請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18367
標題: | 阿拉伯芥粒線體剪切因子之功能性研究 Functional studies of Arabidopsis mitochondrial splicing factors |
作者: | Ya-Wen Hsu 許雅雯 |
指導教授: | 趙光裕(Guang-Yuh Jauh) |
關鍵字: | 阿拉伯芥,粒線體,內含子,剪切因子, Arabidopsis,mitochondria,intron,splicing factor, |
出版年 : | 2014 |
學位: | 博士 |
摘要: | 粒線體是真核生物細胞內的能量生產中心,經由呼吸作用產生足夠能量,以供給細胞及相關胞器執行其正常功能,因此是維持生物個體生命及生長發育所必須。故此,維持粒線體基因的正常表現是相當重要的。其基因表現主要經由後轉譯修飾來調控,例如RNA處理及編輯(processing and editing)、內含子剪切(intron splicing),及RNA穩定性(stability),其中內含子剪切是植物特有的調控機制。參與粒線體內含子剪切需要的蛋白質大部分源自細胞核基因。干擾這些剪切因子(splicing factor)的表現會造成內含子剪切發生問題,進而導致植物的生長發育遭到影響,例如雄性不孕、胚胎致死及營養生長發育遲緩等性狀。本論文中所探討的阿拉伯芥mTER15(mitochondrial transcription termination factor 15)及WTF9(What is This Factor 9)剪切因子即參與粒線體內含子剪切的重要功能。
論文第一部分係利用分子遺傳、細胞學及生物化學方法針對阿拉伯芥mTERF15蛋白進行功能性及分子作用機制的研究。當阿拉伯芥mTERF15基因突變會造成植物生長遲緩及生殖器官缺陷。通常mTERF家族被認為具有與DNA結合能力,但實驗證實mTERF15蛋白無法與DNA結合,而是藉由與RNA結合以調控阿拉伯芥粒線體內nad2基因第三個內含子的剪切,進而影響粒線體呼吸作用complex I的功能,終致植物生長發育遲緩。因此,mTER15是一個參與粒線體內nad2基因第三個內含子剪切的剪切因子,在植物生長發育過程裡扮演相重要的角色。 論文的第二部分則在探討WTF9以及HSP60在粒線體內含子剪切的功能及分子作用機制。在先前的研究發現WTF9會參與粒線體rpl2 以及ccmFC基因的剪切。藉由免疫沉澱法發現HSP60會與WTF9進行蛋白質交互作用。不論在真核或原核細胞內,HSP60被認為是幫助蛋白質摺疊的分子伴護蛋白,但實驗證據顯示HSP60也調控RNA代謝等其他生物功能。這部分研究發現,HSP60與WTF9蛋白皆會與粒線體內ccmFC intron結合並調控rpl2 以及ccmFC基因的剪切效率;此外若阿拉伯芥失去部分的HSP60蛋白即會造成與wtf9突變株相似的外表型,這些結果顯示HSP60可能與WTF9參與rpl2 及ccmFC內含子剪切的過程。 Mitochondria, the central power plant in all eukaryotic cells, provide a majority of energy for normal cellular maintenance and functions. The function of mitochondrial respiratory chain needs proper mitochondrial gene expression which is regulated by extensive post-transcriptional controls including 5’ and 3’ RNA processing, intron splicing, RNA editing, and controlled RNA stability. Of these post-transcriptional processes, intron splicing is an example of acquired nonprokaryotic traits in plant mitochondria and involves in the vast majority of nuclear-encoded proteins. Disruption of these proteins causes the splicing defects of mitochondrial genes and results in the defective plant growth and development such as male sterility, embryo lethality and growth retardation. In this dissertation, three proteins with unique roles in Arabidopsis mitochondrial intron splicing were studied. In the first part, the biological functions and molecular mechanism of Arabidopsis mTERF15 (mitochondrial transcription termination factor 15) in mitochondria are investigated using molecular genetics, cytological and biochemical approaches. The null homozygous mterf15 mutant was found to result in substantial retardation of both vegetative and reproductive development, which was fully complemented by the wild-type genomic sequence. Surprisingly, mitochondrion-localized mTERF15 lacks obvious DNA-binding activity but processes mitochondrial nad2 intron 3 splicing through its RNA-binding ability. Impairment of this splicing event not only disrupted mitochondrial structure but also abolished the activity of mitochondrial respiratory chain complex I. These effects are in agreement with the severe phenotype of the mterf15 homozygous mutant. This study suggests that Arabidopsis mTERF15 functions as a splicing factor for nad2 intron 3 splicing in mitochondria, which is essential for normal plant growth and development. The second part of this dissertation focuses on the function of WTF9 (What is This Factor 9) and its interacting proteins, HSP60s, in mitochondrial intron splicing. Using co-immunoprecipitation, we identified HSP60s as interacting proteins of WTF9. HSP60s are recognized as molecular chaperones to assist protein folding in both eukaryotic and prokaryotic cells. However, accumulating evidence suggests HSP60s also participate in other biological functions such as RNA metabolism and protection. As noncanonical function of HSP60s, we found HSP60s interact with 48 nucleotides of ccmFC intron as WTF9. The seedling phenotype of hsp60-3a-1hsp60-3b-1 mutants with small stature suggests the importance of HSP60s in plant growth and development. Also, the splicing efficiency of rpl2 and ccmFC were reduced in hsp60-3a-1hsp60-3b-1 double mutants. These observations are similar to the findings in wtf9 mutants and suggest that HSP60s are involved in mitochondrial RNA splicing of rpl2 and ccmFC introns. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18367 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 6.62 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。