病毒抑制子HC-Pro對於HEN1功能及甲基轉移酶活性之研究

Abstract

植物基因靜默系統在基因調控、各種生物和發育過程以及諸如防禦病毒等逆境反應中發揮著廣泛的作用。該系統的小核醣核酸 （sRNA）含量藉由調整其合成和代謝率來嚴格調節。3' 末端核醣的 2'-O-甲基化是為小核醣核酸提供穩定性的主要過程，甲基轉移酶 （HEN1） 扮演此至關重要的角色。在本論文中，我們比較並分析被子植物阿拉伯芥HEN1（AtHEN1）和苔蘚植物地錢 (Marchantia polymorpha) HEN1（MpHEN1）的異同處。我們的體內及體外結果顯示 MpHEN1 活性與 AtHEN1 相當，並且它們對雙鏈體微型核醣核酸 (miRNA duplex) 的特異性保持一致。此外，系統發育樹和多重氨基酸序列比對凸顯了不同植物家族HEN1之間保守區域的分子。另外我們也分析在不同物種界中 HEN1 的 MTase 區域以了解蛋白質的相關性。蕪菁嵌紋病毒 (TuMV) 的 P1/HC-Pro 是一種已知的 RNA 靜默抑制因子，也已知可抑制 HEN1 對於 sRNA 的甲基化。在本研究中，我們發現 HC-Pro 可與 AtHEN1物理性交互作用並抑制其甲基化活性。此外，HC-Pro 上的 FRNK 是兩個蛋白相互作用和抑制能力重要的區域。體外 EMSA 數據顯示 TuMV 的 GST-HC-Pro 缺乏 sRNA 雙鏈體結合能力。令人驚訝的是，隨著TuMV HC-Pro 劑量提升可增加對MpHEN1 活性的抑制。未來可針對 HEN1 和不同種病毒 HC-Pros 的相互作用機制研究，增進我們對病毒抑制因子的認識。

Plant gene silencing system plays a broad role in gene regulation, various biological and developmental processes, and stress responses such as defense against viruses. The system's small RNA (sRNA) levels are tightly regulated by adjusting their synthesis and turnover rates. The 2′-O-methylation of the 3′ terminal ribose is a major process that provides stability to the small RNAs which is facilitated by HEN1. In this thesis, we analyzed the Arabidopsis thaliana HEN1 (AtHEN1), an angiosperm as well as Marchantia polymorpha HEN1 (MpHEN1) which is a bryophyte at different levels. Our in vivo and in vitro data have shown MpHEN1 activity being comparable with AtHEN1, and their substrate specificity towards duplex miRNA remained consistent. Furthermore, the phylogenetic tree and multiple alignment highlighted the conserved molecular evolution among HEN1 of different plants family. The MTase domain of HEN1 which is found across different kingdom of species is analyzed to understand the relatedness of the protein. The P1/HC-Pro of the turnip mosaic virus (TuMV) is a known RNA silencing suppressor and is also known to inhibit the HEN1 mediated methylation of sRNAs. In this study, we have found that HC-Pro physically interacts with AtHEN1 inhibiting the methylation activity of the latter. Moreover, the FRNK motif of HC-Pro plays a significant role in this interaction and inhibition ability of the protein. The in vitro EMSA data indicates that GST-HC-Pro of TuMV lacks sRNA duplex-binding ability. Surprisingly, TuMV HC-Pro also inhibits MpHEN1 activity in a dosage-dependent manner. Further investigations on understanding interaction mechanism of HEN1 and various potyviral HC-Pros can advance our knowledge of viral suppressors.