跳到主要內容

臺灣博碩士論文加值系統

(44.200.86.95) 您好!臺灣時間:2024/05/28 08:54
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:楊清淳
研究生(外文):Ching-Chun Yang
論文名稱:B型肝炎病毒的核心蛋白與基因組出細胞核機制的探討
論文名稱(外文):Dissecting the nuclear export mechanism of HBV genome and core particles
指導教授:施嘉和
指導教授(外文):Chiaho Shih
學位類別:博士
校院名稱:國立陽明大學
系所名稱:分子醫學博士學位學程
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:英文
論文頁數:76
中文關鍵詞:B型肝炎病毒核心蛋白出細胞核機制
外文關鍵詞:Hepatitis B virusHBV core particlesNuclear exportCRM1NXF1
相關次數:
  • 被引用被引用:0
  • 點閱點閱:209
  • 評分評分:
  • 下載下載:7
  • 收藏至我的研究室書目清單書目收藏:0
B型肝炎的核心蛋白(HBc)可以在細胞質與細胞核中運輸。臨床上的觀察,發現B型肝炎的核心蛋白停留在細胞質中通常與較嚴重的肝臟發炎有關。核心蛋白在細胞質與細胞核中運輸的機制目前還不是很清楚。細胞中的蛋白離開細胞核主要是透過 NXF1(TAP)主導的信使核糖核酸 (mRNA)出核路徑,或者是由CRM1 (XPO-1)主導的出核路徑。2010 年我們藉由免疫共沉澱的實驗,發現核心蛋白的arginine-rich domain (ARD) 可以跟 NXF1 有物理上的接觸。NXF1已經知道可以與 p15 結合成異源二聚體,並與核糖核酸形成核糖核蛋白複合物(RNP),來擔任主要的核運輸受體。在哺乳動物細胞中,在耦合信使核糖核酸的形成過程與運送出核的連續過程裡,TREX (transcription/export) complex擔任一個很重要的腳色。因此,我們想確定B型肝炎的核心蛋白是否可以利用信使核糖核酸 (mRNA)的出核路徑來輸出細胞核。我們利用了免疫共沉澱的實驗,證明了B型肝炎的核心蛋白也可以與TREX或 NXF1-p15的異源二聚體有專一性且物理上的接觸。當我們降低了TREX或 NXF1-p15的表現量會把B型肝炎的核心蛋白留在細胞核裡。利用RT-qPCR與北方墨點法,我們證明了減少TREX或 NXF1-p15的表現量,細胞質中的B型肝炎最重要的沒有剪接過的核糖核酸(pgRNA)也會明顯地減少。這些結果支持了我們的假設: pgRNA 與核心蛋白可以形成 RNP,利用細胞中信使核糖核酸 (mRNA)的出核路徑來輸出細胞核。pgRNA 的出核機制不需要核心蛋白,核心蛋白的出核機制也不需要pgRNA。

此外,我們進一步也證明了B型肝炎病毒的核心蛋白,也可以利用細胞中CRM1主導的機制來離開細胞核。我們鑑定出在B型肝炎病毒的核心蛋白上,有兩個新的CRM1細胞出核訊息(NES),可以用來控制核心蛋白的出細胞核的運輸。利用許多種的實驗方法來抑制病毒核心蛋白上的出核訊息,會造成病毒核心蛋白聚集在細胞核裡的現象。而且CRM1可以不需要NXF1參與,獨立調節病毒核心蛋白的出核運輸。因此阻斷細胞內B型肝炎病病毒核心蛋白連續不斷地運輸,可以發展成是一個治療用藥的標的。
Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. The mechanism that regulates the subcellular distribution of HBc remains unclear. Nuclear export of cellular proteins can be facilitated by either an NXF1-dependent mRNA export pathway or a CRM1 (XPO-1)-dependent pathway. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

In addition to the NXF1-p15 export pathway, we report here that nuclear export of HBc can also be facilitated by the CRM1-dependent pathway. Two CRM1-mediated nuclear export signals (NESCRM1) are found clustering at the spikes of HBc icosahedral particles. These NESCRM1 can facilitate nucleocytoplasmic shuttling of HBc. Inhibitions of the NES activity by using different approaches invariably resulted in nuclear accumulation of HBc. CRM1 can mediate nuclear export of HBc independent of the NXF1-p15-mediated export pathway. An intracellular loop of repetitive and continuous HBc nucleocytoplasmic shuttling could offer a novel drugable target for hepatitis B.
Contents
中文摘要----i
English Abstract----ii
Contents-----iv
List of Figures----vii

