臺灣博碩士論文加值系統

第一部份 Pinin蛋白在mRNA代謝作用的弁酮膍s

Pinin蛋白最初被發現在上皮細胞的細胞間隙處，在胞橋小體(desmosome)和中間絲(intermediate filament)相接處，被認為與細胞間的結合有關。Pinin除了在胞橋小體(desmosome)這個位置之外，也存在於細胞核中，並且呈核斑結構(nuclear speckle)的形式分佈在細胞核中。核斑結構被認為是儲存或組裝剪接因子(splicing factors)的區域所在，剪接因子於此之後才被招募至mRNA前驅分子(pre-mRNA)轉錄(transcription)進行的位置，執行他們的弁遄A因此Pinin可能參與了剪接作用的調控。
我們實驗室之前的研究發現，Pinin是exon junction complex (EJC)的成分之一，而在mRNA剪接(splicing)的過程中，exon junction complex (EJC)是依附在mRNA上的分子。EJC的弁鈳Q推測是執行釵hmRNA後剪接代謝途徑(post-splicing mRNA metabolism pathway)的作用。基於此，我們希望能更進一步對Pinin做弁鄔吨尷R研究，特別著重在mRNA前驅分子的選擇性剪接(alternative splicing)與無義介導的mRNA降解(nonsense-mediated mRNA decay, NMD)上。於是，藉著RNA干擾技術(RNAi)，我們構築了可以表現短鏈髮夾式核醣核酸(shRNA)以抑制Pinin蛋白表現的干擾載體，pSUPER-siPnn。接著，我們使用E1A做為體內(in vivo)選擇性剪接的報告基因(reporter gene)，藉以評估Pinin在選擇性剪接具備的弁遄C結果發現，將Pinin的表現抑制後，並未對剪接作用的結果有重大影響，顯示Pinin並未參與5端剪接結合點的選擇性。除此之外，我們利用含有提前終止密碼子(premature termination codons, PTC)的beta-globin做為reporter mRNA，藉以評估Pinin在NMD作用中潛在的弁鄔囧丹漶C結果發現，NMD的機制並未受到影響。雖然我們希望透過這個研究找出Pinin在post-splicing mRNA metabolism pathway的潛在弁遄A但目前的結果尚未能完全證實我們最初的前提假設。


第二部分 人類乳突瘤病毒第五型 E2蛋白結合至Polo-like kinase的弁鈺敦Q

愈來愈多的研究顯示，在細胞分裂的過程中，Polo-like kinase (Plk)扮演非常重要而關鍵的調節角色。Plk屬於serine/threonine kinase的一種，最早被發現在Drosophila melanogaster的polo基因突變中，在此種突變的表現型(phenotype)發現無法正常進行有絲分裂(mitosis)。之後，Plk被發現存在於釵h真核生物中，具有四種形式(Plk1-4)，並且參與細胞分裂過程中多重弁鄋瑤楖`，包括entry into mitosis、bipolar spindle formation、chromosome segregation、cytokinesis等。在哺乳類動物中，Plk1被發現其表現量的差異可能與腫瘤的生成有關。比較四種形式的Plk胺基酸序列，在其胺基端(amino-terminal)具有高度保留性的蛋白質激酶弁鈰炩?protein kinase domain)。在Plk執行其弁鉈氶APlk的蛋白質激酶活性高低是一個重要的因素。另外，在其不具催化活性的羥基端(carboxyl-terminal)則含有一個共同的結構性弁鈰炩禲A稱為Polo-box。
在脊椎動物的Plk-family包含釵h成員，在本篇論文，我們挑選了哺乳類動物的Plk1作為研究對象。因為我們之前的研究發現Plk1能與人類乳突瘤病毒第五型(human papillomavirus type 5) E2蛋白(以下簡稱5E2)結合，其弁鄍i能有所關連。所以，我們在HeLa cell中，transiently表現了Plk1與myc tagged 5E2，並將這些細胞進行細胞週期的同步化(synchronization)，藉此獲得不同週期的HeLa cell。接著以免疫螢光染色法檢視Plk1與myc tagged 5E2的表現位置。我們的結果顯示，在5E2表現的情形下，Plk1在有絲分裂的晚期會被recruit到染色體上，顯示Plk1的弁鄏b有絲分裂晚期可能是幫助5E2離開染色體之用。同時，我們觀察到Plk1在有絲分裂早期，表現在spindle pole或kinetochore，在有絲分裂晚期，則表現在central spindle region或contractile ring。

Part I
Pnn (Pinin) is originally characterized based on its localization at epithelial cell junctions, desmosomes, in the convergence of intermediate filaments. In addition to its desmosomal localization, Pnn is also localized within the nucleus with a speckle distribution pattern. The speckles are considered to be the storage/assemble sites for splicing factors before they are recruited to the sites of nascent pre-mRNA transcription.
Our previous report showed that Pnn is a component of the exon junction complex, deposited on mRNA via splicing. EJC has been suggested to function in many post-splicing mRNA metabolism pathways. Thus, we wish to further investigate the potential function of Pnn in mRNA metabolism, specifically in pre-mRNA alternative splicing and in nonsense mediated mRNA decay (NMD). To this end, we used the pSUPER plasmid, from which RNAi-inducing shRNAs could be expressed to suppress Pnn expression. E1A, an in vivo alternative splicing minigene reporter, was then used to evaluate the role of Pnn in alternative splicing. Our results showed no significant difference in the splicing patterns when Pnn is suppressed, indicating that Pnn is not involved in 5’ splice site selection. In addition, the potential function of Pnn in NMD was also examined by measuring the in vivo stability of beta-globin reporter mRNA bearing premature stop codon. The results also showed no significant increase of the reporter level. Although through this project, we hope to obtain evidences implicating Pnn in post-transcriptional mRNA metabolism pathways, our data currently do not support our hypothesis.

