臺灣博碩士論文加值系統

Pnn為本實驗室所發現之蛋白質，其最初被發現於上皮細胞之中
間絲與胞橋小體 (desmosone) 圓盤相連接處，可強化中間絲的結構以及
中間絲與胞橋小體之間的連結，與細胞之間的接合有關。研究顯示，
Pnn存在著兩個形式，一為存在於胞橋小體的Pnn，稱為desmosomeform
(d-form) Pnn；另一存在於細胞核內細胞斑（speckle）中的Pnn，
稱為nucleus-form (n-form) Pnn。過去研究顯示，n-form Pnn與pre-mRNA
splicing有關，並可藉由與其他SR家族蛋白質作用來調控mRNA的剪
接。本研究利用基因刪減方式（RNAi knockdown）將細胞中內生性的
Pnn表現量降低，藉此來探討Pnn與SR蛋白質之間的關係。由免疫螢光
染色及西方點墨法結果顯示，當Pnn減少時，相關之SR家族蛋白質包括
SC-35、SRm300、SRp55及SRp40表現量亦隨之降低，然而並不影響非
SR家族蛋白質如p53、MDM2、ki67的表現量。除此之外，吾人亦發現
Pnn表現量降低會調節pre-mRNA剪接時3’端剪接位置的選擇。由研究
結果可知，Pnn可以調控SR蛋白質的表現並可藉此調控mRNA的選擇性
剪接。本研究結果可使我們更進一步了解Pnn在細胞中的功能。

Pinin (Pnn) was first identified and characterized as a protein closely associated with mature desmosomes of epithelial cells, and several studies have indicated that Pnn can enhance the intermediate filaments organization and the linkage between intermediate filaments and desmosone. Pnn is not only found at desmosome（d-form pnn）, but also localized in speckled domains of the nuclear（ n-form pnn） . In the nucleus Pnn is involved in the pre-mRNA splicing, and regulates mRNA alternative splicing through interaction with members of SR family proteins. To characterize the functional relationship between Pnn and SR proteins, we utilized RNAi in this study to knock-down the endogenous Pnn. We showed that depletion of Pnn expression induces reduced expression of several SR family proteins, including SC-35, SRm300, SRp55, and SRp40, but not that of other nuclear proteins, such as p53, MDM2, and ki67. Additionally, we also demonstrated that depletion of Pnn expression could modulate splice site selection of model reporter minigene in vivo. Our finding is significant in terms of regulation of SR protein cellular concentration because it reveals that Pnn may play a general role
in the control of the cellular amount of family SR proteins through downregulation of its own expression, whereby providing us with a better understanding of the cellular mechanism by which Pnn fulfills its biological function.

前言……………………………………………………………………… 3
1. Pinin ……………………………………………………………… 3
2. pre-mRNA剪接……………………………………………………… 5
3. 研究目標 …………………………………………………………… 6
實驗材料與方法………………………………………………………… 7
1. 細胞培養…………………………………………………………… 7
2. 質體………………………………………………………………… 7
3. 細胞轉染…………………………………………………………… 8
4. 西方點墨法（Western Blotting）…………………………… 11
5. 細胞免疫螢光染色（Immunoflurorescence staining）……12
6. 反轉錄聚合脢鏈鎖反應 （Reverse Transcription -PCR）… 13
結果……………………………………………………………………… 15
1. Pnn單株抗體21D5轉染細胞並不影響Pnn與SR蛋白質之作用…… 15
2. 建構Pnn之RNAi載體……………………………………………… 16
3. Pnn表現量降低會影響其他SR家族蛋白質的表現……………… 17
4. Pnn表現量降低會使SR家族蛋白質之蛋白表現量減少………… 18
5. Pnn表現量降低會改變mRNA剪接方式並降低近端剪接………… 20
討論……………………………………………………………………… 22
附圖……………………………………………………………………… 25
附錄一抗體列表………………………………………………………… 40
附錄二質體列表………………………………………………………… 41
參考文獻………………………………………………………………… 42
Appendix ……………………………………………………………… 46

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