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標題: | 人類粒線體解旋酶Suv3的生化特性 Biochemical characterization of the human mitochondria helicase Suv3 |
作者: | Wei-Hsuan Tang 唐偉軒 |
指導教授: | 袁小琀 |
關鍵字: | 解旋?,RNA降解,粒線體RNA剪接, helicase, RNA turnover,RNA decay,mitochondrial RNA processing, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 粒線體在細胞中的主要功能是產生能量供細胞使用,在真核細胞中是不可或缺的胞器。而粒線體的缺失亦會導致數種人類疾病。為了維持粒線體功能的正常,RNA的降解在粒線體基因表現以及RNA品質控管上扮演重要角色。人類粒線體中,二聚體的解旋酶Suv3與三聚體的外切酶PNPase形成五聚體的RNA降解體,將具有二級結構的RNA解開,並從3'端往5'端分解。雖然單體的Suv3結構已經被報導,但Suv3是如何形成二聚體,並與PNPase交互作用形成RNA降解體,來合作解開並分解RNA仍然未知。
為了瞭解Suv3如何解開雙股RNA,我們從大腸桿菌表達了刪除N端粒線體位置序列(mitochondrial localization sequence)的人類粒線體解旋酶Suv3(Suv3)及C端刪除蛋白(Suv3ΔC)。純化後的Suv3與Suv3ΔC分別以二聚體與單體存在,說明Suv3的C端與形成二聚體有關。我們發現二聚體Suv3相較於單體Suv3ΔC對單股RNA的結合親和力強10倍,並且二聚體Suv3不論ATP或ADP是否存在下,仍可維持與單股RNA結合的能力。而單體Suv3ΔC只有在ATP存在時有較高的單股RNA結合親和力。解旋酶活性測試中,當有ATP時,Suv3可解開帶有3' overhang的雙股RNA,而二聚體Suv3相較於單體Suv3ΔC的解旋酶活性略好。Suv3的二聚體結構不僅促進解旋酶活性,更重要的是與PNPase交互作用。綜合以上實驗結果,Suv3與RNA受質的結合不受ATP或ADP調控,說明在ATP水解後,Suv3會持續與RNA結合並繼續解開雙股RNA,被解開的單股RNA則被與Suv3交互作用的PNPase連續的降解掉。最後總結,二聚體Suv3與三聚體PNPase一起形成一個有效率的粒線體RNA降解體,可以持續的解開並分解粒線體中具有二級結構的RNA。 Mitochondria are indispensable organelles in all eukaryotes as they provide cellular energy in the form of ATP. Defects in mitochondrial functions thus lead to various mitochondria disorder and human diseases. To maintain the normal function of mitochondria, RNA turnover plays an important part in the regulation of mitochondrial gene expression and the surveillance of abnormal RNA molecules. In human mitochondria, dimeric helicase Suv3 and trimeric polynucleotide phosphorylase (PNPase) form a pentameic RNA exosome for unwinding and degrading structured RNA from 3' to 5' ends. Although the structure of monomeric Suv3 was reported, it remains unclear how Suv3 is assembled into a homodimer to interact with PNPase in the mitochondrial RNA exosome for corporative RNA unwinding and degradation. To understand how Suv3 unwinds a RNA duplex, we expressed human Suv3 (residues 44-786) and a C-terminal tail-truncated Suv3, named Suv3ΔC (residues 44-722) in E. coli, both without the N-terminal mitochondrial localization sequence (MLS). The purified recombinant Suv3 and Suv3ΔC formed dimers and monomers, respectively, suggesting that the C-terminal tail of Suv3 mediates protein dimerization. We found that Suv3 had a 10-fold greater RNA-binding affinity compared with Suv3ΔC at pH7.4. Suv3 bound single-stranded RNA with high affinities in the presence or absence of ATP and ADP, while Suv3ΔC bound RNA with a high affinity only in the presence of ATP. Suv3 had a slightly higher ATP-dependent helicase activity than Suv3ΔC in unwinding a duplex RNA with a 3'-overhang. Dimeric conformation of Suv3 is important not only for promoting the ATP-dependent RNA helicase activity but also for interaction with PNPase. Taken together these results suggest that RNA is continuously bound with Suv3 after ATP hydrolysis and RNA unwinding, and the unwound single-stranded RNA is degraded processively by the Suv3-interacting PNPase. We thus conclude that dimeric Suv3 and trimeric PNPase form a robust machinery of mitochondrial RNA exosome for efficient processive RNA unwinding and degradation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74285 |
DOI: | 10.6342/NTU201903334 |
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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