Part II
Polo-like kinases (Plks) are increasingly recognized as key regulators of multiple events during cell division. These serine/threonine kinases were first described in mutants that failed to undergo a normal mitosis in Drosophila melanogaster (polo). Subsequently, Plks have been found in many eukaryotes, and have been shown to have key roles during entry into mitosis, bipolar spindle formation, chromosome segregation, and cytokinesis. It has been shown that alterations in Plk1 expression have been associated with tumorigenesis. Plks share a highly conserved protein kinase domain in the amino-terminal region, and the kinase activity of Plks is critical for various cellular functions of Plks. In addition to the kinase domain, Plks also contain common structural motifs called the Polo box domains in the noncatalytic carboxyl-terminal region, which is also essential for the functions of Plks.
Vertebrate species have several Plk-family members, and we focus on mammalian Plk1, since we discovered probable relationship between Plk1 and human papillomavirus type 5 E2 protein (5E2). We, thus, transiently expressed Plk1 and myc tagged 5E2 in HeLa cells, followed by synchronizing the transfected cells in various cell cycle stages. The localization of Plk1 and 5E2 were then examined by immunostaining. Our results indicated that Plk1 was recruited to chromosomes at late stage of mitosis in the presence of 5E2, indicating that Plk1 may function to dissociate 5E2 from mitotic chromosomes at the end of mitosis.

第一部分中文摘要……………………………………………………i
第二部分中文摘要……………………………………………………iii
第一部分英文摘要……………………………………………………v
第二部份英文摘要……………………………………………………vi
目錄……………………………………………………………………vii
圖目錄…………………………………………………………………xi
表目錄…………………………………………………………………xii

第一部份
一、前言…………………………………………………………………1
1.1 Pinin蛋白……………………………………………………………1
1.2 exon-exon junction complex (EJC)…………………………………2
1.3 nonsense-mediated mRNA decay (NMD)……………………………3
二、材料與方法…………………………………………………………7
2.1質體的構築 (plasmid construction)…………………………………7
2.2勝任細胞 (competent cell)的製備…………………………………9
2.3細胞培養 (cell culture)……………………………………………10
2.4細胞轉染 (transfection)……………………………………………10
2.5細胞萃取液的製備 (preparation of cell lysate)……………………11
2.6西方墨點法 (Western blot)…………………………………………11
2.7 RNA的萃取………………………………………………………13
2.8反轉錄-聚合酶連鎖反應 (Reverse-Transcriptase Polymerase Chain Reaction, RT-PCR)……………………………………………………14
2.9聚合酶連鎖反應 (Polymerase Chain Reaction, PCR)……………14
2.10北方墨點法 (Northern Blot)……………………………………15
三、結果………………………………………………………………18
3.1 以pSUPER RNAi System™構築的pSUPER-siPnn質體能成尼磻薈inin蛋白的表現……………………………………………………18
3.2 Pinin在alternative splicing的弁鈺敦Q…………………………18
3.3抑制Pinin蛋白的表現對nonsense-mediated mRNA decay (NMD)的影響…………………………………………………………………19
四、討論………………………………………………………………20
五、參考文獻…………………………………………………………21
附圖……………………………………………………………………24
附錄……………………………………………………………………28

第二部份
一、前言………………………………………………………………29
1.1 human papillomavirus (HPV)………………………………………29
1.2 HPV E2 protein……………………………………………………29
1.3 Polo-like kinase (Plk)………………………………………………30
二、材料與方法………………………………………………………34
2.1質體的構築 (plasmid construction)………………………………34
2.2細胞培養 (cell culture)……………………………………………34
2.3細胞轉染 (transfection)……………………………………………34
2.4免疫螢光染色法 (immunofluorescence staining)…………………35
2.5 double thymidine blocking…………………………………………36
2.6西方墨點法 (Western blot)…………………………………………37
三、結果………………………………………………………………39
3.1 transiently expressed wt-Plk1與ΔK-Plk1在細胞中的分佈情形…39
3.2檢視Polo-like kinase (Plk1)在Mitosis時的分佈情形……………39
3.3檢視transiently expressed HPV5 E2在HeLa cell中的localization………………………………………………………………39
3.4比較HeLa myc-5E2 stable clone與general HeLa中，Plk1的stability…………………………………………………………………40
四、討論………………………………………………………………41
五、參考文獻…………………………………………………………42
附圖……………………………………………………………………48
附表……………………………………………………………………53

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