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標題: | 線粒體RNA降解體的組成與分析 Mapping the interaction between PNPase and Suv3 in RNA turnover |
作者: | Chiu-Ju Wu 吳秋儒 |
指導教授: | 袁小琀 |
關鍵字: | 核醣核酸降解,線粒體核醣核酸降解體,核醣核酸外切?,解旋?,蛋白質互相作用, RNA turnover,mitochondrial RNA exosome,exoribonuclease,helicase,protein-protein interactions, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 中文摘要
核醣核酸的降解,在基因表現以及核醣核酸品質管控上,扮演著重要的角色。核醣核酸的降解會經由一系列的酵素來進行,包含了核醣核酸內切酶、核醣核酸外切酶以及解旋酶。在核醣核酸的降解過程中,有一個重要的複合體形成,為核醣核酸降解體(RNA degradosome or exosome)。它是一個具有保留性的複合體,在原核生物以及真核生物中皆存在,能降解具有二級結構的核醣核酸。核醣核酸降解體包含由3′端往5′端進行分解核醣核酸的外切酶,以及解開二級結構的解旋酶。在人類線粒體內擔任外切酶的PNPase會和解旋酶Suv3形成線粒體內的核醣核酸降解體。然而在核醣核酸降解體中,我們並不清楚外切酶是如何和解旋酶結合,一起來降解具有二級結構的核醣核酸。所以我們以人類和小鼠的線粒體PNPase和Suv3作為研究的主題,來釐清線粒體內的RNA降解體如何組成降解RNA的複合體。 目前我們已從大腸桿菌中大量表現了老鼠線粒體內mPNPase以及mSuv3,純化的蛋白質能以3比2的比例形成穩定的mPNPase-mSuv3複合體。在人類線粒體內的hPNPase以及hSuv3亦能以3比2的比例形成穩定的複合體。藉由蛋白質刪除區塊實驗,我們發現hPNPase的S1區塊、hSuv3的NTD區塊以及C端區域,對於複合體的結合是重要的,而且hSuv3的C端區域會影響hSuv3是否能形成二聚體。生化分析的結果證實mSuv3藉由水解ATP能鬆開具有二級結構的核醣核酸,並幫助mPNPase分解具有二級結構的核醣核酸。從ATPase活性分析上,我們發現當hPNPase與hSuv3結合後,hSuv3的ATPase活性可被激化,但無論hPNPase是否具有phosphorolysis活性,對於hSuv3的ATPase活性激化上並沒有明顯差異。我們並已篩選出mPNPase的結晶條件,但由於X光繞射後所得的解析度並不高,還需要調整結晶條件。綜合以上的實驗結果,我們可推論哺乳動物線粒體內的PNPase,以S1區塊與Suv3解旋酶直接結合,形成五聚體。而Suv3以NTD區塊以及 C端區域與PNPase結合。Suv3提升PNPase降解具有二級結構的RNA的能力,PNPase亦激化Suv3的解旋酶活性。這兩個蛋白質互相作用,提高彼此的活性,可以快速的合作著來降解線粒體RNA。 Abstract RNA turnover plays an important role in regulating gene expression and RNA quality surveillance. Many enzymes participate in RNA turnover, including endoribonucleases, exoribonucleases and helicases. In the process of RNA turnover, a protein complex, termed RNA degradosome in prokaryotes or exosome in eukaryotes, degrades RNAs with secondary structures. In human mitochondria, the exoribonuclease PNPase interacts with Suv3 helicase and forms the mitochondrial RNA exosome for RNA degradation. However, it remains unclear how PNPase interacts with Suv3 helicase and how they cooperatively degrade RNA with secondary structures. Here using human and mouse recombinant proteins, including PNPase and Suv3, we investigate how these two proteins are assemble into the mitochondrial exosome for RNA degradation. Both human and mouse PNPase and Suv3 were expressed in E. coli, and the purified proteins formed a stable 3-to-2 pentameric complex in which PNPase was a trimer and Suv3 was a dimer. We constructed the truncated mutants of PNPase and Suv3, and found that the S1 domain of hPNPase, and NTD and C-terminal tail of hSuv3 were involved in hPNPase-hSuv3 complex assembly. Moreover, the C-terminal tail of hSuv3 is critical for hSuv3 dimerization. Our biochemical assays further show that the exoribonuclease activity of mPNPase was promoted by mSuv3 in the presence of ATP in the degradation of structured RNA. Conversely, the ATPase activity of hSuv3 was stimulated not only by ssDNA/ssRNA, but also by hPNPase, suggesting that hPNPase directly interacts with hSuv3 to stimulate its ATPase activity. We also crystallized mPNPase, but its resolution (4.3Å) was not sufficient for structural determination. In summary, our results suggest that in mammals, the S1 domain of PNPase, and NTD and C-terminal tail of Suv3 are involved in mitochondria exosome assembly. The exoribonuclease activity of PNPase is promoted by Suv3 in the presence of ATP, and similarly, the ATPase activity of Suv3 is stimulated by its interaction with PNPase in degrading structured RNA. Bulk RNAs are thus degraded efficiently and cooperatively by the PNPase-Suv3 exosome complex in mitochondria. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68558 |
DOI: | 10.6342/NTU201703840 |
全文授權: | 有償授權 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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