Chapter 1 Introduction
1.1 Biology of Hepatitis B virus.----1
1.2 The clinical significance of HBc subcellular localization.----2
1.3 The nuclear export mechanisms of CRM1 and NXF1.----2
1.4 The goal and significance of this study.----3
Chapter 2 Results
2.1 Full-length HBc and HBc arginine rich domain (ARD) can associate with p15 in a ribonuclease sensitive manner.----4
2.2 Full-length HBc and HBc ARD can physically associate with TREX.----6
2.3 Nuclear export of HBc protein depends on TREX and NXF1-p15 complex.----7
2.4 Physical association between HBV pgRNA, TREX and the NXF1-p15 complex.----8
2.5 HBV pgRNA export facilitated by the NXF1-p15 machinery.----10
2.6 HBc is not essential to HBV pgRNA nuclear export.----12
2.7 Potential influence of the NXF1-p15 complex on HBV DNA replication.-----12
2.8 Nuclear export of HBc via CRM1-dependent pathway.----13
2.9 CRM1 dependent NES are clustering on the spike of HBc icosahedral particles.----14
2.10 HBc NESCRM1 can functionally substitute for the NES of HIV-1 Rev.----15
2.11 HBc NESCRM1 can mediate the nucleocytoplasmic shuttling activity in a homokaryon assay.----16
2.12 CRM1 and NXF1 behave independently in nuclear export of HBc.----16

Chapter 3 Discussion
3.1 RNA can enhance HBc protein association with components of the RNA export machinery.----18
3.2 Nuclear export of HBc protein vs. pgRNA.----18
3.3 CRM-1 and PRE in HBV RNA splicing and export?----19
3.4 Building up the pool size of ccc DNA and HBc protein shuttling.----20

Chapter 4 Methods
4.1 Cell culture and transfection.----21
4.2 Plasmids.----21
4.3 Antibodies and inhibitors.----22
4.4 Co-immunoprecipitation (co-IP).----22
4.5 Streptavidin pull down assay.----23
4.6 GST Pull down Assay.----23
4.7 Immunofluorescence analysis (IFA).----23
4.8 HBV luciferase reporter assay, Southern blot analysis and MTT assay.----24
4.9 Northern blot and RT- qPCR analysis of fractionated RNA samples.----25
4.10 RNA immunoprecipitation (RNA-IP) assay.----25
4.11 RT-qPCR primers.----26
4.12 Knockdown by siRNAs.----26
4.13 Docking.----28
4.14 Accession numbers.----28

