利用細胞生物學策略分析 HC-Pro 如何作用並干擾重要核酸靜默反應元件之研究

Investigation of the HC-Pro interaction and interfering with critical components of RNA silencing through cell biology approach

陳芳英(Phuong Anh Tran)

指導教授 ： 林詩舜

國立臺灣大學/生物資源暨農學院/生物科技研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102054

摘要

Potyvirus 的基因靜默抑制子 HC-Pro 在抑制轉錄後基因靜默作用 (PTGS) 中起著重要的作用。然而，目前對於HC-Pro如何抑制微型核酸 (miRNA) 甲基化與 ARGONAUTE 1 (AGO1) 的加载以形成 RISC複合體 (RISC)的作用途徑尚未清楚。我们先前的研究表明HC-Pro 利用自噬途徑中的相關蛋白ATG8a介導降解ARGONAUTE1 (AGO1) 蛋白並抑制HEN1甲基轉移酶活性。本研究利用蛋白的螢光共位 (colocalization) 和福斯特能量共振轉移 (FRET) 研究in vivo細胞內蛋白質與蛋白質相互作用。我們的結果顯示 HC-Pro 与 AGO1 在Suppression body (S-body) 有間接相互作用。另外HC-Pro 和 ATG8a 有colocalization現象和相互作用，再次證明ATG8a 與HC-Pro 間接誘導 AGO1 降解有關。FRET 結果也顯示HC-Pro直接物理相互作用抑制 HEN1 甲基轉移酶活性，而未甲基化的微型核酸 (unMet-miRNA) 無法加載至 RISC 复合體。除此之外，HC-Pro 與Processing body (P-body) 標記物的mRNA-去頭蓋蛋白質2 (DCP2)有colocalization現象和相互作用。實驗結果顯示HC-Pro 在細胞質形成S-body。根據PTGS 分子如AGO1、HEN1、ATG8a 和 DCP2 的焦距大小結果，我們推測S-body會吸引這些蛋白進入。了解 S-body 和 PTGS 分子之間的關係讓我们更加理解 HC-Pro 如何抑制微型核酸途徑，并確認微型核酸被甲基化與加載到 AGO1 的位置。這些研究可闡明HEN1 如何喪失甲基化活性。進一步的研究可以顯示未甲基化的微型核酸如何誘導ATG8a自噬蛋白介導 AGO1 降解。綜合來說，這項研究有助于理解 PTGS 調控與植物和病毒之間的相互作用。

關鍵字

蛋白质-蛋白质相互作用 ； 基因靜默抑制子 ； 轉錄後基因靜默作用

並列摘要

The viral suppressor HC-Pro of potyvirus plays a crucial role in suppressing post transcriptional gene silencing (PTGS). However, how HC-Pro suppresses the microRNA (miRNA) methylation and loading into ARGONAUTE 1 (AGO1) for RNA-induced gene silencing complex (RISC) formation are still unclear. Our previous studies indicated that HC-Pro triggers ARGONAUTE1 (AGO1) degradation through Autophagy-related 8a (ATG8a)-mediated autophagic pathway and HC-Pro also inhibits HEN1 methyltransferase activity through direct interaction. In this project, we investigated the in vivo protein-protein interactions by checking colocalization and Förster resonance energy transfer (FRET) of fluorescently-labeled proteins with the use of confocal microscopy. Interstingly, our results revealed that HC-Pro indirectly interacted with AGO1 in S-body. Additionally, HC-Pro and ATG8a colocalized and interacted, once again confirmed that ATG8a was related to AGO1 degradation that indirectly triggered by HC-Pro. FRET output also indicated that HC-Pro inhibits miRNA methylation activity of HEN1 through direct physical interaction, resulting in un-methylated miRNAs (unMet-miRNA) that cannot be loaded into the RISC complex. Besides, HC-Pro and mRNA-decapping 2 (DCP2), which is processing body (P-body) marker, colocalized and interacted with each other. Moreover, HC-Pro can form the suppression body (S-body) in the cytoplasm. Further investigation of the size of PTGS components, such as AGO1, HEN1, ATG8a and DCP2 foci, suggesting that S-body attracts above mentioned protein into it. Understanding of the relationship between S-body and PTGS elements can give us a better insight on how does the HC-Pro inhibit the miRNA pathway, and confirm the location in which miRNA be methylated and loaded into AGO1. These findings can explain the lost of HEN1 methylation activity. Potentially, further study would unreveal how unMet-miRNA trigger ATG8a-mediated autophagic AGO1 degradation. Overall, this study contributes to the understanding of the PTGS regulation and plant-virus counteraction.

並列關鍵字

Protein-protein interaction ； TuMV ； HC-Pro ； PTGS ； AGO1 ； ATG8a ； DCP2 ； HEN1 ； S-body

參考文獻

Bhaumik, S. R. (2021). Fluorescence resonance energy transfer in revealing protein-protein interactions in living cells. Emerg Top Life Sci., 5, 49-59.

Bologna, N. G., Iselin, R., Abriata, L. A., Sarazin, A., Pumplin, N., Jay, F., Grentzinger, T., Dal Peraro, M., Voinnet, O. (2018). Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway. Mol Cell., 15, 709-719.

Braun, P., Gingras, A. C. (2012). History of protein-protein interactions: from egg-white to complex networks. Proteomics., 12, 1478-1498.

Carthew, R. W., Sontheimer, E. J. (2009). Origins and Mechanisms of miRNAs and siRNAs. Cell, 136, 642-655.

Castillo-González, C., Zhang, X. (2018). Transactivator: A New Face of Arabidopsis AGO1. Dev. Cell, 44, 277-279.