臺灣博碩士論文加值系統

酵母菌常常是我們用來研究其核醣體生合成途徑的模式生物。核醣體蛋白的表現量很高，含高正電及很多非結構區，因此，需要透過嚴謹的調控和回饋機制及伴護子系統，來控制蛋白質的合成量及穩定性。核醣體蛋白Rpl43p結合於核醣體上靠近E-site附近，以往的研究中並沒有深入其功能探究。而在實驗室的先前研究中，發現Rpl43p為puf6Δ的高通量抑制子，但卻不能抑制loc1Δ的生長缺失。但細胞失去Puf6p或Loc1p時，細胞中的Rpl43p量皆會下降，顯示Puf6p和Loc1p的功能和Rpl43p的穩定性及組裝上60S核醣體相關，可能為Rpl43p之伴護蛋白，但是功能上可能有差異性。
在本篇研究中，進一步分析三者間的關係。首先在核醣體rRNA的生合成方面，發現了在Puf6p、Loc1p缺乏的情形之下會產生和Rpl43p缺乏的情形相似。但藉由和運輸蛋白的結合測試以及螢光觀測Rpl43p的細胞位置分布結果，推測Puf6p、Loc1p對於核醣體的影響並非藉由改變Rpl43p在細胞中的分布所造成。進一步檢測RPL43的mRNA含量發現，RPL43成熟的mRNA量會隨著Puf6p、Loc1p的缺乏而降低，未成熟的non-splicing mRNA上升，尤其在Loc1p缺失中特別明顯。而這個缺失的現象其實並不僅僅針對Rpl43，在這個E-site周遭的Rpl2同時也會受其影響，但對其他的核醣體蛋白基因並無影響，因此，Puf6p、Loc1p可能可以專一性的影響RPL43 mRNA的成熟。本研究中也透過各種突變株的選殖建構，分析Rpl43p、Puf6p、Loc1p三者的結合關係。發現了Puf6p是藉由PUF其上的區塊和Rpl43p、Loc1p進行結合，Rpl43p則是藉由N端和Loc1p進行結合；Loc1p則是以N端和Puf6p以及Loc1p結合。

Saccharomyces cerevisiae (budding yeast) is a very common model used in ribosome biogenesis study. Ribosomal proteins are highly expressed. They are tentative to aggregate because of positive charges and unstructured domains. In order to build up a normal-functional ribosome, the qualities of ribosomal proteins need to be rigorously controlled. The regulation and feedback mechanism of these proteins are essential for normal cell growth and viability. Ribosomal protein Large subunit 43 (Rpl43p) is located nearby the E-site of ribosome. The functional domains and characteristic of Rpl43p is not fully identified. In our previous study, we found that Rpl43p is a high copy suppressor of puf6Δ but not loc1Δ. In the absence of PUF6 or LOC1, the level of Rpl43 protein decreased. The data suggests that the functions of Puf6p and Loc1p may correlate with the stability and assembly of Rpl43p with ribosome, but there are differences in between.
In this study, I would like to further dissect the connections among these three proteins. The processing defects of pre-ribosomal RNA in the puf6Δ and loc1Δ is similar to the mutant with depletion of Rpl43p. The results from the in vitro interaction assay with karyopherins and protein distribution of Rpl43 observed by fluorescence microscope suggested that Puf6p and Loc1p are not required for import of Rpl43p. While mature RPL43 mRNA decreased in the puf6Δ and loc1Δ, non-splicing mRNA increased, especially in loc1Δ. This phenomenon is also observed in RPL2, another protein in adjunction with Rpl43 around the E-site. However, Puf6 and Loc1 did not impact the RNA levels of other ribosomal protein genes. Therefore, Puf6 and Loc1 might be required for maturation of RPL43 mRNA. To further dissect the molecular function of each protein and analyze the interaction among Rpl43p, Puf6p and Loc1p, several mutants were constructed. I found that the interaction site of Puf6 to Rpl43p and Loc1p is mainly contributed by its PUF domain; the interaction site of Rpl43p to Loc1p is contributed by its N helix; the N terminal of Loc1p is important for Puf6p and Rpl43p to interaction.

目錄
摘要 iii
Abstract iv
目錄 vi
表目錄 ix
圖目錄 x
一、文獻回顧與探討 1
1.1核醣體簡介 1
1.2 rRNA的成熟過程 1
1.3 核醣體生合成 2
1.3.1 40S生合成 3
1.3.2 60S生合成 3
1.4細胞核內外運輸的調控 4
1.5伴護蛋白 5
1.6和本實驗相關的因子 6
1.6.1 Puf6 6
1.6.2 Loc1 6
1.6.3 Rpl43 7
二、研究動機 8
三、研究材料方法 9
3.1 電泳 9
3.2 DNA電泳膠體純化 9
3.3 限制酶切割 9
3.4 接合作用 9
3.5 突變株的建構 9
3.6 大腸桿菌轉型 10
3.7 蛋白質大量表現以及純化 10
3.8 啤酒酵母轉型化 10
3.9 螢光顯微鏡觀察 11
3.10 生長測試 11
3.11 核醣體圖譜分析法 (polysome profile) 11
3.12 RNA免疫沉澱法 (RNA immuno-precipitation) 12
3.13 DNase處理 12
3.14 反轉錄RNA 12
3.15 qPCR之定量 13
3.16 免疫沉澱 (immuno-precipitation) 13
3.17 胞外結合測試 (In vitro interaction) 13
3.18 酵母菌RNA萃取 14
3.19 北方墨點法分析 15
3.20 ICP 質譜分析 16
四、結果 17
4.1 Rpl43p、Puf6、Loc1p 對於 rRNA 的生合成途徑影響相似 17
4.2 Rpl43p 可以獨立依靠 Kap 蛋白質入核，並且其結合 Kap 蛋白質必須倚靠其 N 端的入核序列以及 helix 結構 18
4.3 Puf6p、Loc1p 影響特異性的影響 RPL43 mRNA 的成熟表現含量 20
4.4 Puf6p、Loc1p 和 Rpl2 的相關性 23
4.5 Puf6、Loc1、Rpl43 三者在 60S 核醣體的結合上先後順序的關聯 24
4.6 Rpl43 蛋白質分析 25
4.6.1 Rpl43 為一個保守性高、與 Zn 結合的核醣體蛋白 25
4.6.2 Rpl43 突變株分析 26
4.7 Puf6p 蛋白質結合位分析 28
4.8 Loc1p 蛋白質結合位分析 29
五、討論 30
5.1於RPL43、PUF6、LOC1的突變株中分析rRNA生合成的裁切情形 30
5.2 Rpl43的入核不依賴Puf6p以及Loc1p 30
5.3 Puf6和Loc1p會影響RPL43 mRNA的含量 31
5.4 Rpl2和Rpl43間的相關性探討 32
5.5 Rpl43p、Puf6p、Loc1p三者關係的探討 32
六、結論 34
七、參考文獻 35

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