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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹世鵬(Shih-Peng Chan) | |
| dc.contributor.author | Yu-De Chu | en |
| dc.contributor.author | 朱育德 | zh_TW |
| dc.date.accessioned | 2021-05-19T17:59:28Z | - |
| dc.date.available | 2021-08-26 | |
| dc.date.available | 2021-05-19T17:59:28Z | - |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-21 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7938 | - |
| dc.description.abstract | 目前已知微小核醣核酸(miRNA)是相當重要的基因表現調控因子,因其可調控超過 70%人類編碼基因之表現與否。然而,目前對於調控微小核醣核酸之生合成與活性的研究相對較少。本篇論文中,我們在線蟲中利用核醣核酸干擾作用(RNAi)抑制 DEAD/H-box 核醣核酸解旋酶家族之蛋白表現,而後觀察其是否有可能參與在微小 核醣核酸的生合成與活性之調控過程中。利用對於微小核醣核酸變化量及功能改 變較敏感的 let-7(mg279)突變株來進行實驗之後,我們發現在線蟲中抑制 DDX-23 及 DDX-17 的表現後會明顯促進 let-7 缺失時所呈現的表現型(phenotype),證明該 蛋白可能參與在 let-7 生合成或是執行功能過程當中。接著,我們亦發現抑制此兩 蛋白之表現後皆明顯造成成熟 let-7 表現量下降,但上游初期 let-7 (primary-let-7) 卻有累積的現象,指出其功能為初期 let-7 加工時所必須。我們後續檢驗了數個不 同的微小核醣核酸以及其初期微小核醣核酸(primary miRNA)之表現量後發現,除 了 let-7 以外,其他的 heterochronic 微小核醣核酸比如 lin-4,miR-48,miR-84 及 miR-241 之加工亦會受到此兩蛋白之影響。最後我們亦證明在特定組織內,非 heterochronic 微小核醣核酸 lsy-6 的功能會因為 DDX-23 及 DDX-17 表現量下降而 降低。因此,我們在此部分提出了一個 DDX-23 會參與在初期微小核醣核酸的加 工步驟過程中的新功能,而且也證明了 DDX-17 在初期微小核醣核酸加工過程中 的角色具有保守性。
在另一方面,在本篇論文中我們亦發現利用核醣核酸干擾作用降低線蟲中數個 hnRNP後只有人類細胞中hnRNP Q的同源蛋白HRP-2可以有效的減輕let-7(n2853) 突變株所表現出來的 let-7 缺失之表現型,證明 HRP-2 在 let-7 調控 lin-41 表現的過 程中扮演重要角色。此外,我們亦發現 HRP-2 可以直接跟微小核醣核酸誘導之核醣核酸靜默複合物(miRISC)形成交互作用,且這樣的交互作用是需要有核醣核酸 於其中當做橋樑來聯結。接著,我們在 lin-41 的訊息核醣核酸之 3’UTR 中位於 let-7 結合位下游但在poly-A 尾巴前找到了一個區域可以直接跟 HRP-2 結合,表示 lin-41 訊息核醣核酸可以是其中一個聯結 HRP-2 以及微小核醣核酸誘導之核醣核酸靜默 複合物的核醣核酸橋樑。然後,我們將此段會結合 HRP-2 的片段從 3’UTR 上切除, 並且接在綠螢光蛋白之核醣核酸後並送入線蟲中去表現。我們發現將此片段去除 的綠螢光蛋白表現量會明顯下降,顯示出此段蛋白對於 let-7 去調控 lin-41 是重要 的。因此我們認為在線蟲中 HRP-2 會降低 let-7 對 lin-41 的調控能力。有趣的是, 我們發現在人類細胞中,我們降低 hnRNP Q 的表現後,let-7 對於人類的 lin-41 同 源基因 TRIM71 的調控能力會有所上升。而且,如同線蟲中的 HRP-2 會結合 lin-41 訊息核醣核酸的 3’UTR 上特定位置一樣,我們在 TRIM71 的訊息核醣核酸之 3’UTR 中亦找到一個特定的區域可以跟 hnRNP Q 結合,且其同樣位於 let-7 結合位及 poly-A尾巴之間。最後我們亦將此區域從3’UTR中去除,結果發現會明顯增加let-7 對 TRIM71 調控的能力。因此,綜合以上所述,我們發現了一個新的 HRP-2 及其 同源蛋白 hnRNP Q 演化上保守的功能,也就是當其結合到 lin-41 或是其同源基因 TRIM71 的訊息核醣核酸 3’UTR 上特定的區域後,會減低 let-7 微小核醣核酸誘導 之核醣核酸靜默複合物對其調控之活性。 | zh_TW |
| dc.description.abstract | As post-transcriptionally gene regulators demonstrated by numerous studies, microRNAs have been predicted to control more than 70% of human coding genes expression. However, studies regarding modulators for miRNA biogenesis and/or function remain relatively fewer. Here, we performed a candidate-based RNAi screen in C. elegans to identify DEAD/H-box proteins involved in miRNA biogenesis and/or function. In a let-7(mg279) sensitized genetic background, knockdown of a homolog of yeast splicing factor Prp28p, DDX-23, or a homolog of human helicases p68 and p72, DDX-17, enhanced let-7 loss-of-function phenotypes, suggesting that these helicases play a role in let-7 processing and/or function. In both ddx-23(RNAi) and ddx-17(RNAi), levels of mature let-7 were decreased while pri-let-7 was found accumulated, indicating that the helicases likely act at the level of pri-let-7 processing. DDX-23 and DDX-17 were also required for the biogenesis of other known heterochronic miRNAs, including lin-4 and the let-7 family members miR-48, miR-84 and miR-241. Their function was not confined to the heterochronic pathway, however, since they were both necessary for down-regulation of cog-1 by the spatial patterning miRNA, lsy-6. Therefore, we present a novel function for C. elegans DDX-23 in pri-miRNA processing, and also suggest a conserved role for DDX-17 in this process. On the other hand, we also show that RNAi knockdown of C. elegans HRP-2, the homolog of mammalian hnRNP Q, relieved the heterochronic phenotypes in let-7(n2853) mutant animals, indicating an involvement of HRP-2 in let-7-lin-41 regulation. In addition, we detected an RNA-dependent interaction between HRP-2 and the Argonaute ALG-1, the core effector protein of miRNA-mediated silencing complex (miRISC). Moreover, we identified an HRP-2 response element in the lin-41 3’UTR at a position, downstream of the two let-7 complementary sites (LCSs), close to the poly(A)-tail. Deletion of this response element caused further down regulation of a GFP reporter carrying the lin-41 3’UTR in a let-7-dependent manner. Thus, we propose that HRP-2 impedes let-7/miRISC activity when binding to the lin-41 3’UTR. Interestingly, we found that depletion of human hnRNP Q also enhanced let-7-mediated down-regulation of TRIM71. Similar to the case in C. elegans, hnRNP Q binds to a response element adjacent to the poly(A)-tail in the TRIM71 3’UTR. Deleting this element from the 3’UTR significantly enhanced let-7 repression. Taken together, our findings uncover a novel evolutionarily conserved function for HRP-2/hnRNP Q to inhibit let-7/miRISC activity when they bind to specific response elements in the lin-41/TRIM71 3’UTRs. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:59:28Z (GMT). No. of bitstreams: 1 ntu-105-F99445120-1.pdf: 83676732 bytes, checksum: 30f1351f0cf6b6f6857b5b78a7f26abc (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書.............................................................................................I
