研究發現有九種人類疾病與丙胺酸(alanine)鏈的擴展相關。其中八種聚丙胺酸(polyA)疾病的蛋白是轉錄因子(transcription factor)，並在發育和分化過程中發揮重要作用。轉錄因子含有細胞核定位訊號（nuclear localization signal; NLS）,可使其定位到細胞核並與DNA序列結合調控轉錄。因此，轉錄因子的核定位是至關重要的。研究指帶有polyA鏈的疾病蛋白主要定位在細胞質而不在細胞核裡。因此，我假設polyA疾病蛋白中的polyA鏈擴展區域帯有核輸出訊號（nuclear export signal; NES）,使polyA疾病蛋白從細胞核輸出到細胞質。所以，細胞質中的疾病蛋白質不能啟動轉錄。為了驗證這一假說，我使用核輸出報告蛋白來代表定位於細胞核內的polyA疾病蛋白。我的實驗結果顯示擴展polyA鏈使得核輸出報告蛋白定位於細胞質。這與polyA疾病蛋白的研究結果一致。再者,我運用光漂白期螢光消退(Fluorescence Loss in Photobleaching; FLIP)核輸出實驗和光激發螢光蛋白(Photoactivatable-GFP; PA-GFP)核輸出實驗來進一步証明在活細胞中存在擴展polyA鏈核輸出現象。我運用谷胱甘肽巯基轉移酶(glutathione-S-transferase; GST)沉澱法証實真核翻譯延長因子1A(eukaryotic translation elongation 1 alpha; EEF1A)是一個擴展polyA鏈的互作因子. 最後，我用小片段干擾RNA(siRNA)誘發的基因沈默確定了EEF1A與擴展polyA鏈的核輸出相關。綜上所述,我的研究証實了擴展polyA鏈是一類新的NES並且其核輸出是由EEF1A參與的未証實的輸出通路所介導的。通過識別其他能調節polyA疾病蛋白核輸出的互作因子，我們將會進一步了解polyA疾病的發病機理。 / The expansion of alanine tracts in disease proteins is found to be associated with nine human diseases. Eight out of nine polyalanine (polyA) disease proteins are transcription factors which play important roles during development and differentiation. Transcription factors contain nuclear localization signals (NLS) that direct them to the nucleus and bind to specific regulatory DNA sequences to regulate transcription. Therefore, nuclear localization is essential for transcription factors to function. Intriguingly, it has been reported that the expanded polyA disease proteins primarily localize in the cytoplasm. Therefore I hypothesize that the expansion of polyA domain in the disease proteins generates a nuclear export signal (NES) which directs nuclear export of polyA disease proteins. As a result, cytoplasmic expanded polyA disease proteins cannot initiate transcription. To test this hypothesis, I first used a nuclear export reporter protein to represent the polyA disease proteins that localized in the nucleus. The insertion of expanded polyA tract conferred cytoplasmic localization of the nuclear export reporter protein as previously reported in polyA disease proteins. I further made use of Fluorescence Loss in Photobleaching (FLIP) Nuclear Export Assay and Photoactivatable-GFP (PA-GFP) Nuclear Export Assay to illustrate the nuclear export of expanded polyA protein in living cells. Finally, by means of glutathione-S-transferase (GST) pull-down assay followed by mass spectrometry, I identified eukaryotic translation elongation factor 1 alpha (EEF1A) as a novel interacting partner of expanded polyA tract and that EEF1A is involved in expanded polyA protein’s nuclear export by means of a siRNA-mediated gene silencing experiment. Taken together, my study identified expanded polyA tract as a novel class of NES and its nuclear export is mediated by an unidentified export pathway involving EEF1A. Through the identification of other factors that modulate nuclear export of polyA disease proteins, we will have a better understanding of the pathogenesis of polyA diseases. / Detailed summary in vernacular field only. / Ng, Ka Lam. / "November 2012." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 108-112). / Abstracts also in Chinese. / ABSTRACT --- p.i / ABSTRACT (Chinese version) --- p.iii / ACKNOWLEDGEMENTS --- p.iv / LIST OF ABBREVIATIONS --- p.v / LIST OF TABLES --- p.ix / LIST OF FIGURES --- p.xi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Polyalanine diseases --- p.1 / Chapter 1.1.1 --- Overview of polyalanine diseases --- p.1 / Chapter 1.1.2 --- Polyalanine tract expansion in transcription factors --- p.1 / Chapter 1.2 --- Nuclear export in eukaryotic cells --- p.5 / Chapter 1.2.1 --- Overview of nucleocytoplasmic transport --- p.5 / Chapter 1.2.2 --- Classical exportins and nuclear export signal --- p.6 / Chapter 1.2.3 --- Non-classical exportins and nuclear export signal --- p.6 / Chapter 1.2.4 --- Role of eukaryotic translation elongation factor alpha 1 in protein nuclear export --- p.7 / Chapter 1.3 --- Aim of study --- p.8 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Molecular cloning of plasmid constructs --- p.9 / Chapter 2.1.1 --- Annealing of double-stranded oligonucleotides --- p.13 / Chapter 2.1.2 --- Phosphorylation at 5' position of double-stranded oligonucleotides --- p.13 / Chapter 2.1.3 --- Polymerase chain reaction --- p.14 / Chapter 2.1.4 --- Restriction enzyme digestion of cloning vector and insert sequence --- p.15 / Chapter 2.1.5 --- Removal of 5' phosphate groups from cloning vector --- p.15 / Chapter 2.1.6 --- DNA gel electrophoresis --- p.16 / Chapter 2.1.7 --- Gel extraction and purification of DNA --- p.16 / Chapter 2.1.8 --- DNA ligation --- p.16 / Chapter 2.1.9 --- Bacterial transformation --- p.17 / Chapter 2.1.10 --- Validation of positive clones by polymerase chain reaction --- p.17 / Chapter 2.1.11 --- Plasmid preparation --- p.19 / Chapter 2.2 --- RNA extraction and reverse transcription polymerase chain reaction --- p.19 / Chapter 2.2.1 --- RNA extraction from cell lysate --- p.19 / Chapter 2.2.2 --- Reverse transcription-polymerase chain reaction --- p.20 / Chapter 2.3 --- Double stranded RNA-mediated gene silencing in Drosophila melanogaster Schneider cell line --- p.21 / Chapter 2.3.1 --- Maintenance of Drosophila melanogaster Schneider cells --- p.21 / Chapter 2.3.2 --- in vitro transcription of RNA --- p.22 / Chapter 2.3.3 --- Double stranded RNA purification --- p.22 / Chapter 2.3.4 --- Double stranded RNA bathing condition --- p.22 / Chapter 2.3.5 --- Transfection condition --- p.23 / Chapter 2.3.6 --- Knockdown efficiency --- p.23 / Chapter 2.4 --- Small interfering RNA-mediated gene silencing in Human Embryonic Kidney 293FT cells --- p.25 / Chapter 2.4.1 --- Maintenance of Human Embryonic Kidney 293FT cells --- p.25 / Chapter 2.4.2 --- Small interfering RNAs used in this study --- p.25 / Chapter 2.4.3 --- Transfection condition --- p.26 / Chapter 2.4.4 --- Knockdown efficiency --- p.27 / Chapter 2.5 --- Expression and purification of recombinant proteins in Escherichia coli --- p.28 / Chapter 2.5.1 --- Bacterial transformation --- p.28 / Chapter 2.5.2 --- Induction of recombinant protein expression --- p.29 / Chapter 2.5.3 --- Purification of recombinant proteins --- p.29 / Chapter 2.6 --- Glutathione S-transferase pull-down assay --- p.30 / Chapter 2.7 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.31 / Chapter 2.7.1 --- Protein extraction from cell lysate --- p.31 / Chapter 2.7.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.31 / Chapter 2.7.3 --- Coomassie Blue staining --- p.32 / Chapter 2.7.4 --- Silver staining --- p.33 / Chapter 2.8 --- Western blotting --- p.33 / Chapter 2.8.1 --- Electro-transfer --- p.33 / Chapter 2.8.2 --- Immunoblotting --- p.34 / Chapter 2.9 --- Fluorescence microscopy --- p.36 / Chapter 2.10 --- Confocal microscopy and time-lapse imaging --- p.36 / Chapter 2.10.1 --- General setting of time-lapse imaging --- p.36 / Chapter 2.10.2 --- Photoactivatable-GFP Nuclear Export Assay --- p.37 / Chapter 2.10.3 --- Fluorescence Lost in Photobleaching Nuclear Export Assay --- p.38 / Chapter 2.11 --- Reagents and Buffers --- p.39 / Chapter 2.11.1 --- Molecular cloning --- p.39 / Chapter 2.11.2 --- RNA extraction and reverse transcription polymerase chain reaction --- p.41 / Chapter 2.11.3 --- Bacterial culture --- p.41 / Chapter 2.11.4 