分析CNBP精胺酸甲基化及其對於CNBP細胞內分佈與RNA交互作用之影響

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 博碩士論文 > Item 310902500/2596

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题名:	分析CNBP精胺酸甲基化及其對於CNBP細胞內分佈與RNA交互作用之影響 Arginine methylation of CNBP and its effect on subcellular localization and RNA interaction.
作者:	張嘉雲 Mariann Chia Yuan,Chang
贡献者:	中山醫學大學:醫學科技學院;生物醫學科學學系碩士班;李娟
关键词:	精胺酸甲基化 CNBP
日期:	2010-07-27
上传时间:	2010-11-05T07:35:18Z (UTC)
摘要:	Cellular nucleic acid binding protein (CNBP)為參與調控轉錄及轉譯之單股核酸結合蛋白質。在結構上，CNBP有七個Cys-Cys-His-Cys (CCHC) 鋅手指(zinc finger domains)和一個arginine/glycine (RG) rich區域。其RG rich區域與蛋白質精胺酸甲基轉移(PRMT)普遍作用的GAR (glycine and arginine rich)區域很相似。實驗室之前的研究顯示，辨識精胺酸甲基化的抗體可辨識全長的FLAG-CNBP蛋白質，然而RG rich deletion(△RG)的FLAG-CNBP則無法被辨識。放射性in vitro蛋白質甲基化的實驗顯示，GST-CNBP重組蛋白質可以被PRMT1甲基化，但是GST-CNBP(△RG)重組蛋白卻無法被甲基化。此外，免疫沉澱(immunoprecipitation)的實驗中，PRMT1會隨著CNBP被共同沉澱下來。本篇研究經由GST pull-down實驗證實，PRMT1和CNBP為直接的交互作用。已知蛋白質精胺酸甲基化涉及許多RNA結合蛋白質在細胞中表現位置的調控，我們想探討HeLa細胞以AdOx抑制蛋白質甲基化後，是否對CNBP在細胞中的表現位置造成影響。透過subcellular fractionation，免疫螢光染色，轉殖入FLAG-CNBP或FLAG-CNBP(△RG)實驗，我們發現不論是細胞處理AdOx或是RG deletion與否，CNBP均主要表現在細胞質。由此可見，CNBP的RG rich區域和蛋白質精胺酸甲基化並不影響CNBP在細胞中的表現位置。進一步，為了探討精胺酸甲基化會否影響CNBP與RNA的結合能力，我們準備了四種CNBP蛋白，分別為：(1)全長、(2) RG rich deletion、(3) RK mutation、 (4)甲基化後的CNBP。RNA親和力層析的實驗顯示，帶有RG rich deletion的CNBP蛋白幾乎喪失與RNA結合的能力，然而帶有RK mutation的CNBP蛋白仍保有部分與RNA結合的能力。此外，相較於沒有甲基化的CNBP蛋白，甲基化後的CNBP蛋白的RNA結合能力有較強的趨勢。這些初步的研究結果有助於了解精胺酸甲基化對於CNBP蛋白的RNA結合能力的調控。 Cellular nucleic acid binding protein (CNBP) contains seven Cys-Cys-His-Cys (CCHC) zinc finger domains and an arginine/glycine (RG) rich region. CNBP is a single-stranded nucleic acid binding protein involved in transcriptional and translational regulations. The RG rich region of CNBP is homologous to the typical protein arginine methyltransferase (PRMT) substrate domain. From previous studies of our laboratory, affinity purified wild type FLAG-CNBP could be recognized by a methylarginine-specific antibody whereas the FLAG-CNBP with an RG deletion could not be recognized. In vitro methylation reaction revealed that recombinant GST-CNBP protein could be methylated by recombinant PRMT1 whereas the RG-deleted GST-CNBP protein could not be modified. Furthermore, PRMT1 co-immunoprecipitated with CNBP. In this study, I confirmed the direct interaction of PRMT1 with CNBP by performing GST pull-down assay. As protein arginine methylation is involved in the regulation of subcellular localization of some RNA-binding proteins, we examined whether the localization of CNBP might be affected by the treatment of an indirect methyltransferase inhibitor AdOx. Subcellular localization of CNBP was analyzed by subcellular fractionation, immunofluorescent analyses, and transfection of plasmids expressing FLAG-CNBP or FLAG-CNBP with an RG deletion. CNBP protein appeared in the cytoplasmic fractions of HeLa cells and the localization was not affected by AdOx treatment. Immunofluorescent assay showed that both endogenous and exogenous CNBP proteins were mainly localized in the cytoplasm and not affected by AdOx treatment. Furthermore, wild type CNBP and the RG deletion CNBP mutant both localized at the cytoplasm suggesting that the RG rich region and protein arginine methylation does not affect CNBP subcellular localization. To test the effects of arginine methylation on the RNA-binding ability of CNBP, we prepared a full-length CNBP, two CNBP mutants (the RG rich deletion and the RK mutation) and in vitro methylated CNBP. RNA affinity chromatography showed that CNBP RG rich deletion mutant had almost no RNA-binding ability whereas CNBP RK mutation mutant maintained a weak RNA-binding ability. Furthermore, methylated CNBP might have a slightly stronger RNA-binding ability than unmethylated full-length CNBP. These preliminary data provided an insight to identifying arginine methylation regulation on CNBP RNA-binding ability.
URI:	https://ir.csmu.edu.tw:8080/handle/310902500/2596
显示于类别:	[生物醫學科學學系暨碩士班] 博碩士論文

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