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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 羅凱尹(Kai-Yin Lo) | |
dc.contributor.author | Ya-Han Ting | en |
dc.contributor.author | 丁亞涵 | zh_TW |
dc.date.accessioned | 2021-06-16T06:48:17Z | - |
dc.date.available | 2024-12-31 | |
dc.date.copyright | 2014-07-29 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-07-24 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57488 | - |
dc.description.abstract | 核醣體生合成是細胞中相當重要的過程,約略有兩百個相關因子參與在此途徑當中。核醣蛋白的生成會在細胞質中,但核醣體的組裝開始於核仁,所以核醣蛋白在組裝前必須先入核。在前人的研究中指出,伴護蛋白或運輸蛋白可以藉由電荷的中和來維持核醣蛋白的穩定性,並且增進其運輸效率。Bcp1已經被證實是PI4P 5-激酶Mss4的運輸蛋白,在本實驗室先前的研究中發現,Bcp1會參與核醣體生合成的途徑,且篩選出了Bcp1突變株的高通量抑制子Rpl23。在本研究中發現,Bcp1會與Rpl23結合,若細胞中缺乏Bcp1時,會造成Rpl23與60S的結合產生問題。另外,Tif6是一個核醣體生合成相關因子,結合在60S上與40S的結合面,防止未成熟的60S進入轉譯步驟,其主要的結合位置就在Rpl23上。當缺乏Bcp1影響到Rpl23時,進而影響到Tif6與60S的結合。然而,當Bcp1突變 (bcp1ts) 時,會滯留於60S上,此時Rpl23雖然於60S上,但亦會使Tif6無法順利結合至60S,影響核醣體的成熟過程。因此,Bcp1應為Tif6結合的一個調控點,當Rpl23被Bcp1協助順利結合至60S後,Bcp1需離開60S,Tif6才能結合至60S上,完成此一步驟。
接著,在本研究中還發現了另一個與Bcp1相關的因子Sqt1,在前人的研究中只知道Sqt1是Rpl10的伴護蛋白,並且主要在細胞質中幫助Rpl10的穩定及和60S的結合。在此我發現Sqt1會與Bcp1及Rpl23有交互作用,並且會進入到細胞核中。在Bcp1及Rpl23的突變株中,會使Sqt1滯留在核醣體上,進而使下游的Nmd3與60S的結合產生問題。另外,改變Sqt1的表現量會影響到Bcp1在細胞中的位置以及含量,顯示Sqt1除了是Rpl10的伴護蛋白外,對於核醣體生合成途徑可能具有其他的功能。 | zh_TW |
dc.description.abstract | The assembly of a ribosome requires about 200 transacting factors that are generally regulated by growth control and developmental signals. Ribosomal proteins are translated in the cytoplasm and must be imported for further assembly with rRNAs. It has been shown that chaperones or karyopherins could maintain the solubility of ribosomal proteins by neutralizing unfavorable positive charges and thus facilitate the transport. Bcp1 has been identified as a factor necessary for the export of Mss4, a PI4P 5-kinase. Here we have also identified Bcp1 as necessary for the maturation of 60S ribosomal subunits. Cells with mutations of Bcp1 are lethal and have deficient 60S levels. To further characterize its functional role, we conducted a high-copy suppressor screen and identified Rpl23 as a suppressor of bcp1 mutant. Bcp1 is shown to have a physical interaction with Rpl23. Depleting Bcp1 causes deficient loading of Rpl23. In addition, Tif6 loses its nucleolar distribution and 60S interaction. Tif6 is a ribosome biogenesis factor of 60S subunits with predominant nuclear and nucleolar distribution; its homolog in higher eukaryotic cells is eIF6. Tif6 is bound on the 60S subunits, and the C-terminus of Rpl23 is the major interaction site. The binding of Tif6 on the 60S subunits blocks association between 60S and 40S subunits by blocking the inter-subunit bridge formation. This blocking prevents inappropriate interaction with 40S subunits before full maturation of 60S. Interestingly, Rpl23 is loaded properly in bcp1ts mutant, which is persistent on the 60S subunits, and Tif6 is also blocked from binding. We conclude that the retention of Bcp1 causes steric hindrance and prevents Tif6 from assessing Rpl23. We find that Bcp1 is a novel 60S biogenesis transacting factor which works as a chaperone of Rpl23. Once Rpl23 is loaded properly by Bcp1, Tif6 is subsequently bound after Bcp1 is released.
