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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹世鵬 | |
| dc.contributor.author | Xiao-Shin Wei | en |
| dc.contributor.author | 韋孝昕 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:25:32Z | - |
| dc.date.available | 2020-09-25 | |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-06 | |
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Ho, J.J., et al., Active stabilization of human endothelial nitric oxide synthase mRNA by hnRNP E1 protects against antisense RNA and microRNAs. Mol Cell Biol, 2013. 33(10): p. 2029-46. 45. Michlewski, G. and J.F. Caceres, Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis. Nat Struct Mol Biol, 2010. 17(8): p. 1011-8. 46. Guil, S. and J.F. Caceres, The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a. Nat Struct Mol Biol, 2007. 14(7): p. 591-6. 47. Kinnaird, J.H., et al., HRP-2, a heterogeneous nuclear ribonucleoprotein, is essential for embryogenesis and oogenesis in Caenorhabditis elegans. Exp Cell Res, 2004. 298(2): p. 418-30. 48. Kabat, J.L., S. Barberan-Soler, and A.M. Zahler, HRP-2, the Caenorhabditis elegans homolog of mammalian heterogeneous nuclear ribonucleoproteins Q and R, is an alternative splicing factor that binds to UCUAUC splicing regulatory elements. J Biol Chem, 2009. 284(42): p. 28490-7. 49. Hassfeld, W., et al., Molecular definition of heterogeneous nuclear ribonucleoprotein R (hnRNP R) using autoimmune antibody: immunological relationship with hnRNP P. Nucleic Acids Res, 1998. 26(2): p. 439-45. 50. Glinka, M., et al., The heterogeneous nuclear ribonucleoprotein-R is necessary for axonal beta-actin mRNA translocation in spinal motor neurons. Hum Mol Genet, 2010. 19(10): p. 1951-66. 51. Chen, H.H., et al., The RNA binding protein hnRNP Q modulates the utilization of exon 7 in the survival motor neuron 2 (SMN2) gene. Mol Cell Biol, 2008. 28(22): p. 6929-38. 52. Svitkin, Y.V., et al., Control of translation and miRNA-dependent repression by a novel poly(A) binding protein, hnRNP-Q. PLoS Biol, 2013. 11(5): p. e1001564. 53. Lyabin, D.N., et al., Identification of proteins specifically interacting with YB-1 mRNA 3' UTR and the effect of hnRNP Q on YB-1 mRNA translation. Biochemistry (Mosc), 2013. 78(6): p. 651-9. 54. Kim, D.Y., et al., hnRNP Q regulates translation of p53 in normal and stress conditions. Cell Death Differ, 2013. 20(2): p. 226-34. 55. Hayes, G.D., C.G. Riedel, and G. Ruvkun, The Caenorhabditis elegans SOMI-1 zinc finger protein and SWI/SNF promote regulation of development by the mir-84 microRNA. Genes Dev, 2011. 25(19): p. 2079-92. 56. Jannot, G. and M.J. Simard, Tumour-related microRNAs functions in Caenorhabditis elegans. Oncogene, 2006. 25(46): p. 6197-6201. 57. Heo, I., et al., Lin28 Mediates the Terminal Uridylation of let-7 Precursor MicroRNA. Molecular Cell, 2008. 32(2): p. 276-284. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53541 | - |
