Part I. Functions of Pinin in mRNA metabolismPart II. Functional significance of the interaction between papillomavirus type 5 E2 protein and human polo-like kinase 1

• Main Authors: I-huan Liao, 廖奕桓
• Other Authors: Chin Li
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/45620580673656330997

碩士 === 國立中正大學 === 分子生物研究所 === 94 === 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.