We also find another Bcp1-related factor Sqt1, which is previously reported as a chaperone of Rpl10. Sqt1 maintains the stability of Rpl10, and facilitates the loading of Rpl10 onto the 60S in the cytoplasm. Here we find that Sqt1 forms a complex with Bcp1 and Rpl23, and transported into the nucleus together. In Bcp1 or Rpl23 mutant, Sqt1 retends on the ribosome, affecting the loading of Nmd3 downstream. Alterring the expression level of Sqt1 decreases the protein level of Bcp1 and change its cellular localization. These data suggest that Sqt1 has another function involved in the ribosome biogenesis pathway. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T06:48:17Z (GMT). No. of bitstreams: 1 ntu-103-R01623006-1.pdf: 35656482 bytes, checksum: 26af6888f3d0adbf22886c46f9a8431c (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 目錄
誌謝 i 摘要 ii Abstract iii 目錄 v 表目錄 viii 圖目錄 ix 附錄目錄 x 一、文獻回顧與探討 1 1.1 核醣體的生合成 1 1.1.1 RNA的生成及剪切 1 1.1.2核醣體組裝 2 1.1.3 40S的組裝 2 1.1.4 60S的組裝 3 1.1.5 核孔複合體 4 1.1.6 物質的進出核運輸調控 4 1.1.7 60S的出核 5 1. Nmd3 5 2. Mex67/Mtr2 6 3. Arx1 7 1.1.8 和本研究相關的60S生合成相關因子介紹 7 1. Tif6 7 2. Rpl10及Sqt1 8 1.2 磷酸肌醇的簡介 9 1.2.1 PI4P 5-kinase Mss4 9 1.2.2 Bcp1 10 1.3 60S合成和磷酸肌醇的關係 10 二、研究動機及研究問題 12 三、研究材料與方法 13 3.1 建構實驗所需的相關質體 13 3.1.1. PCR 13 3.1.2 電泳 13 3.1.3 DNA電泳膠體純化 13 3.1.4 限制酶切割 13 3.1.5 接合作用 14 3.1.6 選殖菌體 14 3.1.7 轉型至啤酒酵母 14 3.2 螢光顯微鏡觀察 14 3.3 生長測試 15 3.4 核醣體圖譜分析法(polysome profile) 15 3.5 Sucrose cushion 15 3.6 免疫沉澱 16 3.7 In vitro interaction 16 四、結果與討論-Bcp1與Rpl23之間的關係探討 17 4.1 結果 17 4.1.1 Bcp1會與Rpl23互相結合 18 4.1.2 Bcp1可以幫助Rpl23的折疊及穩定性 19 4.1.3 Bcp1影響到Rpl23與60S結合的正確性 19 4.1.4 Tif6與60S的結合需要Bcp1 20 4.1.5 RPL23會影響到Bcp1在細胞中正確的分布位置 21 4.1.6 Tif6與60S的結合需要Rpl23 21 4.1.7 停留在60S上的Bcp1會阻擋Tif6和60S之間的結合 22 4.2 結論 23 4.3 討論 23 4.3.1 Bcp1是Rpl23的伴護蛋白 23 4.3.2 Bcp1影響Rpl23與60S的結合 24 4.3.3 Bcp1為Tif6與60S結合的調控點 25 五、結果-Sqt1與Bcp1、Rpl23之間的關係探討 26 5.1 結果 26 5.1.1 Sqt1與Bcp1和Rpl23 有結合能力 26 5.1.2 測試三者間的存在是否影響其他蛋白的穩定性 27 5.1.2 Bcp1及Rpl23會影響到Sqt1與60S之間的結合 27 5.1.3 缺乏正確的Bcp1及Rpl23時會影響到Nmd3與60S之間的結合 28 5.1.4 缺乏Sqt1時會影響到Bcp1在細胞中的分布 29 5.2 結論 30 5.3 討論 30 5.3.1 Sqt1-Bcp1-Rpl23複合體 30 5.3.2 Nmd3於bcp1ts、tet:BCP1 及GAL:RPL23A rpl23b∆中減少和pre-60S的結合 31 5.3.3 Nmd3、Tif6於bcp1ts 及GAL:RPL23A rpl23b∆中減少和pre-60S的結合但卻累積於Lsg1-pre-60S免疫沉澱複合體 32 5.3.4 在bcp1ts時,Nmd3在免疫沉澱的pre-60S中的含量下降,但在多核醣體圖譜中的分布並無明顯改變 33 5.3.5 Bcp1位置在GAL:RPL10及rpl10ts中並不相同 33 六、參考文獻 35 | |
dc.language.iso | zh-TW | |
dc.title | Bcp1、Rpl23及Sqt1形成的小複合體於核醣體生合成的功能研究 | zh_TW |
dc.title | Functional characterization of the small complex containing Bcp1, Rpl23, and Sqt1 in ribosome biogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃偉邦(Wei-Pang Huan),陳美瑜(Mei-Yu Chen),徐駿森(Chun-Hua Hsu),冀宏源(Hung-Yuan Chi) | |
dc.subject.keyword | Bcp1,Rpl23,Tif6,Sqt1,Nmd3,核醣體生合成, | zh_TW |
dc.subject.keyword | Bcp1,Rpl23,Tif6,Sqt1,Nmd3,Ribosome biogenesis, | en |
dc.relation.page | 70 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-07-25 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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