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研究生:沈郁芳
研究生(外文):Yu-Fang Shen
論文名稱:ERK訊息路徑磷酸化與調控訊息RNA去頭蓋蛋白之分析
論文名稱(外文):Identification and Functional Characterization ofERK-Signal-Mediated Dcp1a Phosphorylation.
指導教授:張瀞仁張瀞仁引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:62
中文關鍵詞:訊息核醣核酸去頭蓋去頭蓋複合物Dcp1aDcp1a轉譯後修飾前脂肪細胞分化
外文關鍵詞:mRNA decappingdecapping complexDcp1aDcp1a posttranslational modificationpreadipocyte early differentiation
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摘要
訊息核醣核酸新陳代謝在調控基因的表現中為重要的一環。而去除訊息核醣核酸5’端頭蓋在一般訊息核醣核酸新陳代謝和某些特殊的代謝途徑上,都扮演重要的角色。哺乳纇細胞中Dcp1a和Dcp2所形成的去頭蓋複合物,其詳細機制和如何調控去頭蓋複合物的活性目前還不清楚。和酵母菌的Dcp1p比較起來,老鼠Dcp1a擔任輔因子而且在C端多了一段富含脯胺酸序列。因此,本研究想要了解在哺乳纇細胞的去頭蓋複合物中,Dcp1a到底扮演什麼角色。首先我們發現老鼠Dcp1a會和去頭蓋複合物中其他的成員DDX6和Edc3有交互作用,但是不包含Dcp2、Lsm4和PatL1△N′。接著我們去比對他們作用的區域,DDX6和Edc3都是作用於Dcp1a C端富含脯胺酸的序列上,但是當我們把這段序列去除,Dcp2卻可以和Dcp1a N端高度保留性EVH1區域作用。另外我們也觀察到Dcp1a在3T3-L1細胞早期分化時會受到磷酸化作用,而這活性是受到ERK訊息路徑的調控,但是它在細胞中的位置是不會受到磷酸化的影響。我們發現一旦Dcp1a被磷酸化,它會藉由促進和Dcp2的交互作用進一步刺激去頭蓋複合物的活性,但是它和其他的蛋白質例如:DDX6、Edc3和Edc4作用則不受影響。我們利用質譜儀找到了兩個磷酸化的點分別是絲胺酸315和絲胺酸319,這兩個點都位於Dcp1a C端富含脯胺酸序列中。我們利用點突變的技術,將315和319這兩個點都突變成天門冬胺酸或丙胺酸,去模擬有磷酸化或是沒有磷酸化的Dcp1a。接著把它們用免疫共沉澱的方法沉澱下來以進行in vitro去頭蓋的實驗,實驗發現,模擬有磷酸化狀態的Dcp1a可以藉由加強和Dcp2的交互作用而增強去頭蓋複合物的去頭蓋活性。我們的研究指出Dcp1a C端富含脯胺酸序列和轉譯後修飾,在調節去頭蓋複合物中擔任重要的一環。


ABSTRACT
Messenger RNA turnover plays an important role in the regulation of gene expression. Removal of the 5′ mRNA cap is an important step in both general mRNA turnover and specific mRNA decay pathways. The detailed Dcp1–Dcp2 decapping complex assembly in mammals and the regulation of decapping activity are still unclear. Compared to yeast Dcp1p, mouse Dcp1a which serves as a cofactor of decapping contains a large extra extension in the C-terminus. In this study, we aimed to explore the possible role of Dcp1a in decapping complex assembly in mammalian cells. First, we found mouse Dcp1a interacts with DDX6 and Edc3 but not Dcp2, Lsm4 or PatL1△N′. In our mapping analysis, Dcp1a interacts with DDX6 and Edc3 through its proline-rich C-terminal extension. Interestingly, when this region was deleted, the conserved EVH1 domain located in Dcp1a N-terminus could immunoprecipitate with Dcp2. On the other hand, in vivo and in vitro kinase assays confirmed the previous observation in 3T3-L1 preadipocyte early differentiation, which indicated that Dcp1a was phosphorylated by ERK signaling. The subcellular localization of Dcp1a was not affected by its phosphrylation status. In contrast, we observed Dcp1a phosphorylation appeared to activate decapping complexes by enhancing the interaction with Dcp2. Interaction between Dcp1a and other decapping regulators such as DDX6, Edc3 and Edc4 was not affected by its phosphorylation. Furthermore, two ERK-mediated phosphorylated sites at Ser 315 and Ser 319 that are located in Dcp1a C-terminus were identified by mass spectrometry analysis and demonstrated by site-direct mutagenesis combined with in vivo and in vitro kinase assay. Protein complexes immunoprecipitated by S315D/S319D phosphorylation-mimic mutant contained higher amount of Dcp2 and showed higher decapping efficiency than those by S315A/S319A mutant. Our findings suggest that the C-terminal domain of mouse Dcp1a and its post-translational modification play some roles in regulation of decapping machinery assembly.


