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

第一部份
1.Ouyang, P. & Sugrue, S. P. Characterization of pinin, a novel protein associated with the desmosome-intermediate filament complex. J Cell Biol 135, 1027-42 (1996).
2.Brandner, J. M., Reidenbach, S. & Franke, W. W. Evidence that "pinin", reportedly a differentiation-specific desmosomal protein, is actually a widespread nuclear protein. Differentiation 62, 119-27 (1997).
3.Brandner, J. M., Reidenbach, S., Kuhn, C. & Franke, W. W. Identification and characterization of a novel kind of nuclear protein occurring free in the nucleoplasm and in ribonucleoprotein structures of the "speckle" type. Eur J Cell Biol 75, 295-308 (1998).
4.Wang, P., Lou, P. J., Leu, S. & Ouyang, P. Modulation of alternative pre-mRNA splicing in vivo by pinin. Biochem Biophys Res Commun 294, 448-55 (2002).
5.Li, C., Lin, R. I., Lai, M. C., Ouyang, P. & Tarn, W. Y. Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1. Mol Cell Biol 23, 7363-76 (2003).
6.Le Hir, H., Izaurralde, E., Maquat, L. E. & Moore, M. J. The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 19, 6860-9 (2000).
7.Le Hir, H., Moore, M. J. & Maquat, L. E. Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions. Genes Dev 14, 1098-108 (2000).
8.Le Hir, H., Gatfield, D., Izaurralde, E. & Moore, M. J. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J. 20, 4987-97 (2001).
9.Lu, S. & Cullen, B. R. Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells. Rna 9, 618-30 (2003).
10.Nott, A., Meislin, S. H. & Moore, M. J. A quantitative analysis of intron effects on mammalian gene expression. Rna 9, 607-17 (2003).
11.Lejeune, F. & Maquat, L. E. Mechanistic links between nonsense-mediated mRNA decay and pre-mRNA splicing in mammalian cells. Curr Opin Cell Biol 17, 309-15 (2005).
12.Tange, T. O., Nott, A. & Moore, M. J. The ever-increasing complexities of the exon junction complex. Curr Opin Cell Biol 16, 279-84 (2004).
13.Tange, T. O., Shibuya, T., Jurica, M. S. & Moore, M. J. Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core. Rna 11, 1869-83 (2005).
14.Maquat, L. E. & Carmichael, G. G. Quality control of mRNA function. Cell 104, 173-6 (2001).
15.Hillman, R. T., Green, R. E. & Brenner, S. E. An unappreciated role for RNA surveillance. Genome Biol 5, R8 (2004).
16.Morrison, M., Harris, K. S. & Roth, M. B. smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans. Proc Natl Acad Sci U S A 94, 9782-5 (1997).
17.Maquat, L. E. Nonsense-mediated mRNA decay: a comparative analysis of different species. Curr Genomics 5, 175-190 (2004).
18.Nagy, E. & Maquat, L. E. A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. Trends Biochem Sci 23, 198-9 (1998).
19.Maquat, L. E. & Li, X. Mammalian heat shock p70 and histone H4 transcripts, which derive from naturally intronless genes, are immune to nonsense-mediated decay. Rna 7, 445-56 (2001).
20.Brocke, K. S., Neu-Yilik, G., Gehring, N. H., Hentze, M. W. & Kulozik, A. E. The human intronless melanocortin 4-receptor gene is NMD insensitive. Hum Mol Genet 11, 331-5 (2002).
21.Leeds, P., Peltz, S. W., Jacobson, A. & Culbertson, M. R. The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev 5, 2303-14 (1991).
22.Hodgkin, J., Papp, A., Pulak, R., Ambros, V. & Anderson, P. A new kind of informational suppression in the nematode Caenorhabditis elegans. Genetics 123, 301-13 (1989).
23.Lykke-Andersen, J., Shu, M. D. & Steitz, J. A. Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon. Cell 103, 1121-31 (2000).