致謝...............................................................................................................II 中文摘要........................................................................................................III English abstract.............................................................................................V 目錄...............................................................................................................1 Chapter 1 Introduction ................................................................................11 1.1 microRNAs.............................................................................................11 1.1.1 Discovery of miRNAs...........................................................................11 1.1.2 miRNA family......................................................................................12 1.1.3 Functions of miRNAs...........................................................................13 1.1.4 Biogenesis of miRNAs..........................................................................14 1.2 The let-7 miRNA....................................................................................14 1.2.1 Regulation of let-7 biosynthesis..........................................................14 1.2.2 The role of let-7 in maintaining stem cell stemness.............................16 1.2.3 The evolutionary conserved let-7/lin-41 regulatory pathway..............17 1.3 The DExD/H-box RNA helicases ............................................................18 1.3.1 Functions of the DExD/H-box RNA helicases.......................................18 1.3.2 The implication of DExD/H-box RNA helicases in the miRNA pathway.......................................................................................................19 1.4 Heterogeneous nuclear proteins (hnRNPs) .............................................20 1.4.1 Functions of hnRNPs...........................................................................20 1.4.2 The potential roles of hnRNPs in the miRNA pathway..........................21 1.5 The animal model—Caenorhabditis elegans...........................................22 1.5.1 The advantages of using C. elegnas as animal model..........................22 1.5.2 The heterochronic gene pathway and let-7 miRNA in C. elegans.........22 1.5.3 Controlling of vulval development by miR-84-mediated repression of let-60/RAS..................................................................................................24 1.5.4 The lsy-6 miRNA-mediated repression of cog-1.................................25 1.6 Project aims...........................................................................................26 Chapter 2 Materials and Methods.................................................................27 2.1 Caenorhabditis elegans..........................................................................27 2.1.1 Strains................................................................................................27 2.1.2 Culture...............................................................................................28 2.1.3 RNA-interference (RNAi) ....................................................................28 2.2 Construction of plasmids.......................................................................30 2.2.1 C. elegans RNAi clones.......................................................................30 2.2.2 C. elegans expressing plasmids...........................................................30 2.2.3 TRIM71 3’UTR fused luciferase plasmids..............................................32 2.2.3 hnRNP Q1 plasmids.............................................................................32 2.2.4 Ago2-GFP plasmid..............................................................................33 2.3 Microscopy analysis................................................................................34 2.4 RNA isolation.........................................................................................34 2.4.1 From C. elegans..................................................................................34 2.4.2 From human cell cultures....................................................................35 2.5 Northern blot analysis............................................................................35 2.6 RT-PCR and RT-qPCR.............................................................................37 2.6.1 Poly-A tail-based reverse transcription and qPCR................................37 2.6.2 Random priming-based reverse transcription and qPCR......................41 2.7 Lysate preparation.................................................................................45 2.7.1 For C. elegans western blot analysis....................................................45 2.7.2 For C. elegans immunoprecipitation experiment.................................46 2.7.3 For mammalian cell lines western blot analysis....................................46 2.7.4 For mammalian cell lines immunoprecipitation experiment.................47 2.8 Western blot analysis..............................................................................47 2.9 Co-immunoprecipitation........................................................................48 2.10 RNA-immunoprecipitation (RIP)...........................................................49 2.11 In vitro transcription............................................................................50 2.12 Biotinylated RNA affinity selection pull-down.......................................54 2.13 Generation of transgenic C. elegans.....................................................55 2.13.1 Conventional microinjection..............................................................55 2.13.2 Mos1 mediated single copy transgene insertion (MosSCI)...................55 2.14 Integration of extra-chromosomal array in C. elegans..........................56 2.14 Cell culture and transfection................................................................57 2.15 Lenti-virus infection............................................................................58 2.16 Luciferase assay...................................................................................60 2.17 DAPI staining.......................................................................................61 Chapter 3 Results........................................................................................63 3.1 The role of DDX-23 in primary microRNA processing in Caenorhabditis elegans.......................................................................................................63 3.1.1 The potential roles for several DEAD-box RNA helicases in let-7-mediated gene regulation............................................................................63 3.1.2 DDX-23 and DDX-17 promote let-7 maturation.................................67 3.1.3 DDX-23 and DDX-17 support the processing of primary let-7............67 3.1.4 DDX-23 and DDX-17 facilitate regulation of let-60/RAS by miR-84...68 3.1.5 DDX-23 and DDX-17 support pri-miR-84 biogenesis.........................70 3.1.6 DDX-23 and DDX-17 are also required for other heterochronic miRNAs processing...................................................................................................71 3.1.7 Expression of miRNA pathway components is not affected by depletion of DDX-23 or DDX-17................................................................................71 3.1.8 DDX-23 is localized to the nucleus and functions with miRNAs in multiple tissues...........................................................................................73 3.2 HRP-2/hnRNP Q impede let-7-mediated repression of lin-41/TRIM71 in Caenorhabditis. elegans and humans...........................................................75 3.2.1 HRP-2 functions upstream of lin-29...................................................75 3.2.2 Depletion of HRP-2 causes precocious lin-29 expression....................77 3.2.3 Depletion of HRP-2 enhances let-7-mediated repression of lin-41.....78 3.2.4 Depletion of HRP-2 does not alter let-7 level......................................79 3.2.5 C. elegans HRP-2 interacts with ALG-1 in an RNA-dependent manner........................................................................................................80 3.2.6 HRP-2 directly interacts with lin-41 3’UTR..........................................80 3.2.7 HRP-2 binds to lin-41 3’UTR at a position between LCS and poly-(A)..81 3.2.8 Deletion of HRE enhances let-7-mediated repression of lin-41...........84 3.2.9 HRP-2 locates in both nucleus and cytoplasmic fractions.....................85 3.2.10 hnRNP R does not involved in let-7-mediated repression of TRIM71........................................................................................................87 3.2.11 hnRNP Q blocks repression of TRIM71 by let-7..................................88 3.2.12 hnRNP Q is associated with miRISCs..................................................90 3.2.13 hnRNP Q binds to an element at a position near poly-A tail in TRIM71 3’UTR...........................................................................................................92 3.2.14 QRE in TRIM71 3’UTR is required for hnRNP Q binding......................93 3.2.15 Deletion of QRE enhances let-7-mediated repression of TRIM71.......94 Chapter 4 Discussion...................................................................................96 4.1 Helicase part..........................................................................................96 4.2 hnRNP part..........................................................................................102 Chapter 5 Table.........................................................................................108 Table 1. Using let-7(n2853) mutant to screen for hnRNP(s) involved in let-7/lin-41 pathway.......................................................................................108 Chapter 6 Figures......................................................................................109 Figure 1. The vulval bursting phenotype caused by RNAi knockdown of DEAD-box RNA helicase genes.............................................................................109 Figure 2. The retarded seam cell development caused by RNAi knockdown of several DEAD-box helicases.......................................................................111 Figure 3. Depletion of DDX-17 or MOG-4 causes discontinued clusters of seam