生命科學院: 植物科學研究所指導教授: 趙光裕; 謝旭亮呂官杰Leu, Kuan-ChiehKuan-ChiehLeu2017-03-032018-07-062017-03-032018-07-062016http://ntur.lib.ntu.edu.tw//handle/246246/274930長久以來RNA被認為是連結遺傳資訊DNA及最終蛋白質產物之間的關鍵分子；而隨著日益精進的研究，訊息RNA在轉譯成蛋白質之前仍可藉由後轉錄修飾作用，進而創造許多不同的成熟訊息RNA且豐富了所轉譯出蛋白質的多樣性。在真核生物中，細胞核的RNA代謝主要有以下調控方式:轉錄、RNA剪接、5’端修飾、3’端加工及多聚腺苷酸化；但在真核生物細胞的胞器中，則具有其它特殊的RNA代謝作用包含了：3’端及5’端的加工及RNA的編輯作用。因此，此論文便以不同的研究方法與角度，去探討兩個分別參與細胞核內與粒線體內RNA代謝的基因，是如何去影響RNA表現，並進一步控制植物生長發育的過程。 論文第一部份是探討一個阿拉伯芥中特殊的P型PPR (Pentatricopeptide repeat)蛋白質PPME (P-type PPR modulating editing)。當T-DNA突變株破壞了PPME的基因功能時會導致ppme突變株的植物生長遲緩、植株矮小及產生形狀皺縮及低萌芽能力的種子。不同於其它阿拉伯芥中的P型PPR蛋白質僅參與的胞器內mRNA加工作用，ppme突變失去了粒線體內nad1基因上898及937位置的RNA編輯效果，並造成粒線體呼吸傳遞鍊的活性損壞。在此研究中我們首次發現阿拉伯芥P型PPR蛋白質參與了RNA編輯作用的調控。 第二部份是探討阿拉伯芥另一個位於細胞核參與mRNA代謝╱3’端多聚腺苷酸化的關鍵蛋白質CstF64。在mRNA成熟過程中，3’端修飾作用是使mRNA成熟的關鍵步驟。當CstF64發生突變時會導致植物無法存活；而在較弱的cstf64突變株呈現許多生長發育的缺陷，例如不正常的葉型發育及晚開花的現象等。後續分析了cstf64-1突變株的RNA圖譜，發現了許多長短不一的成熟mRNA存在於細胞中，顯示CstF64可能參與mRNA3’端修飾作用的調節。之後分析中，我們發現了mRNA上所存在的CstF64所對應的鍵結序列（cis-element）AAG/NAAA，且該序列與cstf64-1突變株中造成長短不一mRNA性狀的基因群緊密相關；我們推論CstF64的功能可能是與真正poly(A)訊號附近的其他的AAG/NAAA結合，使參與mRNA3’端poly(A) 修飾作用的複合體蛋白質能辨認真正的poly(A)訊號，以在正確的位置完成poly(A) 修飾作用產生正常的mRNA分子。RNA is the major functional molecule that connecting the genetics information (DNA) and the final product (protein). There are many post-transcriptional modifications on mRNAs before translation and are important to generate the complexities of proteins. The RNA metabolisms in eukaryotes include transcription, RNA splicing, 5’ capping, 3’ processing, and polyadenylation in the nuclear genome. On the other hand, there are other unique RNA metabolic processes such as 5’ and 3’ end processing, and RNA editing in plant organelles genomes. How these metabolic processes are controlled is the critical question in RNA studies. In the first portion of this dissertation, the biological and molecular functions of Arabidopsis P-type PPR protein, PPME, were addressed. The null T-DNA mutant of ppme exhibited severe developmental defects, including retarded seedling growth, draft, seed abortion, shriveled seeds, and reduced seed viabilities. Besides, the RNA editing of mitochondrial nad1-898 and nad-937 was lost, leading to amino-acid shifts within NAD1 protein that resulted in the reduced complex I activity in the ppme mutant. Different from other P-type PPR members, the P-type PPR protein, PPME, was the first identified that functioned as an editing factor to modulate RNA editing in the mitochondrial nad1 transcripts and was important for plant development. In the second part of this dissertation, the important processing factor, Arabidopsis CstF64 in the nucleus, was studied. The cstf64 T-DNA insertional mutants are defective in post-embryonic development. Interestingly, there are many genes exhibited variable mRNA 3’ end length in the cstf64-1 knockdown mutant in our RNA-sequencing profile and the specific cis-elements AAG/NAAA was isolated from those genes showing variable mRNA 3’ end lengths in the cstf64-1 mutant. This AAG/NAAA motifs were randomly distributed within the regions nearby poly(A) sites in those genes with variable mRNA 3’ end length. Therefore, we speculate that Arabidopsis CstF64 may serve as barriers to block non-specific poly(A) signal usages by directly binding to the AAG/NAAA variants during transcription, and this mechanism was different from those in mammals and important for other trans-factors to recognize the major poly(A) signal.4470071 bytesapplication/pdf論文公開時間: 2019/7/26論文使用權限: 同意有償授權(權利金給回饋本人)RNA代謝粒線體RNA加工RNA編輯阿拉伯芥PPRCstF64RNA editingRNA processingPolyadenylationArabidopsisthesis10.6342/NTU201601010http://ntur.lib.ntu.edu.tw/bitstream/246246/274930/1/ntu-105-D98b42010-1.pdf