| dc.description.abstract | 微小核醣核酸(miRNAs)為一小段非編碼的核醣核酸，可和目標訊息核糖核酸的3端不轉譯區結合，進而抑制目標基因的轉譯或是使其被降解。異質核醣核酸蛋白(Heterogeneous nuclear ribonucleoproteins , hnRNPs)為一核醣核酸結合蛋白的家族之一，參與許多核醣核酸的代謝路徑，其中包含參與miRNA的生合成以及功能當中，例如人類hnRNP A1被發現能夠抑制let-7 miRNA的生合成。從線蟲到人類中let-7以及let-7對於lin-41的調控具有保守性，在本篇論文中，我們降低了在線蟲中已知的hnRNP來觀察這些hnRNP對於let-7的調控是否有影響。在線蟲幼蟲晚期階段，let-7會降低LIN-41的表現，間接活化被LIN-41抑制的成蟲專屬轉錄因子LIN-29，進而促進細胞終端分化並且進入成蟲細胞階段。let-7的功能缺失造成線蟲生殖孔不正常發育、皮下接縫細胞(hypodermal seam cells)終端分化異常以及LIN-29所活化的膠原蛋白基因col-19表現量降低。在let-7亞效等位基因（hypomorphic allele）線蟲突變種let-7(n2853)中降低HRP-2能夠抑制這些let-7異時基因性狀(heterochronic phenotypes)，但並不能抑制lin-29無效等位基因(null allele)線蟲突變種lin-29(n333)的性狀缺失。HRP-2的減少並不影響let-7的表現量。這些觀察顯示HRP-2參與在lin-29的上游來影響let-7對於目標基因的調控，並且不會影響let-7的生合成。此外，我們發現降低HRP-2也能抑制let-60/Ras 功能增強所導致的多生殖孔性狀，let-60/Ras被let-7旁系同源(paralogous)miRNA miR-84調控，進而決定生殖孔前驅細胞的分化命運，然而，我們的結果顯示HRP-2對於生殖孔的調控是miR-84-independent的。HRP-2除了先前研究發現參與在選擇性剪接的功能之外，我們的研究也發現HRP-2能夠參與miRNA的功能進而調控基因的表現，至少參與了let-7的功能。但在人類Huh7細胞中降低線蟲HRP-2的人類同源蛋白hnRNP Q，對於let-7的表現量以及let-7對TRIM71/LIN41的調控沒有影響。有趣的是，在細胞中降低另一個HRP-2的同源蛋白hnRNP R能夠顯著的降低TRIM71/LIN41的表現量，但可能是透過3端不轉譯區以外未知的機制來調控TRIM71/LIN41。總結以上結果，我們推測HRP-2能調控let-7在線蟲異時基因let-7-lin-41-lin-29調控路徑中的功能，但在人類Huh7細胞中HRP-2同源蛋白hnRNP Q/R卻不影響具保守性的let-7對TRIM71/LIN41調控關係。 | zh_TW |
| dc.description.abstract | MicroRNAs (miRNAs) are small non-coding RNAs that regulate genes by binding to the 3′ untranslated region (3′ UTR) of target mRNAs and trigger translational repression and/or mRNA degradation. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins involved in many RNA metabolisms, including miRNA biogenesis and functions. For example, human hnRNP A1 has been implicated in inhibiting biogenesis of the let-7 miRNA. let-7 and the regulation of lin-41 by let-7 are conserved from nematodes to humans. In this study, we knocked down several known C. elegans hnRNPs by RNAi and examined the effects on let-7-mediated regulation. In C. elegans, let-7 down-regulates LIN-41 in the late larval stages and indirectly activates the adult-specific transcription factor LIN-29 that is suppressed by LIN-41, and hence promotes cell terminal differentiation and adult cell fates. Dysfunction of let-7 results in abnormal vulva development, attenuated terminal differentiation of hypodermal seam cells and low expression of col-19, an adult collagen gene directly