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.43 / Chapter 2.11.5 --- Immunoblotting --- p.46 / Chapter 2.11.6 --- Expression and purification of recombinant proteins in Escherichia coli --- p.48 / Chapter 2.11.7 --- Cell culture --- p.49 / Chapter 2.11.8 --- Fluorescence microscopy --- p.50 / Chapter Chapter 3 --- Expanded Polyalanine Tract Possesses Nuclear Export Activity / Chapter 3.1 --- Expanded polyalanine tract conferred cytoplasmic localization to the nuclear export reporter protein --- p.51 / Chapter 3.1.1 --- Principle of the Rev Nuclear Export Assay --- p.52 / Chapter 3.1.2 --- Selection of polyalanine tract for the Rev Nuclear Export Assay --- p.55 / Chapter 3.1.3 --- The expanded polyalanine tract conferred cytoplasmic localization to the nuclear export reporter protein --- p.57 / Chapter 3.2 --- Expanded polyalanine tract conferred nuclear export activity on the nuclear export reporter protein by Fluorescence Loss in Photobleaching Nuclear Export Assay --- p.60 / Chapter 3.2.1 --- Principle of the Fluorescence Loss in Photobleaching Nuclear Export Assay --- p.60 / Chapter 3.2.2 --- Fluorescence intensity loss in the nuclear compartment indicates that expanded polyalanine tract possesses nuclear export activity --- p.62 / Chapter 3.3 --- The nuclear export of expanded polyalanine tract-containing nuclear export reporter protein was confirmed by Photoactivatable-GFP Nuclear Export Assay --- p.65 / Chapter 3.3.1 --- Expression of photoactivatable-GFP constructs in HEK293 cell line --- p.65 / Chapter 3.3.2 --- Principle of photoactivatable GFP (PA-GFP) Nuclear Export Assay --- p.67 / Chapter 3.3.3 --- The nuclear export activity of expanded polyalanine tract-containing nuclear export reporter protein was captured by Photoactivatable-GFP Nuclear Export Assay --- p.67 / Chapter 3.4 --- Discussion --- p.70 / Chapter Chapter 4 --- Identification of Genes That Are Involved in Nuclear Export of Expanded Polyalanine Tract-Containing Protein / Chapter 4.1 --- Screening of genes involved in nuclear export of expanded polyalanine tract containing nuclear export reporter protein in Drosophila melanogaster Schneider cells using a double-stranded RNA-mediated gene knockdown approach --- p.75 / Chapter 4.2 --- Screening of genes involved in nuclear export of expanded polyalanine tract in HEK293 cells using small interfering RNA-mediated gene knockdown approach --- p.80 / Chapter 4.3 --- Identification of expanded polyalanine tract-interacting partners by glutathione S-transferase pull-down assay --- p.84 / Chapter 4.3.1 --- Expression and purification of polyalanine tract as a glutathione S-transferase fusion protein --- p.91 / Chapter 4.3.2 --- Glutathione S-transferase pull-down assay identified three interacting proteins of expanded polyalanine tract --- p.91 / Chapter 4.4 --- Eukaryotic transcription elongation factor alpha 1 was involved in nuclear export of expanded polyalanine tract-containing nuclear export reporter protein --- p.95 / Chapter 4.4.1 --- Eukaryotic translation elongation factor 1 interacted with expanded polyalanine tract --- p.95 / Chapter 4.4.2 --- Knockdown of EEF1A expression reduced nuclear export of expanded polyalanine tract-containing nuclear export reporter protein --- p.97 / Chapter 4.5 --- Discussion --- p.101 / Chapter Chapter 5 --- General Discussion and Conclusion --- p.104 / References --- p.108
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328691 |
| Date | January 2013 |
| Contributors | Ng, Ka Lam., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xix, 112 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
Page generated in 0.0029 seconds