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標題: | DEAD-box 核糖核酸解旋酶VBH-1/DDX3在let-7功能中所扮演的角色 The role of DEAD-box RNA helicase VBH-1/DDX3 in let-7 microRNA function |
作者: | Chiao-Nung Chen 陳巧儂 |
指導教授: | 詹世鵬(SHIH-PENG CHAN) |
關鍵字: | microRNA,let-7,DDX3,VBH-1,miRISC,AGO2, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | MicroRNAs(miRNAs)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。miRNAs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miRNA生合成及作用時,RNA-protein的交互作用及RNA的構型重組,都可能需要屬於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,發現會加強由let-7基因突變所造成的異常性狀。顯示VBH-1可能在let-7相關基因調節中扮演正向調控的角色。但降低VBH-1表現並不會減少let-7的表現量。因此,先前所看到降低VBH-1表現,導致增強的let-7缺陷性狀並不是因為let-7的表現量減少,而可能是與微型核醣核酸沈默複合體(miRISC)的功能有關。利用gfp螢光報告基因帶有已知為let-7目標基因lin-41的3' UTR來探討VBH-1對於let-7調控目標基因的可能影響,發現降低VBH-1表現會使得原本受let-7調節抑制的螢光略為回復。本篇我們利用anti-sense oligonucleotide pull-down assay探討let-7是否會跟VBH-1有交互作用,發現anti-let-7反應中可以偵測到Argonaute蛋白與VBH-1。已知在線蟲的體側表皮接縫細胞(seam cell)的分裂與分化過程中,需要較高的miRNA以及Argonaute活性。在我們基因轉殖的線蟲中也發現GFP-VBH-1大量表現於seam cell中,這支持我們的假說,亦即VBH-1可能參與miRISC的功能。而我們在線蟲共同免疫沈澱也可以看到VBH-1與ALG-1有交互作用。另一方面,我們也探討人類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,我們利用共同免疫沈澱法發現DDX3和AGO2之間存在RNA independent的交互作用。並且以免疫螢光染色法可以觀察到DDX3與AGO2在細胞質會有相同表現的位置,而這個位置可能為P bodies(Processing bodies)。另一方面,當我們利用siRNA降低人類細胞中DDX3的表現量時,會減低let-7對3’ UTR帶有 let-7 binding site的luciferase reporter的負向調控。綜合以上結果可知,VBH-1/DDX3會和Argonaute蛋白交互作用並且有助於miRISC的功能。 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 miRNA biogenesis and functioning, remodeling of RNA structures or RNA-protein interactions may require the RNA helicase activity of the DEAD-box proteins. In this study, we found that depletion of the DEAD-box protein VBH-1 by RNAi in Caenorhabditis elegans induced heterochronic phenotypes associated with let-7 mutations, 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. Thus, the induction of let-7 mutant phenotypes by vbh-1(RNAi) may not stem from lower levels of let-7 but probably from a failure in miRISC function. In addition, we used a gfp reporter fused with the 3’ UTR of lin-41, which is a known 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, consistent with the seen phenotypes upon vbh-1(RNAi). Moreover, we detected both the Argonaute ALG-1 and VBH-1 in an anti-let-7 oligonucleotide pull down assay, suggesting an interaction between VBH-1 and the miRISC machinery. Regulation of proliferation and differentiation of the epithelial seam cell requires active let-7/miRISC activity, where we detected a high level of VBH-1 expression by monitoring a stable GFP::VBH-1 strain prepared in this work. We also found an interaction between ALG-1 and VBH-1 in C. elegans by co-immunoprecipitation. These results support our hypothesis that VBH-1 is involved in the miRISC function. Interestingly, we also detected an interaction between human DDX3, the ortholog of C. elegans VBH-1, and the Argonaute protein AGO2 by co-immunoprecipitation. By immunofluorescence assays, we observed concentrated DDX3 signals colocalized with AGO2 at distinct cytoplasmic foci. On the other hand, we found that DDX3 knockdown by siRNA derepressed the control of a luciferase reporter carrying let-7 binding sites in the 3' UTR. Taken together, our results suggest that VBH-1/DDX3 interacts with the Argonaute and facilitates miRISC function. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49523 |
DOI: | 10.6342/NTU201602926 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
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