李芳仁Lee, Fang-Jen S.臺灣大學:分子醫學研究所蔡松蒲Tsai, Sung-PuSung-PuTsai2010-05-042018-07-092010-05-042018-07-092009U0001-2407200910280800http://ntur.lib.ntu.edu.tw//handle/246246/178734在真核細胞中，核醣核酸結合蛋白調控著信使核醣核酸(mRNA)的轉錄和轉錄後事件(post-transcriptional events)，例如剪接(splicing)、編輯(editing)、運輸出核(export)、穩定性(stability)、位置(localization)和轉譯(translation)。之前實驗室發現酵母菌核醣核酸結合蛋白Rbp1p，分布在細胞質中，為具有抑制酵母菌生長的負調控因子。此外，在葡萄糖剝奪(glucose depletion)、熱壓力(heat shock)等環境壓力下，Rbp1p分別會座落到細胞質中的細胞質處理小體(P-bodies)及壓力體(stress granules; SGs)。利用二維電泳分析，發現在這兩種環境壓力下Rbp1p等電點圖譜皆有往酸性端移動之現象。推測在處在環境壓力下Rbp1p含有不同的轉譯後修飾。藉由質譜儀分析Rbp1p轉譯後修飾，發現了八個磷酸化的位置，並且利用定點突變(site-directed mutagenesis)的技術將它們突變為丙氨酸，觀察到這些突變型的Rbp1p不會影響抑制酵母菌生長的能力。另外，也發現在環境壓力下，這些突變型的Rbp1p依然可以座落到細胞質處理小體。同時在酵母菌雙雜交實驗發現S646A和S649A突變型的Rbp1p和野生型的Rbp1p結合的能力會減弱。藉著共同免疫沉澱實驗，我們發現了Ssa2p為Rbp1p的結合蛋白，並且在熱壓力的環境下結合的能力會下降。此外，在ssa2突變株中大量表現Rbp1p，Rbp1p抑制酵母菌生長的能力增強了。In eukaryotic cells, RNA binding proteins regulate mRNA transcription and post-transcriptional events, such as splicing, editing, export, stability, localization, and translation. Our laboratory has demonstrated that RNA binding protein1, Rbp1p, appears punctate in cytoplasm and as a negative growth regulator. In addition, Rbp1p localizes to P-bodies and stress granules in cytoplasm upon glucose deprivation and heat shock, respectively. Using two-dimensional electrophoresis analysis, the iso-electric point pattern of Rbp1p shows spots toward acidic pore after glucose deprivation and heat shock treating. The results imply that Rbp1p contains different post-translational modifications in stress conditions. By mass spectrometry analysis, we identify eight phosphorylation sites of Rbp1p (Ser459, Ser462, Ser463, Ser524, Ser526, Thr637, Ser646, and Ser649), and using site-directed mutagenesis approach to substitute alanine for serine/threonine. S646A and S649A mutants of Rbp1p decrease the ability of interaction with Rbp1p by yeast two-hybrid assay. Under glucose deprivation, however, phosphorylation mutants of Rbp1p can still localize to P-bodies, and these mutants cannot affect ability of growth inhibition. In co-immunoprecipitation experiments, I identify that Ssa2p is one of associated proteins with Rbp1p, and the interaction ability diminishes at heat shock. Besides, overexpression of Rbp1p in ssa2Δ strain enhances the growth-inhibition ability. It implies that Ssa2p can inhibit Rbp1p negative regulator growth.中文摘要..................................................IVBSTRACT...................................................VNTRODUCTION...............................................1ESULT....................................................12. Properties of phospho-mutant of Rbp1p..................12I. To characterize Rbp1p under glucose deprivation.......14II. To characterize Rbp1p under heat shock...............15ISCUSSION................................................19ATERIALS AND METHODS.....................................22rowth of yeast...........................................22onstruction of plasmids..................................22east transform...........................................22east cell lysates, Western blot and immunoprecipation....23ample preparation for two-dimensional electrophoresis....24wo-dimensional electrophoresis...........................25east two-hybrid analysis.................................25ABLES....................................................27able 1. Yeast strains used in this thesis................27able 2. Plasmids used in this thesis.....................28able 3. Primers used in this thesis......................29able 4. Antibodies used in this thesis...................30able 5. Summarize candidates of Rbp1p associated protein under heat shock..........................................31IGURES...................................................33 Figure 1. Structural organization of Rbp1p and its phosphorylation mutants...................................33 Figure 2. Overecxpression of Rbp1p phospho-mutants in rbp1Δ strain.............................................34 Figure 3. Rbp1p-S646A and Rbp1p-S649A affect self-association ability..................................35 Figure 4. Rbp1p shows different patterns between glucose-containing medium and glucose-deprivation medium by two-dimensional electrophoresis analysis..................36 Figure 5. Rbp1p localization to P-bodies is not affected by mutation at Ser459, Ser462, Ser463, Ser524 and Ser526....................................................37 Figure 6. Figure 6. Rbp1p localization to P-bodies is not affected by mutation at Thr637, Ser646, and Ser649....38 Figure 7. Glucose deprivation affects Rbp1p interacting with its associated proteins..............................39 Figure 8. Rbp1p shows different patterns between 30℃ and 42℃ medium by two-dimensional electrophoresis analysis..........................................................40 Figure 9. The localization of Rbp1p under heat shock is not affected by mutation at Ser459, Ser462, Ser463, Ser524 and Ser526................................................41 Figure 10. The localization of Rbp1p under heat shock is not affected by mutation at Thr637, Ser646, and Ser649....42 Figure 11. Heat shock affects Rbp1p interacting with its associated proteins.......................................43 Figure 12. Identification of Rbp1p interacting proteins under heat shock..........................................44 Figure 13. Overexpression of Rbp1p in ssa2Δ strain enhances the growth-inhibition ability....................45 Figure 14. The amount of pSer524 Rbp1p is altered under glucose deprivation.......................................46 Appendix 1. Overecxpression of Rbp1p in scj1, kns1, and hmt1 are unable to affect the growth-inhibition ability .........................................................47 Appendix 2. Rbp1p shows different patterns between wild type and ksp1 strains.....................................48EFERENCE.................................................49application/pdf2003651 bytesapplication/pdfen-US酵母菌核醣核酸蛋白Rbp1phttp://ntur.lib.ntu.edu.tw/bitstream/246246/178734/1/ntu-98-R94448007-1.pdf