activated by LIN-29. We found that knockdown of hrp-2 suppressed the let-7 heterochronic phenotypes in animals carrying a hypomorphic let-7(n2853) allele, but not those with a null lin-29(n333) allele. Depletion of HRP-2 did not change let-7 levels. These observations suggest that HRP-2 plays a role in let-7-mediated gene regulation in the heterochronic gene pathway, likely upstream of lin-29, without affecting let-7 biogenesis. On the other hand, hrp-2 knockdown suppressed the multiple vulva (Muv) phenotype caused by gain-of-function mutations of let-60/Ras, a direct target of the let-7 paralogous miRNA miR-84 that determines vulval precursor cell differentiation. However, we found that the suppression is miR-84-independent. Nevertheless, our findings suggest that HRP-2, in addition to its previously reported function in alternative splicing, controls gene expression via miRNA-mediated regulation, at least for let-7. In human Huh7 cells, depletion of hnRNP Q, the homolog of C. elegans HRP-2, did not affect let-7 levels or the regulation of TRIM71/LIN41 by let-7. Interestingly, knockdown of the other HRP-2 homolog, hnRNP R, significantly reduced TRIM71/LIN41 expression, however, by an unknown mechanism outside regulation through the 3’ UTR. Taken together, we propose that HRP-2 controls let-7-mediated regulation in the let-7-lin-41-lin-29 axis in the C. elegans heterochronic gene pathway, while its homologous proteins hnRNP Q/R in human Huh7 cells may not affect the conserved let-7-TRIM71/LIN41 regulation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:25:32Z (GMT). No. of bitstreams: 1 ntu-104-R02445121-1.pdf: 2397463 bytes, checksum: 14ddbcf0c41fde8be7bc2efccab9cb42 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 口試委員會審定書...# 誌謝...i 中文摘要...ii ABSTRACT...iii 目錄...v Chapter 1Introduction...1 1.1 microRNA的生合成及其功能...1 1.2 let-7 調控線蟲發育過程中的細胞分化... 1 1.3 let-7家族成員miR-84調控線蟲生殖孔的發育...3 1.4 let-7可調控癌症相關基因...3 1.5 let-7與目標基因之間的相互調控...4 1.6 hnRNPs可能參與miRNA之調控...4 1.7 HRP-2以及人類同源蛋白hnRNP Q/R的功能... 5 Chapter 2 Materials and methods ...7 2.1 線蟲品系(strains) ...7 2.2 線蟲生長同步化...7 2.3 RNAi...7 2.3.1 RNAi clone...7 2.3.2 Feeding RNAi...8 2.4 線蟲細胞萃取液製備...8 2.5 線蟲RNA萃取...8 2.6 北方墨點法...9 2.6.1 實驗流程 ...9 2.6.2 探針製備 ...9 2.6.3 探針引子序列...9 2.7 西方墨點法...10 2.7.1 膠體製備...10 2.7.2 電泳、轉印及抗體雜和...10 2.8 Real time quantitative RT-PCR... 11 2.8.1 Tailing qRT-PCR ...11 2.8.2 Random priming ...13 2.8.3 TanMan microRNA assay(invitrogen)... 