CONTENTS
摘要……………………………………………………………………………..……….i
Abstract………………………………………………………………………...………iii
Contents………………………………………………………………………..………..v
Abbreviations………………………………………………………………….……….vii
I. Introduction
Messenger RNA Decay Pathways ………………………………………………..1
The mRNA decapping machinery ………………………………………………..3
Decapping enzyme ………………………………………………………….4
Regulators of decapping ……………………………………………………….5
Processing bodies …………………………………………….…………………...8
II. Materials and Methods
Plasmid constructs ………………………………………………………………..11
Site-directed mutagenesis ………………………………………………………...13
Cell culture ……………………………………………………………………….15
Coimmunoprecipitation Assays ………………………………………………….15
SDS-PAGE and Western Blotting ………………………………………………..16
Indirect Immunofluorescence and confocal microscopy ………………………...16
In vitro transcription ……………………………………………………………..17
In vitro decapping assay …………………………………………………………18
Expression and purification of recombinant protein .............................................19
In vitro phosphorylation assay ..............................................................................20

III. Results
Mouse Dcp1a interacts with DDX6, EDC3, but not Dcp2 ………………………21
Mapping of interacting domains of Dcp1a with DDX6, Edc3, and Dcp2 ……….21
ERK signaling pathway mediates Dcp1a phosphorylation ………………………23
Dcp1a phosphorylation enhances the activity of decapping complex …………...23
Dcp1a phosphorylation increases its interaction with Dcp2 …………………….24
Two phosphorylated sites by ERK signal at Ser 315 and Ser 319 are identified ..25
S315D/S319D mimic-phosphorylation Dcp1a mutant increases the interaction
with Dcp2 and enhances the decapping activity ...................................27
IV. Discussion ………….…………………………………………………………...…29
V. Figure
Figure 1. Mouse Dcp1a interacts with DDX6, Edc3, but not Dcp2 ……………..36
Figure 2. Mapping of interacting domains of Dcp1a with DDX6, Edc3, and
Dcp2 ……………………………………………………………………38
Figure 3. ERK signaling pathway mediates Dcp1a phosphorylation …………….41
Figure 4. Dcp1a phosphorylation enhances the activity of decapping complex ....42
Figure 5. Dcp1a phosphorylation enhances its interaction with Dcp2 …………...44
Figure 6. Two phosphorylated sites by ERK signal at Thr 197 and Ser 315 are
identified by mass spectrometry ……………………………………...…46
Figure 7. Phosphorylation assay of Dcp1a mutations in vivo and in vitro ………48
Figure 8. S315D/S319D mimic-phosphorylation Dcp1a mutant increases the
interaction with Dcp2 and enhances the decapping activity …………….50
Figure 9. This project proposes the model for decapping complex ……………..52
VI. Appendix
Appendix 1.Four kinds of mRNA decay pathway ………………………………53
Appendix 2. Messenger RNA decpping machinery and two kinds of dacapping
Enzyme …………………………………………………………….55
Appendix 3. Dcp2 together with its activators (Dcp1, Edc3, Rck, Heals, and Pat1)
……………………………………………………………………...56
Appendix 4. Schematic diagrams of yDcp1, hDcp1a and hDcp1b ………………..57
VII. Refferences ……………………………………………………………………....5 8


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