臺灣博碩士論文加值系統

基於這兩點原因: 第一，真核生物其核醣體生成過程中，核醣蛋白需要進到細胞核作組裝。以及第二，比對原核生物和真核生物核醣蛋白的序列，結果發現真核生物的核醣蛋白多出一段序列，這段多出來的序列稱作Expansion segment，簡稱ES。因此本篇論文的研究動機是探討ES上是否帶有進核序列 (Nuclear localization signal, NLS)，成為真核核醣蛋白進核的主要原因。經過生物資訊程式分析，得到的結果可以分為三種類型: 第一類: NLS分布在ES上，第二類: NLS沒有分布在ES上，第三類: 是不帶有任何典型的NLS，此類包括RPS4, RPS20, RPL10, RPL17, RPL18以及RPL31。我們有興趣的是，這六個不帶有典型NLS的核醣蛋白是否需要進入細胞核，參與核醣體的組裝？實驗結果發現RPS4, RPL17以及RPL31都有明顯進核的情況，其中RPS20分布在細胞質，而RPL10和RPL18卻呈現分布在細胞膜的形態。有些文獻指出，RPL10的分布位在細胞質，是最後一步組裝成large subunit的核醣蛋白。重複幾次實驗去確認RPL10在細胞內的分布情況，結果發現，RPL10大部分還是呈現分布細胞膜的形態，但在少數細胞，以及正在進行細胞分裂的細胞中有觀察到RPL10進核情況。因此我們推測RPL10在細胞內的分布情況也許跟細胞週期有關。另外其他些文獻指出，RPL10在胺基酸37-42是一段可能的 NLS，因此我們將其做點突變，並觀察L10K39A/K40A進核情況。實驗結果發現，只有大約1.11%的進核比例。另外探討RPL10分布在細胞膜的原因，我們利用生物資訊程式去分析RPL10序列，看是否有和細胞膜結合的區域，結果發現，RPL10序列中有六個PDZ domain binding motif (PBM)，我們挑選位在ES上的PBM做點突變，觀察L10D183K進核情況，實驗結果發現，進核的比例明顯提高，大約是78.35%。因此我們進一步利用GST pull down assay分析，找出是否有另一個蛋白和RPL10有交互作用，導致RPL10呈現分布在細胞膜的形態。

In the eukaryotic ribosome biogenesis, the ribosomal proteins must be imported into the nucleus for assembly. In general, proteins which are capable of entering the nucleus usually contain a nuclear localization signal (NLS). In this study, we first used all available NLS prediction programs to search the presence of NLS in all eukaryotic ribosomal proteins, and found RPS4, RPS20, RPL10, RPL17, RPL18 and RPL31 do not contain any predicted NLS. To verify the nature of such ribosomal proteins, we used a flag-tagging approach to see whether these proteins are able to reach nucleus or not. The results show that FLAG-S4, FLAG-L17, and FLAG-L31 have made into the nucleus, whereas FLAG-S20 resides at cytoplasm; furthermore, of particularly interest is that FLAG-L10 and FLAG-L18 have been located at the plasma membrane. Accordingly, we have continued to investigate the nature of RPL10 with respect to it cellular distribution. The mutant FLAG-L10D183K carrying a deletion of a putative PDZ domain binding motif (PBM) at the C-terminal end allows the protein to be transported from the membrane to the nucleus, suggesting that the nuclear entry of RPL10 could be membrane-binding dependent. Interestingly, during examination of the cellular distribution of FLAG-L10, we also found that on rare occasions, FLAG-L10 can be located within the nucleus and nucleoli in dividing cells. This implies that the nuclear import of RPL10 could be cell cycle dependent. By transfecting a FLAG-L10 gene into synchronized cells, we have found that FLAG-L10 reaches the nucleus and nucleoli during the G1 phase. To understand the mechanism behind such an unusual phenomenon of nuclear uptake, we intend to use a GST-pull down assay to search for possible proteins that may facilitate the nuclear import or involve in the plasma membrane association of RPL10.

