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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐立中(Li-Chung Hsu) | |
| dc.contributor.author | Chia-Ching Lin | en |
| dc.contributor.author | 林佳靜 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:14:11Z | - |
| dc.date.available | 2024-08-28 | |
| dc.date.copyright | 2019-08-28 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-07-31 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78715 | - |
| dc.description.abstract | 先天性免疫反應之於宿主的重要性在於其能有效地清除病原體及修復受傷的組織，然失去調控的免疫反應則會導致許多疾病，例如:癌症、敗血症及代謝疾病等。目前已知，許多機制已被證實能有效地調節免疫反應，其中包含轉錄後調控(post-transcriptional regulation/PTR)。針對免疫相關基因的表現，轉錄後調控是一種快速且有效率的方式。第七終端尿醯苷轉移酶(Terminal uridyltransferase 7，TUT7)，亦稱為ZCCHC6；目前的研究已知TUT7藉由轉移尿醯苷至其標的核醣核酸(RNA)的方式，調控核糖核酸(RNA)的穩定度及生合成。然而，TUT7在先天性免疫系統中所扮演的角色仍未釐清。於本論文中，我們發現以脂多醣(LPS)刺激第四型類鐸受體(TLR4)的活化，會誘發TUT7的表現。再者，利用慢病毒攜帶短片段RNAi (shTut7)或是特定基因剔除鼠(Tut7-/-)減少或去除巨噬細胞裡TUT7的表現並施以LPS，導致部分的細胞激素表現失衡，如:細胞介白素6 (interleukin-6/IL-6)。另外，我們更進一步證實TUT7利用其尿醯苷轉移之能力，將調節型核糖核酸內切酶(Regulatory riboendonuclease-1/Regnase-1)的RNA進行尿醯化的修飾以減少其蛋白質產生，進而對IL-6 mRNA達到保護作用，最終使IL-6得以適時地產生作用。另外，與先前的研究一致，我們也發現尿醯化的Regnase-1 mRNA具有較短的腺苷鏈。綜合前述，我們證實了TUT7藉由尿醯化調節Regnase-1 mRNA穩定度降低Regnase-1蛋白質的表現，以此使IL-6可以適時地參與TLR4所誘導之免疫反應。 | zh_TW |
| dc.description.abstract | Innate immune responses play a crucial role for the host to effectively clear pathogens and repair damaged tissues, but their dysregulation has been associated with numerous illnesses, such as cancer, sepsis and metabolic disorders. Different mechanisms have been demonstrated to elaborately regulate inflammatory response, including post-transcriptional regulation (PTR), which is a fast and effective way to modulate the expression of inflammatory-related genes. Terminal uridyltransferase 7 (TUT7), also named ZCCHC6, is known to be involved in the regulation of RNA stability or microRNA biogenesis by the addition of uridine tails to the end of their RNA substrates. Nevertheless, the role of TUT7 in the innate immune response remains unclear. Here we reveal that TUT7 is induced upon TLR4 activation, and selectively controls the expression of a subset of inflammatory cytokines. Depletion of TUT7 in macrophages leads to dysregulation of several cytokines production including IL-6 upon LPS challenge. We further demonstrate that TUT7 mediates IL-6 expression by controlling its mRNA stability through Regnase-1 after LPS stimulation. Mechanistically, TUT7 directly interacts with and uridylates Regnase-1 for degradation following stimulation LPS, which required the stem-loop structure in its 3’-UTR. Consistently, Regnase-1 mRNAs possess uridylated ends and shorter poly(A) tails in TUT7-proficient macrophages after LPS treatment, whereas Regnase-1 transcripts carries long poly(A) length without uridylation in Tut7-/- cells. Taken together, our findings reveal the functional role of TUT7 in sculpting TLR4-driven responses by modulating the mRNA stability of certain inflammatory mediators. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:14:11Z (GMT). No. of bitstreams: 1 ntu-108-D00448008-1.pdf: 7288823 bytes, checksum: b37da61a33ae8dbdabc3e2d63c61442e (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | Contents