cells..................................................................................................112 Figure 4. Adult hermaphrodites show disrupted seam cell syncytia or unfused seam cells upon RNAi against several RNA helicase genes...........................113 Figure 5. col-19::GFP expression is down-regulated upon RNAi against several RNA helicase genes....................................................................................115 Figure 6. RNAi knockdown of ddx-23, ddx-17 and mog-4 reduced mature let-7 levels................................................................................................117 Figure 7. DDX-23 and DDX-17 are required for pri-let-7 processing.........118 Figure 8. Inactivation of ddx-23 and ddx-17 enhance the MUV phenotype in let-60 gain-of-function mutant................................................................120 Figure 9. Reduced expression of ddx-23 and ddx-17 increases accumulation of pri-miR-84............................................................................................122 Figure 10. DDX-23 and DDX-17 are required for biogenesis of several heterochronic miRNAs................................................................................123 Figure 11. Reduction of DDX-23 or DDX-17 does not affect the expression of machineries for miRNA biogenesis and function.........................................124 Figure 12. DDX-23 and DDX-17 are required for efficient regulation of cog-1::gfp expression by ectopic lsy-6 miRNA in the uterus..............................125 Figure 13. The spatial expression pattern of DDX-23.................................126 Figure 14. The model for DDX-23 and DDX-17 in miRNA biogenesis.........127 Figure 15. Depletion of HRP-2 relieved let-7(n2853) retarded seam cell development..............................................................................................128 Figure 16. HRP-2 genetically acts upstream of LIN-29................................130 Figure 17. RNAi knockdown of HRP-2 promotes precocious LIN-29 expression at early stage.............................................................................................131 Figure 18. Reduction of HRP-2 enhances let-7-mediated repression of lin-41..............................................................................................................133 Figure 19. hrp-2(RNAi) does not alter let-7 and miRISC levels....................135 Figure 20. HRP-2 interacts with miRISC in an RNA-dependent manner.......136 Figure 21. The let-7 binding site is not required for HRP-2 to interact with lin-41 mRNA 3’UTR....................................................................................137 Figure 22. HRP-2 is associated with a 14-nt U-rich motif in lin-41 3’UTR..138 Figure 23. Deletion of HRP-2 response element (HRE) enhances let-7-mediated lin-41 repression........................................................................140 Figure 24. HRP-2 distributes either in nucleus and cytoplasm in C. elegans......................................................................................................142 Figure 25. hnRNP R does not functions as HRP-2 in C. elegans in let-7-medeated lin-41/TRIM71 repression..........................................................144 Figure 26. hnRNP Q impedes let-7-mediated repression of TRIM71...........146 Figure 27. hnRNP Q is associated and co-localized with miRISC in P-bodies.......................................................................................................148 Figure 28. hnRNP Q binds to an element in TRIM71 3’UTR..........................150 Figure 29. The hnRNP Q response element (QRE) is required for hnRNP Q binding......................................................................................................151 Figure 30. Deletion of hnRNP Q response element increases repression of TRIM71 by let-7.........................................................................................153 Chapter 7 References.................................................................................154 Appendix 1................................................................................................170 Appendix 2................................................................................................172 Appendix 3. Brief summary of RACK-1 project...........................................173 Appendix 4. RACK-1 paper........................................................................174 Appendix 5. Helicase DDX-23 paper..........................................................189 | |
| dc.language.iso | en | |
| dc.title | 鑑定新穎微小核醣核酸生合成及功能之調控因子 | zh_TW |
| dc.title | To identify novel modulators of miRNA biogenesis and function | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),譚婉玉(Woan-Yuh Tarn),陳俊宏(Chun-Hong Chen),朱家瑩(Chia-Ying Chu) | |
| dc.subject.keyword | 微小核醣核酸,let-7,DDX-23,生合成,HRP-2,hnRNP Q,lin-41/TRIM71, | zh_TW |
| dc.subject.keyword | miRNA,let-7,DDX-23,biogenesis,HRP-2,hnRNP Q,lin-41/TRIM71, | en |
| dc.relation.page | 174 | |
| dc.identifier.doi | 10.6342/NTU201601014 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2016-07-21 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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