References----68

List of Figures
Figure 1. An HBV transcription map is illustrated.----29
Figure 2. Full-length HBc 183 protein can physically and specifically associate with a p15-Flag and NXF1 protein in a ribonuclease (RNase)-sensitive manner by coimmunoprecipitation assay (co-IP) and Western blot analysis (WB).----30
Figure 3. Only the chimera protein K128T-HBc ARD, but not K128T, can physically associate with the p15-Flag and NXF1 protein.----31
Figure 4. Biotin-HBc ARD synthetic polypeptide can in vitro pull down purified recombinant proteins of GST-NXF1 and GST-p15 by using streptavidin T1 beads.----32
Figure 5. A cartoon illustrates that either NXF1 or p15 can bind directly to HBc ARD.----33
Figure 6. HBV core protein (HBc) can physically and specifically associate with the cellular NXF1-p15 complex via the NES (nuclear export signal) motif of HBc arginine rich domain (ARD).----34
Figure 7. The association between GST-HBc ARD and a TREX component ALY was ribonuclease (RNase)-sensitive and DNase-resistant in a GST pull down assay.----35
Figure 8. The full length HBc protein can be physically associated with endogenous ALY.----36
Figure 9. Full-length HBc 183 protein cannot associate with another TREX component BAT1/DDX39 in the co-IP assay.----37
Figure 10. A cartoon summarizes the putative associations among HBc, RNA, ALY, TREX components, and others (other known or unknown cellular factors).----38
Figure 11. Three different subcellular distribution patterns of HBc are cartoon illustrated.----39
Figure 12. Perturbation of the TREX complex and NXF1-p15 nuclear export machinery by siRNA treatments can result in accumulation of HBc protein in the nucleus by IFA.----40
Figure 13. Physical association between HBV RNA and NXF1-p15 complex was revealed by the RNA-immunoprecipitation assay (RNA-IP).----41
Figure 14. Physical association between HBV pgRNA and TREX complex was revealed by the RNA-immunoprecipitation assay (RNA-IP).----42
Figure 15. Both NXF1 and p15 can contribute to the efficient nuclear export of HBV pgRNA by reporter assay.----43
Figure 16. The knockdown efficacies of siRNAs.----44
Figure 17. Both NXF1 and p15 can contribute to the efficient nuclear export of HBV core+ RNA by Northern blot analysis.----45
Figure 18. The knockdown efficacies of CRM1 siRNAs.----46
Figure 19. Both NXF1 and p15 can contribute to the efficient nuclear export of HBV core+ RNA by RT-qPCR.----47
Figure 20. Treatment with siRNA specific for CRM-1 resulted in no effect on the N/C ratio of HBV pgRNA.----48
Figure 21. HBV core protein (HBc) exhibited no significant effect on nuclear export of the 3.5 kb pgRNA by RT-qPCR.----49
Figure 22. HBV core protein (HBc) exhibited no significant effect on nuclear export of the 3.5 kb pgRNA by Northern blot analysis.----50
Figure 23. Interference with the NXF1-p15 complex reduced HBV DNA synthesis.----51
Figure 24. A graphic summary of the nuclear export of HBV pgRNA and HBc.----52
Figure 25. CRM1 inhibitors increased nuclear accumulation of HBc.----53
Figure 26. HBc can be associated with exogenous CRM1 protein.----54
Figure 27. CRM1-mediated nuclear export signals (NES) are predicted on the spike of HBc icosahedral capsid particles.----55
Figure 28. The characterization of CRM1-mediated nuclear export signals (NES) on HBc spike.----56
Figure 29. Alignments of multiple consensus sequences of HBc spike region from different hepadnaviruses.----58
Figure 30. HBc 46-113 of the spike domain of HBc capsid particles can functionally replace a prototype NES of the HIV Rev protein.----59
Figure 31. Homokaryon analysis revealed the existence of CRM1 dependent NES-like signals in HBc.----61
Figure 32. HBV replication has no effect on the expression levels of the endogenous NXF1 and CRM1 proteins.----63
Figure 33. Nuclear export of HBc is mediated via the CRM1 and NXF1 machinery.----64
Figure 34. CRM1 and NXF1 machineries mediate independently nuclear export of HBc.----66
Figure 35. HBc protein contains two distinct nuclear export domains mediated by the CRM1-dependent and NXF1-dependent pathways, respectively.----67
1 Tuttleman, J. S., Pourcel, C. & Summers, J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47, 451-460 (1986).

2 Huang, J. & Liang, T. J. A novel hepatitis B virus (HBV) genetic element with Rev response element-like properties that is essential for expression of HBV gene products. Molecular and cellular biology 13, 7476-7486 (1993).

3 Huang, Z. M. & Yen, T. S. Hepatitis B virus RNA element that facilitates accumulation of surface gene transcripts in the cytoplasm. Journal of virology 68, 3193-3199 (1994).

4 Donello, J. E., Loeb, J. E. & Hope, T. J. Woodchuck hepatitis virus contains a tripartite posttranscriptional regulatory element. Journal of virology 72, 5085-5092 (1998).

5 Smith, G. J., 3rd, Donello, J. E., Luck, R., Steger, G. & Hope, T. J. The hepatitis B virus post-transcriptional regulatory element contains two conserved RNA stem-loops which are required for function. Nucleic acids research 26, 4818-4827 (1998).

6 Otero, G. C., Harris, M. E., Donello, J. E. & Hope, T. J. Leptomycin B inhibits equine infectious anemia virus Rev and feline immunodeficiency virus rev function but not the function of the hepatitis B virus posttranscriptional regulatory element. Journal of virology 72, 7593-7597 (1998).

7 Heise, T. et al. The hepatitis B virus PRE contains a splicing regulatory element. Nucleic acids research 34, 353-363, doi:10.1093/nar/gkj440 (2006).

8 Su, T. S. et al. Hepatitis B virus transcript produced by RNA splicing. Journal of virology 63, 4011-4018 (1989).

9 Summers, J. & Mason, W. S. Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell 29, 403-415 (1982).

10 Bruss, V. & Ganem, D. The role of envelope proteins in hepatitis B virus assembly. Proceedings of the National Academy of Sciences of the United States of America 88, 1059-1063 (1991).

11 Yuan, T. T., Tai, P. C. & Shih, C. Subtype-independent immature secretion and subtype-dependent replication deficiency of a highly frequent, naturally occurring mutation of human hepatitis B virus core antigen. Journal of virology 73, 10122-10128 (1999).