摘要 i Abstract ii Contents 1 Introduction 9 Innate immunity 9 Regulation of TLR4-mediated cytokines expression 10 TLR4 Signal transduction 11 Regulation of TLR4-driven immune response by translation and post-transcriptional modification (PTM) 13 Post-transcriptional regulation (PTR) in TLR4-triggered inflammation 14 Regnase-1 and its physiological functions in innate immunity 15 The terminal RNA uridyltransferases (TUTases/TUTs) family 17 ZCCHC6/TUTase 7/TUT7 20 TUT7 in the innate immunity 23 TUT7 modulates the expression of cytokines upon LPS challenge 23 Specific aim 25 Materials and Methods 26 Plasmids 26 Reagents and Antibodies 27 Mice 29 Cell cultures and preparation of bone marrow-derived macrophages 30 RNA extraction and Real-time quantitative PCR 30 shRNA-based gene silencing and lentiviral infection 31 mRNA stability assay 32 Immunoblotting 33 Immunoprecipitation 34 In vitro dephosphorylation 34 Dual-Luciferase reporter assay 35 Enzyme-Linked Immunosorbent Assays (ELISA) 36 In vitro transcription and In vitro uridylation 37 RNA-immunoprecipitation 38 Preparation of 3'-ligation rapid amplification of cDNA ends (RACE) library 39 RNA sequencing 40 Mass spectrometry (MS) analysis 41 Immunofluorescence 42 The endotoxemia mice model 43 Statistical analysis 43 Results 44 TUT7 expression is induced by various TLR ligands in BMDMs 44 Depletion of TUT7 modulates cytokines production in response to LPS in RAW 264.7 macrophages 45 Depletion of TUT7 influences the expression of TLR4-driven pro-inflammatory cytokines in response to various TLR ligands in BMDMs 46 TUT7 is involved in a subset of inflammatory cytokine expression in response to LPS in macrophages 49 TUT7 regulates Il6 mRNA stability through the stem-loop structure of Il6 3’-UTR 50 TUT7 controls Il6 mRNA destabilization through Regnase-1 54 TUT7 alters Regnase-1 mRNA stability via its 3’-UTR 56 TUT7 binds to and uridylates Regnase-1 58 TUT4 and TUT7 regulate Regnase-1 expression through different mechanisms 61 The exonuclease, Dis3L2, regulates LPS-induced cytokines expression like TUT7 61 Deletion of TUT7 reduces SerpinB2 expression 63 Discussion 64 Figures and Tables 73 Figure 1. Schematic diagram of the human TUTases. 73 Figure 2. TUT7 is induced by various TLRs ligands in BMDMs. 74 Figure 3. Deficiency of TUT7 alters the mRNA expression of pro-inflammatory cytokines in response to LPS in RAW 264.7 macrophages. 75 Figure 4. Deficiency of TUT7 affects the protein levels of pro-inflammatory cytokines in response to LPS in RAW 264.7 macrophages. 77 Figure 5. Deficiency of TUT7 does not influence activation of mitogen-activated protein kinases (MAPKs) and IKK in RAW 264.7 macrophages. 78 Figure 6. The nucleotidyltransferase activity of TUT7 is required for TUT-modulated LPS-induced Il6 and Il12b expression. 79 Figure 7. Generation of conventional Tut7-deleted mice. 80 Figure 8. Depletion of Tut7 has no significant effect on in vitro differentiation of bone marrow cells into macrophages, body weight, and spleen weight. 81 Figure 9. TUT7 deficiency in BMDMs alters the mRNA expression of LPS-induced pro-inflammatory cytokines. 82 Figure 10. TUT7 Deficiency regulates the production of LPS-induced pro-inflammatory cytokines in BMDMs. 83 Figure 11. Depletion of TUT7 does not change activation of mitogen-activated protein kinases and IKK in BMDMs. 84 Figure 12. Deletion of TUT7 alters the mRNA expression of pro-inflammatory cytokines in response to TLR-ligands in BMDMs. 85 Figure 13. Generation of mice whose Tut7 gene is specifically deleted in type I interferon-responsive cells. 87 Figure 14. TUT7 deficiency alters the expression of LPS-induced pro-inflammatory cytokine mRNAs in BMDMs isolated from Tut7Δ mice. 88 Figure 15. TUT7 deficiency alters innate immune response-related gene expression profile in response to LPS in BMDMs. 89 Figure 16. Depletion of TUT7 influences Il6 mRNA stability but not transcription. 90 Figure 17. TUT7 regulates Il6 mRNA stability via the stem-loop structure in its 3’UTR56-104. 91 Figure 18. TUT7 downregulates Regnase-1 mRNA and protein expression. 92 Figure 19. TUT7 does not influence Zc3h12c expression. 93 Figure 20. LPS induces Regnase-1 phosphorylation in RAW 264.7 macrophages. 