24.Maquat, L. E. Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat Rev Mol Cell Biol 5, 89-99 (2004).

第二部份
1.Harwood, C. A., McGregor, J. M., Proby, C. M. & Breuer, J. Human papillomavirus and the development of non-melanoma skin cancer. J Clin Pathol 52, 249-53 (1999).
2.Jenson, A. B., Geyer, S., Sundberg, J. P. & Ghim, S. Human papillomavirus and skin cancer. J Investig Dermatol Symp Proc 6, 203-6 (2001).
3.Wentzensen, N., Vinokurova, S. & von Knebel Doeberitz, M. Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract. Cancer Res 64, 3878-84 (2004).
4.Burd, E. M. Human papillomavirus and cervical cancer. Clin Microbiol Rev 16, 1-17 (2003).
5.Deau, M. C., Favre, M., Jablonska, S., Rueda, L. A. & Orth, G. Genetic heterogeneity of oncogenic human papillomavirus type 5 (HPV5) and phylogeny of HPV5 variants associated with epidermodysplasia verruciformis. J Clin Microbiol 31, 2918-26 (1993).
6.Haller, K., Stubenrauch, F. & Pfister, H. Differentiation-dependent transcription of the epidermodysplasia verruciformis-associated human papillomavirus type 5 in benign lesions. Virology 214, 245-55 (1995).
7.Steger, G. & Corbach, S. Dose-dependent regulation of the early promoter of human papillomavirus type 18 by the viral E2 protein. J Virol 71, 50-8 (1997).
8.Band, V., De Caprio, J. A., Delmolino, L., Kulesa, V. & Sager, R. Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells. J Virol 65, 6671-6 (1991).
9.Munger, K. et al. Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 8, 4099-105 (1989).
10.Stirdivant, S. M. et al. Human papillomavirus type 16 E7 protein inhibits DNA binding by the retinoblastoma gene product. Mol Cell Biol 12, 1905-14 (1992).
11.Ronco, L. V., Karpova, A. Y., Vidal, M. & Howley, P. M. Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes Dev 12, 2061-72 (1998).
12.Park, J. S. et al. Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis. J Biol Chem 275, 6764-9 (2000).
13.Berg, M. & Stenlund, A. Functional interactions between papillomavirus E1 and E2 proteins. J Virol 71, 3853-63 (1997).
14.Van Tine, B. A. et al. Human papillomavirus (HPV) origin-binding protein associates with mitotic spindles to enable viral DNA partitioning. Proc Natl Acad Sci U S A 101, 4030-5 (2004).
15.You, J., Croyle, J. L., Nishimura, A., Ozato, K. & Howley, P. M. Interaction of the bovine papillomavirus E2 protein with Brd4 tethers the viral DNA to host mitotic chromosomes. Cell 117, 349-60 (2004).
16.Lehman, C. W., King, D. S. & Botchan, M. R. A papillomavirus E2 phosphorylation mutant exhibits normal transient replication and transcription but is defective in transformation and plasmid retention. J Virol 71, 3652-65 (1997).
17.Lai, M. C., Teh, B. H. & Tarn, W. Y. A human papillomavirus E2 transcriptional activator. The interactions with cellular splicing factors and potential function in pre-mRNA processing. J Biol Chem 274, 11832-41 (1999).
18.Fenton, B. & Glover, D. M. A conserved mitotic kinase active at late anaphase-telophase in syncytial Drosophila embryos. Nature 363, 637-40 (1993).
19.Glover, D. M., Hagan, I. M. & Tavares, A. A. Polo-like kinases: a team that plays throughout mitosis. Genes Dev 12, 3777-87 (1998).
20.Glover, D. M., Ohkura, H. & Tavares, A. Polo kinase: the choreographer of the mitotic stage? J Cell Biol 135, 1681-4 (1996).
21.Kitada, K., Johnson, A. L., Johnston, L. H. & Sugino, A. A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5. Mol Cell Biol 13, 4445-57 (1993).
