| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 84 === mRNA的位移作用是早期胚胎發育時細胞分化的重要機制。在非洲有爪水生蛙卵母細胞中，Vgl mRNA的位移現象是目前被研究最為清楚的系統；Vgl mRNA在卵母細胞生長期間，會由平均分佈的形式漸漸的朝植物極運動並固著在植物極上，由於vgl蛋白質只在植物極表現，這似乎表示Vgl mRNA在位移的過程中，同時受到了轉譯抑制作用，所以不會在植物極以外的部位表現蛋白質。目前，雖然已發覺在卵母細胞中，抑制 maternal mRNA轉譯之masking蛋白質─p56及p54會與Vgl定位訊號RNA產生專一性的接合，但尚不清楚mRNA的位移現象與轉譯作用的調控是否有所關聯。
在本論文中，利用微量注射技術與共軛焦點顯微鏡，發覺綠螢光蛋白質(green fluorescent protein;GFP)之mRNA在非洲有水生蛙卵母細胞中，轉譯出的綠螢光蛋白質可穩定存在並分佈於整個卵母細胞中;但在3'端未轉譯區多加了Vgl定位訊號RNA的GFP-VglLS chimeric mRNA則只能在植物極表現綠螢光蛋白質。因為上述兩種mRNA的唯一的差別是Vgl定位訊號RNA的存在與否，所以mRNA的位移與轉譯抑制兩種作用受到同一cis-actingelement─Vgl定位訊號RNA的調控。在 In Vitro 蛋白質轉譯實驗中，部份純化之卵母細胞masking蛋白質對含 Vgl定位訊號之chimeric mRNA有較佳而顯著的轉譯抑制作用。而經去磷酸化處理的部份純化之masking蛋白質，在失去與RNA接合的能力後，則亦會同時失去對GFP-VglLS chimeric mRNA的轉譯抑制作用。這結果顯示，可能就是masking蛋白質和 Vgl定位訊號RNA的專一性結合而導致蛋白質的轉譯受抑制。我將提出一工作假說來解釋mRNA的定位與轉譯抑制的關係。
Messenger RNA localization is an important mechanism of differentiation in both germline and somatic cells. The Vgl mRNA is the best characterized system to study mRNA localization in Xenopus oocytes so far. This mRNA is expressed globally and then translocated to and anchored at the vegetal pole during oogenesis. The Vgl protein is also locally expressed at the vegetal pole, which may imply that the translation of Vgl mRNA is inhibited in other part of the oocyte. Nevertheless, it is not known whether the translation and mRNA localization processes are regulated coordinately. It has been shown that the p56 and p54 masking proteins, which can inhibit the translation of maternal mRNA, can interact with Vgl localization signal (LS) RNA specifically.
Using confocal microscopy, I showed that the mRNA of green fluorescent protein (GFP) was translated globally in Xenopus oocytes, but the chimeric mRNA of GFP and VglLS-RNA can only be expressed at the vegetal pole of oocytes. Furthermore, the crude preparation of p56 and p54 inhibited the translation of both the chimeric mRNA and the GFP mRNA in vitro, but the translation of the chimeric mRNA is preferentially suppressed. Dephosphorylated masking proteins, which failed to bind VgILS, also failed to suppress the translation of this chimeric mRNA. Therefore, the VglLS-RNA is sufficient to suppress the translation, possibly mediated by the interacting with p56 and p54. In other words, the VgILS is a cis-acting element for both mRNA localization and translation suppression in Xenopus oocytes.
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