12 Le Pogam, S. & Shih, C. Influence of a putative intermolecular interaction between core and the pre-S1 domain of the large envelope protein on hepatitis B virus secretion. Journal of virology 76, 6510-6517 (2002).

13 Kian Chua, P., Lin, M. H. & Shih, C. Potent inhibition of human Hepatitis B virus replication by a host factor Vps4. Virology 354, 1-6, doi:10.1016/j.virol.2006.07.018 (2006).

14 Shih, C. et al. Control and Eradication Strategies of Hepatitis B Virus. Trends in microbiology 24, 739-749, doi:10.1016/j.tim.2016.05.006 (2016).

15 Chu, C. M. & Liaw, Y. F. Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis. Gastroenterology 92, 220-225 (1987).

16 Hsu, H. C. et al. Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection. Journal of hepatology 5, 45-50 (1987).

17 Naoumov, N. V. et al. Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection. Gastroenterology 99, 1248-1253 (1990).

18 Li, H. C. et al. Nuclear export and import of human hepatitis B virus capsid protein and particles. PLoS pathogens 6, e1001162, doi:10.1371/journal.ppat.1001162 (2010).

19 Yang, C. C., Li, H. C. & Shih, C. A Homokaryon Assay for Nucleocytoplasmic Shuttling Activity of HBV Core Protein. Methods in molecular biology 1540, 53-58, doi:10.1007/978-1-4939-6700-1_5 (2017).

20 Yang, C. C., Huang, E. Y., Li, H. C., Su, P. Y. & Shih, C. Nuclear Export of Human Hepatitis B Virus Core Protein and Pregenomic RNA Depends on the Cellular NXF1-p15 Machinery. PloS one 9, e106683, doi:10.1371/journal.pone.0106683 (2014).

21 Johnson, L. A. & Sandri-Goldin, R. M. Efficient nuclear export of herpes simplex virus 1 transcripts requires both RNA binding by ICP27 and ICP27 interaction with TAP/NXF1. Journal of virology 83, 1184-1192, doi:10.1128/JVI.02010-08 (2009).

22 Juillard, F. et al. Epstein-Barr virus protein EB2 contains an N-terminal transferable nuclear export signal that promotes nucleocytoplasmic export by directly binding TAP/NXF1. Journal of virology 83, 12759-12768, doi:JVI.01276-09 [pii]
10.1128/JVI.01276-09 (2009).

23 Dong, X. et al. Structural basis for leucine-rich nuclear export signal recognition by CRM1. Nature 458, 1136-1141, doi:10.1038/nature07975 (2009).

24 Muller-McNicoll, M. & Neugebauer, K. M. How cells get the message: dynamic assembly and function of mRNA-protein complexes. Nature reviews. Genetics 14, 275-287, doi:10.1038/nrg3434 (2013).

25 Natalizio, B. J. & Wente, S. R. Postage for the messenger: designating routes for nuclear mRNA export. Trends in cell biology 23, 365-373, doi:10.1016/j.tcb.2013.03.006 (2013).

26 Emerman, M. & Malim, M. H. HIV-1 regulatory/accessory genes: keys to unraveling viral and host cell biology. Science 280, 1880-1884 (1998).

27 Sherer, N. M. et al. Evolution of a species-specific determinant within human CRM1 that regulates the post-transcriptional phases of HIV-1 replication. PLoS pathogens 7, e1002395, doi:10.1371/journal.ppat.1002395 (2011).

28 Bodem, J., Schied, T., Gabriel, R., Rammling, M. & Rethwilm, A. Foamy virus nuclear RNA export is distinct from that of other retroviruses. Journal of virology 85, 2333-2341, doi:10.1128/JVI.01518-10 (2011).

29 Schmid, M., Gonzalez, R. A. & Dobner, T. CRM1-dependent transport supports cytoplasmic accumulation of adenoviral early transcripts. Journal of virology 86, 2282-2292, doi:10.1128/JVI.06275-11 (2012).

30 Reed, R. & Cheng, H. TREX, SR proteins and export of mRNA. Current opinion in cell biology 17, 269-273, doi:10.1016/j.ceb.2005.04.011 (2005).

31 Sakuma, T. et al. Murine leukemia virus uses NXF1 for nuclear export of spliced and unspliced viral transcripts. Journal of virology 88, 4069-4082, doi:10.1128/JVI.03584-13 (2014).

32 Conway, J. F. et al. Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy. Nature 386, 91-94, doi:10.1038/386091a0 (1997).