INDEX
ABSTRACT - 1 -
中文摘要 - 2 -
1. INTRODUCTION - 3 -
1.1 THE DIFFERENCE OF RIBOSOME ASSEMBLY IN EUKARYOTES AND PROKARYOTES - 3 -
1.2 EXPANSION SEGMENT (ES) IS EXTENSIVELY FOUND IN EUKARYOTES - 3 -
1.3 NUCLEAR LOCALIZATION SIGNAL (NLS) IS A TICKET FOR NUCLEAR ENTRY IN EUKARYOTES - 4 -
1.4 CHARACTERISTICS OF RPL10 - 4 -
1.5 RPL10 MAY DISPLAY A NUCLEAR PATTERN - 6 -
1.6 RPL10P/QSR1P IS REQUIRED FOR 40S AND 60S SUBUNITS JOINING - 6 -
1.7 RPL10 HAS A CYTOPLASM PATTERN - 7 -
1.8 NMD3P IS A CRM1P-DEPENDENT ADAPTER PROTEIN FOR THE EXPORT OF THE 60S SUBUNIT - 8 -
1.9 THE EXPORT OF 60S SUBUNIT INVOLVES IN RPL10 - 9 -
1.10 PDZ DOMAIN- BINDING MOTIF (PBM) - 10 -
1.11 RPL10 MAY EXHIBIT A MEMBRANE PATTERN - 11 -
2. AIMS - 13 -
3. MATERIALS AND METHODS - 15 -
3.1 CONSTRUCTION OF PLASMIDS - 15 -
3.2 TRANSFORMATION - 16 -
3.3 TA-CLONING - 17 -
3.4 CELL CULTURE - 18 -
3.5 TRANSFECTION - 18 -
3.6 IMMUNOFLUORESCENCE STAINING - 20 -
3.7 WESTERN BLOTTING - 22 -
3.8 DOUBLE THYMIDINE BLOCK SYNCHRONIZATION OF HELA CELLS - 23 -
3.9 PROPIDIUM IODIDE (PI) STAINING - 24 -
3.10 SITE-DIRECTED MUTAGENESIS USING - 25 -
3.11 GST FUSION PROTEINS - 26 -
3.12 PROTEIN EXPRESSION AND PURIFICATION - 27 -
3.13 GST PULL-DOWN ASSAY - 28 -
3.14 THROMBIN CLEAVAGE - 29 -
3.15 SILVER STAIN - 30 -
4. RESULTS - 32 -
4.1 CHARACTERIZATION OF THE EUKARYOTIC RIBOSOMAL PROTEINS - 32 -
4.2 CONSTRUCTION OF HUMAN RIBOSOMAL PROTEINS S4, S20, L10, L17, L18 AND L31 WITH A FLAG-TAG AT THE N-TERMINAL - 33 -
4.3 THE SIX HUMAN RIBOSOMAL PROTEINS EXHIBITED DIVERSE SUBCELLULAR LOCALIZATIONS - 34 -
4.4 THE SUBCELLULAR LOCALIZATION OF FLAG-L10 IS CELL CYCLE-DEPENDENT - 35 -
4.5 RPL10 CONTAINS A PUTATIVE NLS - 36 -
4.6 THE PBM DOMAIN LOCATED WITHIN THE ES OF RPL10 MAY BE THE REASON FOR PLASMA MEMBRANE-ASSOCIATION - 37 -
4.7 GST PULL-DOWN ASSAY PROVIDES USEFUL INSIGHTS INTO THE PARTNERS THAT INTERACT WITH RPL10 THROUGH ITS PBM - 38 -
5. DISCUSSION - 41 -
6. LEGENDS - 46 -
7. TABLES - 60 -
8. REFERENCE - 62 -
9. APPENDIX - 65 -

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吳京穎 (2004). 真核核醣蛋白L17和L26與內質網之交互作用. 國立陽明大學遺傳學研究所碩士論文.

陳映潔 (2004). 真核核醣蛋白L35與內質網之交互作用. 國立陽明大學遺傳學研究所碩士論文.

李逸芳 (2005). 人類核醣體蛋白質L7氨基端所含之鹼性胺基酸群對細胞核/細胞質間運輸過程中所扮演的腳色. 國立陽明大學遺傳學研究所碩士論文.