14 2.9 細胞培養 ...15 2.9.1 細胞株 ...15 2.9.2 細胞培養環境...15 2.10 細胞轉染...15 2.11 基因沉默...16 2.11.1 siRNA基因沉默...16 2.11.2 shRNA 基因沉默...17 2.12 Luciferase assay...18 2.13 細胞萃取液製備...19 Chapter 3 Results...21 3.1 線蟲HRP-2可能影響let-7對於lin-41的調控...21 3.1.1 降低HRP-2能可有效抑制let-7(n2853)突變種線蟲中生殖孔爆裂的性狀 ...21 3.1.2 降低HRP-2能可有效抑制let-7(n2853)突變種線蟲中seam cell異常分化的性狀...21 3.1.3 降低HRP-2能可有效抑制let-7(n2853)突變種線蟲中COL-19無法表現的性狀 ...22 3.2 降低HRP-2不影響let-7以及lin-4表現...22 3.3 HRP-2可能影響到生殖孔的發育 ...23 3.4 探討人類hnRNP Q是否可能影響let-7對TRIM71/LIN41的調控 ...24 3.5 人類hnRNP R能夠影響TRIM71表現... 25 Chapter 4 Discussion... 27 4.1 HRP-2可能藉著和lin-41 3’ UTR結合來調控let-7對lin-41的作用 ...27 4.2 HRP-2可能透過其他機制調控生殖孔發育...28 4.3 Hek293細胞可能較適合用來探討hnRNP Q對於let-7的調控... 28 4.4 hnRNP R對於TRIM71的調控機制未來值得探討...29 Chapter 5 Figures 30 Figure 1降低HRP-2可有效抑制let-7(n2853)突變種的性狀缺失...30 Figure 2降低HRP-2D可有效抑制let-7(n2853)線蟲產生的seam cells異常分化... 31 Figure 3 降低HRP-2可有效抑制let-7(n2853)產生的COL-19異常表現... 32 Figure 4降低HRP-2不影響let-7的表現...33 Figure 5降低HRP-2表現不會影響到lin-4的表現...34 Figure 6 HRP-2對於生殖孔發育的調控 ...36 Figure 7 降低hnRNP Q1能抑制TRIM71的蛋白質表現...37 Figure 8 降低hnRNP Q1不影響let-7的表現...38 Figure 9 hnRNP Q1可能會調控let-7對TRIM71的作用...39 Figure 10 hnRNP Q可能會調控TRIM71 3’UTR... 40 Figure 11 hnRNP Q可能不會使TRIM71以及LIN28B表現量下降...41 Figure 12 hnRNP R會影響TRIM71的蛋白質以及mRNA的表現... 42 Figure 13 降低hnRNPR不影響let-7a以及let-7g的表現量 ...43 Figure 14 hnRNP R可能不影響let-7的功能...44 Figure 15 降低hnRNP R不影響TRIM71 3’ UTR的表現... 45 REFERENCE ...46 附錄... 51 附圖 一線蟲heterochronic gene pathway 與seam cell 分化的關係 ...51 附圖 二 線蟲生殖孔發育過程 ...52 附圖 三 hnRNP Q mRNA map以及siRNA、shRNA作用位置... 53 附圖 四 psicheck2-1-TRIM71 3’ UTR map...54 附表 一 let-7(n2853)各個hnRNP RNAi 後的seam cell數目統計數據 ...56 附表 二 let-7(n2853) seam cell數目統計數據...58 附表 三 lin-29(n333) seam cell數目統計數據...60 附表 四 let-7(n2853) hrp-2(RNAi) northern blot定量分析原始數據... 62 附表 五 let-60(mt2124)、GR1689 let-60(n1046)IV;mir-84(tm1304)X、GR1690 let-60(n1046)IV;mir-84++(mgIs45)I 生殖孔數目...63 附表 六以qRT-PCR測試shRNA(TRCN0000275206)降低hnRNP Q1後hnRNP Q1、TRIM71以及LIN28B mRNA的表現 ...64 附表 七 以luciferase assay測量TRIM71 3’UTR在Huh7細胞中的表現量... 65 附表 八以qRT-PCR測試shRNA降低hnRNP R後TRIM71、LIN28B以及HMGA2 mRNA的表現...66 附表 九以Taqman qRT-PCR測量shRNA降低hnRNP R後let-7a以及let-7g的表現量 ...67 | |
| dc.language.iso | zh-TW | |
| dc.subject | 異質核醣核酸蛋白 | zh_TW |
| dc.subject | 微小核醣核酸 | zh_TW |
| dc.subject | let-7 | en |
| dc.subject | hnRNP | en |
| dc.title | 線蟲HRP-2及其人類同源蛋白hnRNP Q/R在微小核醣核酸功能中扮演的角色 | zh_TW |
| dc.title | The roles of Caenorhabditis elegans HRP-2 and its human homolog hnRNP Q/R in miRNA function | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 譚婉玉,陳俊宏 | |
| dc.subject.keyword | 微小核醣核酸,異質核醣核酸蛋白, | zh_TW |
| dc.subject.keyword | let-7,hnRNP, | en |
| dc.relation.page | 65 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-06 | |
| 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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