33 Bottcher, B., Wynne, S. A. & Crowther, R. A. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 386, 88-91, doi:10.1038/386088a0 (1997).

34 Wynne, S. A., Crowther, R. A. & Leslie, A. G. The crystal structure of the human hepatitis B virus capsid. Molecular cell 3, 771-780 (1999).

35 Yu, X., Jin, L., Jih, J., Shih, C. & Zhou, Z. H. 3.5A cryoEM structure of hepatitis B virus core assembled from full-length core protein. PloS one 8, e69729, doi:10.1371/journal.pone.0069729 (2013).

36 Guzik, B. W. et al. NXT1 (p15) is a crucial cellular cofactor in TAP-dependent export of intron-containing RNA in mammalian cells. Molecular and cellular biology 21, 2545-2554, doi:10.1128/MCB.21.7.2545-2554.2001 (2001).

37 Herold, A., Klymenko, T. & Izaurralde, E. NXF1/p15 heterodimers are essential for mRNA nuclear export in Drosophila. Rna 7, 1768-1780 (2001).

38 Lanford, R. E. & Butel, J. S. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell 37, 801-813 (1984).

39 Steven, A. C. et al. Structure, assembly, and antigenicity of hepatitis B virus capsid proteins. Advances in virus research 64, 125-164, doi:10.1016/S0065-3527(05)64005-5 (2005).

40 Newman, M., Suk, F. M., Cajimat, M., Chua, P. K. & Shih, C. Stability and morphology comparisons of self-assembled virus-like particles from wild-type and mutant human hepatitis B virus capsid proteins. Journal of virology 77, 12950-12960 (2003).

41 Zhou, Z. et al. The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans. Nature 407, 401-405, doi:10.1038/35030160 (2000).

42 Luo, M. L. et al. Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly. Nature 413, 644-647, doi:10.1038/35098106 (2001).

43 Sun, J. et al. Deregulation of cofactor of BRCA1 expression in breast cancer cells. Journal of cellular biochemistry 103, 1798-1807, doi:10.1002/jcb.21568 (2008).

44 Stuffers, S., Sem Wegner, C., Stenmark, H. & Brech, A. Multivesicular endosome biogenesis in the absence of ESCRTs. Traffic 10, 925-937, doi:10.1111/j.1600-0854.2009.00920.x (2009).

45 Stieler, J. T. & Prange, R. Involvement of ESCRT-II in hepatitis B virus morphogenesis. PloS one 9, e91279, doi:10.1371/journal.pone.0091279 (2014).

46 Beck, J. & Nassal, M. Hepatitis B virus replication. World journal of gastroenterology : WJG 13, 48-64 (2007).

47 Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E. & Kutay, U. Nuclear export of microRNA precursors. Science 303, 95-98, doi:10.1126/science.1090599 (2004).

48 Ou, J. H. Molecular biology of hepatitis B virus e antigen. Journal of gastroenterology and hepatology 12, S178-187 (1997).

49 Yuan, T. T., Faruqi, A., Shih, J. W. & Shih, C. The mechanism of natural occurrence of two closely linked HBV precore predominant mutations. Virology 211, 144-156, doi:10.1006/viro.1995.1387 (1995).

50 Nassal, M. The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. Journal of virology 66, 4107-4116 (1992).

51 Hautbergue, G. M. et al. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA. Current biology : CB 19, 1918-1924, doi:10.1016/j.cub.2009.09.041 (2009).

52 Moore, M. J. & Proudfoot, N. J. Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136, 688-700, doi:10.1016/j.cell.2009.02.001 (2009).

53 Chang, C. T. et al. Chtop is a component of the dynamic TREX mRNA export complex. The EMBO journal 32, 473-486, doi:10.1038/emboj.2012.342 (2013).

54 Verheggen, C. & Bertrand, E. CRM1 plays a nuclear role in transporting snoRNPs to nucleoli in higher eukaryotes. Nucleus 3, 132-137, doi:10.4161/nucl.19266 (2012).

55 Hatton, T., Zhou, S. & Standring, D. N. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. Journal of virology 66, 5232-5241 (1992).

56 Kudo, N. et al. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proceedings of the National Academy of Sciences of the United States of America 96, 9112-9117 (1999).

57 Sun, Q. et al. Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1. Proceedings of the National Academy of Sciences of the United States of America 110, 1303-1308, doi:10.1073/pnas.1217203110 (2013).

