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Title: | 線蟲體內DEAD-box核醣核酸解旋酶VBH-1和MTR-4在let-7功能中所扮演的角色 The role of Caenorhabditis elegans DEAD-box RNA helicases VBH-1 and MTR-4 in let-7 microRNA function |
Authors: | Guan-Rong Chen 陳冠融 |
Advisor: | 詹世鵬 |
Keyword: | 線蟲,微型核醣核酸,let-7,VBH-1,MTR-4,DDX3, C. elegans,miRNA,let-7,VBH-1,MTR-4,DDX3, |
Publication Year : | 2014 |
Degree: | 碩士 |
Abstract: | 微型核醣核酸 (mircoRNAs) 是微小單股具調節性的核醣核酸,通常在後轉錄階段 (post-transcriptional level) 控制基因的表現。微型核醣核酸影響非常廣泛層面的生物作用機制,他們的功能缺失可導致許多人類疾病與癌症。在微型核醣核酸生合成及作用時,許多步驟中發生了核醣核酸分子間或本身分子內交互作用的變化,以及核醣核酸蛋白複合體的構型重組,可能需要屬於DExD/H-box蛋白家族核醣核酸解旋酶 (RNA helicase) 活性的參與。我們在線蟲中利用RNA干擾的方式去除VBH-1表現會加強由let-7亞效等位基因突變株所造成的異常性狀。顯示VBH-1可能在let-7相關的基因調節中扮演正向調控的角色。當減低VBH-1表現時並不會減少let-7的表現量及其他數個參與在heterochronic pathway中的微型核醣核酸的表現量,如lin-4, miR-48 and miR-241。相較之下,我們發現減低VBH-1時卻發現會增加初始primary let-7的生合成,而在前驅物precursor let-7 及成熟mature let-7的增加的量並不太明顯。因此,先前所看到降低VBH-1表現導致增加let-7缺陷性狀至少不是因為let-7的表現量減少,而可能是因為微型核醣核酸沈默複合體 (miRISC) 的功能有關。接下來我們利用帶有已知為let-7目標基因LIN41的三端非編碼區的gfp螢光報告基因來探討VBH-1對於let-7調控目標基因的可能影響。我們發現降低VBH-1表現會使得原本受let-7調節抑制的螢光表現略為回復,這符合之前所觀察到的線蟲性狀且支持我們的假說。為了更進一步探討VBH-1是否參與在miRISC的功能的功能,我們想要在線蟲中表現一個帶有三個HA及myc標記的VBH-1蛋白並找尋和miRISC組成蛋白之間可能的交互作用。然而,可能因為外加的VBH-1過量表現造成細胞毒性,目前我們並沒有辦法建立穩定表現外加VBH-1的線蟲品系。因此,我們先探討人類VBH-1的同源性蛋白DDX3和Argonaute 蛋白Ago2之間的交互作用。有趣的是,我們利用共同免疫沉澱法發現DDX3和Ago2之間存在不需要的RNA的交互作用。此外,當利用siRNA的方式降低DDX3表現時,可以使得在三端不轉錄區帶有let-7結合位的螢光素酶 (luciferase)報告基因的抑制效果降低。由以上的結果可得知,VBH-1/DDX3會和Argonaute交互作用並且有助於miRISC功能。
在這篇論文中,我們也發現另一個TRAMP複合物中的DExD/ H-box蛋白MTR-4表現降低時,會導致pre-let-7和mature let-7的表現量減少,並且會造成pri-let-7的累積,顯示MTR-4可能於核內進行的初始微型核醣核酸之處理有關,其詳細作用機制則有待進一步闡明。 MicroRNAs (miRNAs) are small non-coding regulatory RNAs that usually regulate gene expression at the post-transcriptional level. They are involved in a broad spectrum of biological processes and their dysfunction is frequently associated with a variety of human cancers. In the multiple processes of miRNA biogenesis and function, rearrangements of RNA structures and conformational changes of ribonucleoprotein complexes may require the RNA helicase activity of the DExD/H-box protein family. We found that depletion of the DExD/H-box protein VBH-1 by RNAi in Caenorhabditis elegans enhanced the phenotypes caused by the hypomorphic let-7 (n2853) mutant, suggesting that VBH-1 may play a positive role in let-7 miRNA-mediated gene regulation. Depletion of VBH-1 did not decrease the levels of let-7 miRNA or several other miRNAs involved in the heterochronic pathway, such as lin-4, miR-48 and miR-241. Instead, we found that depletion of VBH-1 caused a significant increase in the primary let-7 levels while the increase in the precuosor and mature let-7 levels was modest to subtle. Thus, the enhancement of let-7 mutant phenotype by vbh-1(RNAi) may not be due to lower levels of let-7 miRNA and is probably linked to miRISC function. We used a gfp reporter fused with the 3’ UTR of lin-41, a known and well-studied let-7 target, to determine the effect of vbh-1(RNAi) on let-7 regulation. We found that vbh-1(RNAi) relieved the let-7 repression on the reporter, which is consistent to the phenotype assay and supports our hypothesis. To further investigate the involvement of VBH-1 in miRISC function, we planned to express 3xHA-myc-VBH-1 in Caenorhabditis elegans and determine possible interactions between VBH-1 and miRISC components. Unfortunately, stable lines of transgenic animals were never successfully established, perhaps due to putative cellular toxicity of over-expressed exogenic VBH-1. Hence, we instead examined possible interactions between human DDX3, the ortholog of Caenorhabditis elegans VBH-1, and the Argonaute protein Ago2. Interestingly, we detected an RNA-independent DDX3-Ago2 interaction by co-immunoprecipitation. In addition, we found that DDX3 knockdown by siRNA derepressed the control on let-7 binding sites in the 3’ UTR of a luciferase reporter. Collectively, our results suggest that VBH-1/DDX3 interacts with Argonaute and may facilitate miRISC function. In this thesis, we also show that depletion of another DExD/H-box protein MTR-4, a component of the TRAMP complex (Trf4/Air2/Mtr4p polyadenylation complex), reduced the levels of pre-let-7 and mature let-7 miRNA and caused accumulation of pri-let-7, suggesting that MTR-4 may be involved in the primary miRNA processing in the nucleus. The mechanism of action awaits further elucidation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56165 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 微生物學科所 |
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