker of putative P bodies). It was found that proteins that involved in the 5’ to 3’ mRNA decay all localize to the same cytoplasmic foci, we called as dDcp1-containing bodies. However, we can not exclude the possibility that these foci are sites for protein storage rather than mRNA decay. To answer this question, a series of experiments such as dsRNA silencing the mRNA decay factors, translational inhibitor treatment, and the visible mRNA decay intermediates are required. Finally, we may identify these dDcp1-containing bodies are the “processing bodies” in Drosophila cells.