58 Monecke, T., Dickmanns, A. & Ficner, R. Allosteric control of the exportin CRM1 unraveled by crystal structure analysis. The FEBS journal, doi: 10.1111/febs.12842., doi:10.1111/febs.12842 (2014).

59 la Cour, T. et al. Analysis and prediction of leucine-rich nuclear export signals. Protein engineering, design & selection : PEDS 17, 527-536, doi:10.1093/protein/gzh062 (2004).

60 Prieto, G., Fullaondo, A. & Rodriguez, J. A. Prediction of nuclear export signals using weighted regular expressions (Wregex). Bioinformatics 30, 1220-1227, doi:10.1093/bioinformatics/btu016 (2014).

61 Monecke, T. et al. Crystal structure of the nuclear export receptor CRM1 in complex with Snurportin1 and RanGTP. Science 324, 1087-1091, doi:10.1126/science.1173388 (2009).

62 Askjaer, P., Jensen, T. H., Nilsson, J., Englmeier, L. & Kjems, J. The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP. The Journal of biological chemistry 273, 33414-33422 (1998).

63 Meyer, B. E. & Malim, M. H. The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes & development 8, 1538-1547 (1994).

64 Williams, B. J. et al. The prototype gamma-2 herpesvirus nucleocytoplasmic shuttling protein, ORF 57, transports viral RNA through the cellular mRNA export pathway. The Biochemical journal 387, 295-308, doi:10.1042/BJ20041223 (2005).

65 Tunnicliffe, R. B., Hautbergue, G. M., Wilson, S. A., Kalra, P. & Golovanov, A. P. Competitive and cooperative interactions mediate RNA transfer from herpesvirus saimiri ORF57 to the mammalian export adaptor ALYREF. PLoS pathogens 10, e1003907, doi:10.1371/journal.ppat.1003907 (2014).

66 Viphakone, N. et al. TREX exposes the RNA-binding domain of Nxf1 to enable mRNA export. Nature communications 3, 1006, doi:10.1038/ncomms2005 (2012).

67 Jackson, B. R. et al. An interaction between KSHV ORF57 and UIF provides mRNA-adaptor redundancy in herpesvirus intronless mRNA export. PLoS pathogens 7, e1002138, doi:10.1371/journal.ppat.1002138 (2011).

68 Roth, J. & Dobbelstein, M. Export of hepatitis B virus RNA on a Rev-like pathway: inhibition by the regenerating liver inhibitory factor IkappaB alpha. Journal of virology 71, 8933-8939 (1997).

69 Popa, I., Harris, M. E., Donello, J. E. & Hope, T. J. CRM1-dependent function of a cis-acting RNA export element. Molecular and cellular biology 22, 2057-2067 (2002).

70 Zang, W. Q. & Yen, T. S. Distinct export pathway utilized by the hepatitis B virus posttranscriptional regulatory element. Virology 259, 299-304, doi:10.1006/viro.1999.9777 (1999).

71 Huang, Z. M. & Yen, T. S. Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts. Molecular and cellular biology 15, 3864-3869 (1995).

72 Huang, C. et al. A structured RNA in hepatitis B virus post-transcriptional regulatory element represses alternative splicing in a sequence-independent and position-dependent manner. The FEBS journal 278, 1533-1546, doi:10.1111/j.1742-4658.2011.08077.x (2011).

73 Chi, B. et al. A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18. Nucleic acids research doi: 10.1093/nar/gku350, doi:10.1093/nar/gku350 (2014).

74 Chen, H. L., Huang, J. Y., Chen, C. M., Chu, T. H. & Shih, C. MicroRNA-22 can reduce parathymosin expression in transdifferentiated hepatocytes. PloS one 7, e34116, doi:10.1371/journal.pone.0034116 (2012).

75 Yuan, T. T., Sahu, G. K., Whitehead, W. E., Greenberg, R. & Shih, C. The mechanism of an immature secretion phenotype of a highly frequent naturally occurring missense mutation at codon 97 of human hepatitis B virus core antigen. Journal of virology 73, 5731-5740 (1999).

76 Selth, L. A., Gilbert, C. & Svejstrup, J. Q. RNA immunoprecipitation to determine RNA-protein associations in vivo. Cold Spring Harbor protocols 2009, pdb prot5234, doi:10.1101/pdb.prot5234 (2009).

77 Chi, B. et al. Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA. Nucleic acids research 41, 1294-1306, doi:10.1093/nar/gks1188 (2013).

78 Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome research 14, 1188-1190, doi:10.1101/gr.849004 (2004).
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top