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研究生:陳信舟
研究生(外文):Hsin-Chou Chen
論文名稱:Ntc77影響剪接體活化的功能性區域
論文名稱(外文):A Functional Domain of Ntc77 in Spliceosome Activation
指導教授:鄭淑珍鄭淑珍引用關係
指導教授(外文):Soo-Chen Cheng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:剪接體剪接體活化Prp19複合體
外文關鍵詞:spliceosomespliceosome activationPrp19p-associated complex
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剪接反應是由一個叫做剪接體的核醣核酸蛋白複合體所催化的兩步驟轉酯反應,將前訊息核醣核酸中介入子序列移除的過程。在剪接體活化過程中,U4小核醣核酸蛋白複合體會因重組作用而離開剪接體,Prp19複合體就在此時結合至剪接體,並進行剪接體的活化。之前實驗室研究發現Prp19複合體成員之一Ntc77的抗體會抑制剪接反應進行,但並不影響Prp19複合體與剪接體的結合,因此推測Ntc77的功能可能是在Prp19複合體結合至剪接體後,參與在剪接體活化過程之中。Ntc77約為82kD,序列中包含了15個TPR主模。為了找出Ntc77參與在剪接體活化的功能性區域,我將NTC77的刪除突變株藉由GAL啟動子大量表現的方式,尋找能夠具有顯性抑制特性的突變株。只有ntc77∆N-3刪除突變株的蛋白大量表現時具有顯性抑制的性狀,並且伴隨前訊息核醣核酸的累積,顯示其剪接缺陷。免疫沉澱分析顯示Prp19複合體的形成及穩定度並不受ntc77∆N-3影響。純化後含有Ntc77∆N-3的Prp19複合體對回復免疫去除Prp19複合體後酵母菌細胞萃取液的剪接活性,僅具極低效率。但Ntc77∆N-3複合體結合至剪接體的能力卻正常,其所形成的剪接體,也只有少量Lsm會因剪接體活化時構型重組而脫離,因此我們結論Ntc77的前三個TPR主模對於剪接體活化應扮演重要角色。
Splicing removes intron from precursor messenger RNA via a two-step transesterification reation catalyzed by a large ribonucleoprotein complex, called the spliceosome. The Prp19p-associated complex (NTC) is incorporated into the spliceosome after dissociation of U4 snRNP and plays an important role in spliceosome activation. Previous studies have shown that antibodies against Ntc77 inhibited the splicing reaction but did not inhibit binding of NTC to the spliceosome, suggesting that Ntc77 might function in spliceosome activation after binding of NTC to the spliceosome. Ntc77 is a protein of 82-kD and contains 15 TPR repeats. To identify the domain of Ntc77 involved in spliceosome activation, a strategy of isolating dominant negative mutants was taken. A series of deletion mutants were constructed and placed under the control of GAL-promoter to regulate the expression of the mutant genes. Only ntc77∆N-3 (deletion from the N-terminus to the third TPR repeat) showed severe growth phenotype upon overexpression of the protein. Unspliced precursor mRNA accumulated correspondingly, indicating a splicing defect. Immunoprecipitation studies revealed that the integrity of NTC was not affected in ntc77∆N-3. NTC containing the wild-type or ∆N-3 of Ntc77 was affinity purified and studied for their complementation activity of NTC-immunodepleted extracts. The results show that NTC containing Ntc77∆N-3 could efficiently associate with the spliceosome, but only poorly complemented the NTC-depleted extract for splicing, consistent with the dominant negative phenotype of nct77∆N-3 mutant. Dissociation of Lsm from the spliceosome was also retarded, indicating failure in spliceosome activation. We conclude that the first three TPR repeats are responsible for the activity of Ntc77 in spliceosome activation.
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