DEAD-box Protein 6的分子生物學探討

Abstract

對生物而言，受控制的基因表現是至關重要的。基因表現的控制部分有賴後轉錄(Post-transcriptional)調控者，包括DEAD-box protein 6 (DDX6)。DDX6最廣為所知的是它參與RNA代謝(RNA turnover)以及轉譯抑制(translational repression)。本研究中，我們關注DDX6在基因表現調控中所扮演的角色與其在細胞內的分佈、分佈的調控機制、及其生物意義。於第一章，我們闡述了DDX6於基因表現及其調控中的意義，並由此展開DDX6的基礎生化性質、在細胞內的功能、以及其對表型的(phenotypic)影響。第二章包括我們對於DDX6的細胞核質(nucleocytoplasmic)分佈的研究。深入地檢視現行的假說，並以分子生物以及蛋白結構的證據予以反駁。我們的結果更顯示DDX6可以藉由另外兩種並存的機制分布到細胞核與細胞質之中。第三章描述我們對於DDX6的基因表現調控的研究。我們利用整合性的功能性基因體學的方法，來探討DDX6對於核糖核酸 (RNA)及蛋白質表現的調控以及其規模與侷限。我們首先指出DDX6在細胞內能結合信使核糖核酸(messenger RNA)以及長片段非編碼核糖核酸(long noncoding RNA)。不若一般的期待，我們發現在HeLa細胞株中DDX6並不顯著地影響整體的核糖核酸及蛋白質表現量。相反地，我們觀察到DDX6調控基因表現雜訊(gene expression noise)的證據。因此，我們的實驗結果提供對於DDX6的基因表現調控的新觀點。第四章描述了我們對於DDX6的入核機制更進一步的實驗結果。這些結果或能引導未來的研究。

Well-tuned gene expression is essential for life and is in part secured by the post-transcriptional regulators. DEAD-box protein 6 (DDX6) is one such regulator. DDX6 is best known for its participation in RNA turnover and translational repression. In this study, we focus on the roles of DDX6 in regulating gene expression and on the subcellular localization of DDX6, and its regulation, as well as its functional implications. In the first chapter, we elaborated a logical sequence to locate the niche of DDX6 in the context of gene expression and regulation. On this basis, we further expounded the fundamental biochemical properties of DDX6, its functional roles in the cells, and their phenotypic consequences. The second chapter includes our investigation on the governing mechanism of the nucleocytoplasmic distribution of DDX6. We scrutinized the working hypothesis and refuted it with molecular and structural evidence. We further demonstrated that two parallel mechanisms direct the nucleocytoplasmic distribution of DDX6. The third chapter includes our investigation on the regulation of gene expression by DDX6. We adopted an integrative functional genomics approach to investigate the size and the extent of the DDX6-mediated regulation on RNA and protein expression. We first demonstrated that DDX6 binds both the messenger RNA and the long noncoding RNA. In contrast our original expectation, we did not observe significant regulation over the RNA or the protein expression levels by DDX6 in the HeLa cell line. Notwithstanding, we provided evidence that DDX6 regulates gene expression noise. Thus, our results provide a new perspective on the gene regulation by DDX6. The fourth chapter includes the further experimental results related to the nuclear entry mechanism of DDX6. The results described therein may guide further investigations in the future.