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標題: | BCAS2構造和RNA剪接功能的關係 Structure and Function Relationship for BCAS2 RNA Splicing |
作者: | Meng-Hsuan Chou 周孟萱 |
指導教授: | 陳小梨(Show-Li Chen) |
關鍵字: | BCAS2,果蠅,Delta-Notch 訊息傳遞,核醣核酸剪切, BCAS2,Drosophila,Delta-Notch signaling,splicing, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | BCAS2 (Breast carcinoma amplified sequence 2) 是一個約26-kD的核蛋白,被發現在MCF-7及BT-20等乳癌細胞株中表現量有上升現象。在早期的研究中指出,BCAS2與雌激素受體有直接的交互作用,並可做為雌激素受體在調控基因表現時的輔助因子。本實驗室過去的研究發現BCAS2也是抑癌蛋白p53的負調控者,但除了透過p53,BCAS2還可能藉由其他方式調控細胞生長。我們實驗室的研究亦指出BCAS2的確是參與核醣核酸剪切體 hPrp19/CDC5L complex的核心成員之一,並且參與核醣核酸的剪切。當果蠅體內BCAS2相似蛋白 (dBCAS2) 的表現在全身被默化時,會造成果蠅無法發育為成蟲;而利用組織特異性啟動子在果蠅翅膀降低dBCAS2的表現,則會造成翅膀的殘缺,這些證據顯示BCAS2在發育過程中的重要性。另外,在dBCAS2被默化的果蠅翅膀中異位表現人類的BCAS2 (hBCAS2) 可以挽救翅膀殘缺的情形,顯示人類與果蠅的BCAS2扮演相似的角色。
本實驗室先前已經證明BCAS2能調控Delta基因轉譯的過程中核醣核酸的剪切,進而調控Delta-Notch訊息傳遞路徑。因此本篇論文想要進一步去探討BCAS2在Delta基因轉譯核醣核酸剪切過程中所扮演的角色,以及它如何去調控Delta-Notch訊息傳遞路徑的活性。我們發現Delta-Notch訊息傳遞路徑的活性會因為dBCAS2表現量減少而下降。當dBCAS2被默化時,會降低Delta基因在轉譯的過程中核醣核酸剪切的效率,進而導致Delta表現量下降、影響Delta-Notch 訊息的傳遞,但Notch基因在轉譯的過程中核醣核酸剪切的效率並不受到dBCAS2默化影響。同時我們證明BCAS2所調控Delta-Notch訊息傳遞路徑的活性會因為dBCAS2表現量減少而下降不是來自抑癌蛋白p53所造成的細胞凋亡。另外我們也證明在BCAS2的C端捲曲螺旋結構中的其中四個胺基酸點突變會影響到BCAS2與核醣核酸剪切體其他成員之間的交互作用,進而影響Dlk1基因核醣核酸的剪切。在果蠅體內大量表現這些點突變的BCAS2不會產生任何異常現象。另外,在dBCAS2被默化的果蠅翅膀中大量表現這些點突變的BCAS2,他們挽救翅膀殘缺的能力都如同野生型BCAS2,除了翅脈L5有縮短及分岔等微小缺陷。因此我們認為這四個點突變會影響BCAS2與核醣核酸剪切體其他成員之間的交互作用,但核醣核酸剪切體仍可以形成並具有功能。 Previously, we found that breast carcinoma amplified sequence 2 (BCAS2), a 26-kD nuclear protein, is a p53 negative regulator and is involved in cell growth regulations. We also proved that BCAS2 is indeed a component of hPrp19/CDC5L complex and functions in pre-mRNA splicing. Ubiquitous silencing of dBCAS2 and tissue-specific knockdown of dBCAS2 in the wings result in larval lethality and wing deformity respectively, suggesting the importance of dBCAS2 in Drosophila development. Ectopic expression of hBCAS2 in dBCAS2-depleted fly can rescue the lethality and wing deformity, indicating that hBCAS2 plays a similar role to dBCAS2. Our previous study showed that BCAS2 may be involved in Delta-Notch signaling. Here, we further confirmed that BCAS2 could affect Delta-Notch signaling via regulating Delta pre-mRNA splicing. Deprivation of dBCAS2 down-regulates the expression of Delta ligand by impeding splicing efficiency of Delta but not Notch, hence diminishing the transcription of Delta-Notch signaling target genes. We also found that the reduction of Delta-Notch signaling caused by dBCAS2 depletion is apoptosis-independent. Furthermore, four point mutations located in BCAS2 C-terminal coiled-coil domain could alter the hPrp19 complex affinity and Dlk1 alternative splicing pattern, indicating that these specific amino acids maybe associated with the affinity to hPrp19 complex and affect the alternative splicing of Dlk1. Moreover, the transgenic flies containing each point mutated hBCAS2 gene showed no defect in the wing morphology as the wild-type flies. Using these point mutated hBCAS2 to rescue the dBCAS2 depleted flies, the results showed that all of the four point mutated hBCAS2 could rescue the wing shape defect as wild type hBCAS2 flies did. Notably, the point mutant-rescued flies have minor defect at wing longitudinal veins L5, which appeared truncated and forked. Surprisingly, over-expression of Delta could fully rescue the defect in wing margin caused by BCAS2 depletion while the wing margin was intact as wild type when we over-expressed of Delta only. Therefore, BCAS2 can regulate Drosophila wing formation though Delta-Notch signaling pathway. In sum, BCAS2 can regulate Delta-Notch signaling pathway through Delta RNA splicing. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56291 |
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顯示於系所單位: | 微生物學科所 |
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