臺灣博碩士論文加值系統

MSP58/MCRS1 過去報導與許多蛋白質有交互作用關係，如核仁蛋白 p120、Mi-2β及轉錄因子 Daxx、STRA13、Nrf1、SWI/SNF 染色質重組複合體次單元 BRG1 或是 RNA 結合蛋白 FMR等，這些發現說明了 MSP58 在細胞核以及核仁內為重要的轉錄調節者。在本研究我們探討 MSP58 調控核醣體 DNA (rDNA) 基因轉錄所扮演的角色，利用本實驗室設計與製作的 MSP58 多株抗體進行免疫螢光染色，我們發現 MSP58 呈現微小球顆粒狀分布在核仁內，細胞質內的分佈較少，並且 MSP58 會與核仁轉錄因子 UBF 結合且共位。透過報告基因分析法，我們發現細胞過度表現 MSP58 會抑制 rDNA 啟動子的轉錄活性，然而利用 siRNA 抑制內生性 MSP58 的表現則會提升 rDNA 轉錄活性。此外，以酵母菌雙雜交技術我們發現一些 MSP58 的新穎結合蛋白。由以上發現指出 MSP58 蛋白質複合體於細胞內參與核醣體 DNA 基因的轉錄調控。

MSP58/MCRS1 has been reported to interact with several proteins, such as nucleolar protein p120, Mi-2β; transcription factors Daxx, STRA13, Nrf1, SWI/SNF chromatin remodeling subunit BRG1, and also RNA-binding protein FMR. These findings imply that MSP58 proteins are crucial for transcriptional regulation in nuclei and nucleoli. In this study, we examined the role of MSP58 in the transcriptional modulation of the ribosomal genes. Initially, we generated and characterized the polyclonal antibodies against MSP58. The main localization of MSP58 was consistent in nuclei and microspherule speckles of nucleoli with faint cytoplasmic staining. Interestingly, MSP58 was associated and co-localized with endogenous nucleolar transcription factor UBF in the nucleoli. Analysis by luciferase reporter assay revealed that ectopic expression of MSP58 suppressed transcriptional activity of the rDNA promoter, whereas knocking-down MSP58 expression by siRNA treatment up-regulated ribosomal RNA transcription. In addition, we identified several nucleolar proteins as novel MSP58-interacting proteins by yeast two-hybrid screening. These results indicated that MSP58 complexes are involved in the regulation of ribosomal gene transcription in vivo.

第一章 緒論1
第一節 核醣體 DNA 基因轉錄調控及相關訊息傳遞路徑2
第二節 58-kDa Microspherule Protein (MSP58) 之介紹5
第三節 核仁蛋白之介紹6
第四節 核仁與癌症的關係7
第五節 研究動機與目的9
第二章 實驗方法與材料11
第一節 細胞培養11
第二節 質體之建構12
第三節 持續表現 MSP58 或新穎核仁蛋白細胞株之製備18
第四節 全細胞液抽取與蛋白質定量20
第五節 西方墨點法 (Western Blot)22
第六節 免疫螢光染色法 (Immunofluorescence)29
第七節 共同免疫沈澱法 (Co-immunoprecipitation Assay)31
第八節 全量 RNA 抽取與 RNA 反轉錄 cDNA33
第九節 及時定量反轉錄-聚合酶連鎖反應 (Real-time qPCR)35
第十節 報告基因分析法 (Reporter Gene Assay)37
第十一節 酵母菌雙雜交分析法 (Yeast two-hybrid Assay)38
第三章 實驗結果45
第一節 利用酵母菌雙雜交技術發現 MSP58 的新穎結合蛋白質45
第二節 利用共同免疫沈澱法驗證 MSP58、UBF 及新穎核仁蛋白為一複合體45
第三節 過度表現 EGFP-MSP58 與核仁上游結合因子 UBF 共位46
第四節 內生性的 MSP58 及新穎核仁蛋白與核仁上游結合因子共位47
第五節 MSP58 具有負向調控 rDNA 轉錄活性的功能47
第六節 新穎核仁蛋白具有負向調控 rDNA 轉錄活性的功能48
第四章 討論49
第五章 參考文獻54
自述74

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