22.Elia, A. E. et al. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell 115, 83-95 (2003).
23.Lowery, D. W., Mohammad, D. H., Elia, A. E. & Yaffe, M. B. The Polo-box domain: a molecular integrator of mitotic kinase cascades and Polo-like kinase function. Cell Cycle 3, 128-31 (2004).
24.Jang, Y. J., Ma, S., Terada, Y. & Erikson, R. L. Phosphorylation of threonine 210 and the role of serine 137 in the regulation of mammalian polo-like kinase. J Biol Chem 277, 44115-20 (2002).
25.Pak, D. T. & Sheng, M. Targeted protein degradation and synapse remodeling by an inducible protein kinase. Science 302, 1368-73 (2003).
26.Barr, F. A., Sillje, H. H. & Nigg, E. A. Polo-like kinases and the orchestration of cell division. Nat Rev Mol Cell Biol 5, 429-40 (2004).
27.Golsteyn, R. M. et al. Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5. J Cell Sci 107 ( Pt 6), 1509-17 (1994).
28.Simmons, D. L., Neel, B. G., Stevens, R., Evett, G. & Erikson, R. L. Identification of an early-growth-response gene encoding a novel putative protein kinase. Mol Cell Biol 12, 4164-9 (1992).
29.Ma, S., Liu, M. A., Yuan, Y. L. & Erikson, R. L. The serum-inducible protein kinase Snk is a G1 phase polo-like kinase that is inhibited by the calcium- and integrin-binding protein CIB. Mol Cancer Res 1, 376-84 (2003).
30.Donohue, P. J., Alberts, G. F., Guo, Y. & Winkles, J. A. Identification by targeted differential display of an immediate early gene encoding a putative serine/threonine kinase. J Biol Chem 270, 10351-7 (1995).
31.Xie, S. et al. Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2. Cell Cycle 1, 424-9 (2002).
32.Habedanck, R., Stierhof, Y. D., Wilkinson, C. J. & Nigg, E. A. The Polo kinase Plk4 functions in centriole duplication. Nat Cell Biol 7, 1140-6 (2005).
33.do Carmo Avides, M., Tavares, A. & Glover, D. M. Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes. Nat Cell Biol 3, 421-4 (2001).
34.Ohkura, H., Hagan, I. M. & Glover, D. M. The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes Dev 9, 1059-73 (1995).
35.Sanchez, Y. et al. Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms. Science 286, 1166-71 (1999).
36.Roshak, A. K. et al. The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase. Cell Signal 12, 405-11 (2000).
37.Yuan, J. et al. Cooperative phosphorylation including the activity of polo-like kinase 1 regulates the subcellular localization of cyclin B1. Oncogene 21, 8282-92 (2002).
38.Descombes, P. & Nigg, E. A. The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts. EMBO J. 17, 1328-35 (1998).
39.Carmena, M. et al. Drosophila polo kinase is required for cytokinesis. J Cell Biol 143, 659-71 (1998).
40.Smits, V. A. et al. Polo-like kinase-1 is a target of the DNA damage checkpoint. Nat Cell Biol 2, 672-6 (2000).
41.Wolf, G. et al. Polo-like kinase: a novel marker of proliferation: correlation with estrogen-receptor expression in human breast cancer. Pathol Res Pract 196, 753-9 (2000).
42.Yuan, J. et al. Polo-like kinase, a novel marker for cellular proliferation. Am J Pathol 150, 1165-72 (1997).
43.Takai, N. et al. Expression of polo-like kinase in ovarian cancer is associated with histological grade and clinical stage. Cancer Lett 164, 41-9 (2001).
44.Kneisel, L. et al. Expression of polo-like kinase (PLK1) in thin melanomas: a novel marker of metastatic disease. J Cutan Pathol 29, 354-8 (2002).
45.Wolf, G. et al. Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer. Oncogene 14, 543-9 (1997).