94 Figure 21. Depletion of TUT7 influences Regnase-1 mRNA stability. 95 Figure 22. The nucleotidyltransferase activity of TUT7 is required for its regulation of Regnase-1 mRNA expression after TLR4 activation. 96 Figure 23. TUT7-modulated mRNA expression of a subset of TLR4-triggered inflammatory cytokines is dependent on Regnase-1. 97 Figure 24. TUT7 regulates the mRNA stability of Regnase-1 via its 3’-UTR1-358 in response to LPS in RAW 264.7 macrophages. 99 Figure 25. TUT7 downregulates Regnase-1 expression via 3’-UTR100-250. 100 Figure 26. TUT7-regulated Regnase-1 expression requires the stem-loop structure in its 3’-UTR100-250. 102 Figure 27. TUT7 associates with Regnase-1. 104 Figure 28. TUT7 uridylates Regnase-1. 105 Figure 29. TUT7 mediates oligo-uridylation on Regnase-1 3’end in response to LPS. 106 Figure 30. 3’end sequences of Il6 from LPS-treated BMDMs. 108 Figure 31. Deletion of TUT7 does not affect Tut4 expression in response to LPS in RAW 264.7 macrophages and BMDMs. 109 Figure 32. Deletion of TUT4 decreases the mRNA and protein expression of Regnase-1 in response to LPS in RAW 264.7 macrophages. 110 Figure 33. TUT7 associates with Dis3L2, and depletion of Dis3L2 impacts the mRNA expression of LPS-induced Il6, Il12b, and Regnase-1 in BMDMs. 111 Figure 34. TUT7 does not associate with Dcp1. 113 Figure 35. The sera concentrations of cytokines are decreased in Tut7-/- mice after challenge with low-dose LPS. 114 Figure 36. Depletion of TUT7 alters the mRNA expression of SerpinB2. 115 Figure 37. Model for the regulation of TUT7-mediated Reganse-1 expression 116 Supplementary figure 1. The TLR4 signaling pathway. 117 Supplementary figure 2. TUT7 is induced by various TLR ligands in BMDMs and RAW 264.7 macrophages. 119 Supplementary figure 3. Alignments of the conserved residue of catalytic core of TUT7. 120 Supplementary figure 4. TUT7 regulates Il6 mRNA expression via its 3’-UTR. 121 Table 1. TUTases 123 Table 2. Complete blood counts of Tut7+/+ and Tut7-/- mice 124 Table 3. Downregulated genes in Tut7-/- BMDMs upon LPS treatment. 125 Table 4. Upregulated genes in Tut7-/- BMDMs upon LPS treatment. 129 Table 5. Identification of TUT7-associated proteins in RAW264.7 macrophages upon LPS treatment. 132 Table 6. Primer list 133 References 137 | |
| dc.language.iso | en | |
| dc.subject | 尿醯化 | zh_TW |
| dc.subject | 先天性免疫 | zh_TW |
| dc.subject | 第四型類鐸受體 | zh_TW |
| dc.subject | 轉錄後調控 | zh_TW |
| dc.subject | 第七終端尿醯?轉移? | zh_TW |
| dc.subject | 調節型核糖核酸內切? | zh_TW |
| dc.subject | Innate immune responses | en |
| dc.subject | Toll-like receptor 4 (TLR4) | en |
| dc.subject | post-transcriptional regulation (PTR) | en |
| dc.subject | uridylation | en |
| dc.subject | regulatory ribonuclease 1 (Regnase-1) | en |
| dc.subject | Terminal uridyltransferase 7 (TUT7) | en |
| dc.title | 終端尿醯苷轉移酶TUT7在類鐸受體4引發發炎反應扮演之角色及機制探討 | zh_TW |
| dc.title | The functional role of terminal uridyltransferase 7 in TLR4-mediated inflammatory response | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 詹世鵬(Shih-Peng Chan),蔡欣祐(Hsin-Yue Tsai),曾炳輝(Ping-Hui Tseng),李建國(Chien-Kuo Lee) | |
| dc.subject.keyword | 先天性免疫,第四型類鐸受體,轉錄後調控,第七終端尿醯?轉移?,調節型核糖核酸內切?,尿醯化, | zh_TW |
| dc.subject.keyword | Innate immune responses,Toll-like receptor 4 (TLR4),post-transcriptional regulation (PTR),Terminal uridyltransferase 7 (TUT7),regulatory ribonuclease 1 (Regnase-1),uridylation, | en |
| dc.relation.page | 147 | |
| dc.identifier.doi | 10.6342/NTU201902241 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-07-31 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| dc.date.embargo-lift | 2024-08-28 | - |
| 顯示於系所單位： | 分子醫學研究所 | |
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