請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65554完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹世鵬(Shih-Peng Chan) | |
| dc.contributor.author | Meng-Wei Yeh | en |
| dc.contributor.author | 葉孟瑋 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:50:08Z | - |
| dc.date.available | 2014-09-19 | |
| dc.date.copyright | 2012-09-19 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-07-20 | |
| dc.identifier.citation | 1.Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116(2):281-97.
2.Medina, P.P. and F.J. Slack. microRNAs and cancer: an overview. Cell Cycle 2008;7(16):2485-92. 3.Nimmo, R.A. and F.J. Slack. An elegant miRror: microRNAs in stem cells, developmental timing and cancer. Chromosoma 2009;118(4):405-18. 4.Stefani, G. and F.J. Slack. Small non-coding RNAs in animal development. Nat Rev Mol Cell Biol 2008;9(3):219-30. 5.Filipowicz, W., S.N. Bhattacharyya, and N. Sonenberg. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 2008;9(2):102-14. 6.Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. An RNA-directed nuclease mediates posttranscriptional gene silencing in Drosophila cells. Nature 404, 293–296 (2000). 7.Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 (2001). 8.Mourelatos, Z.(2002) miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16, 720–728 9.Lee, Y. S., K. Nakahara, J. W. Pham, K. Kim, Z. He, E. J. Sontheimer, and R. W.Carthew. 2004. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117:69-81. 10.Caudy, A. A., Myers, M., Hannon, G. J. & Hammond, S. M. Fragile X-related protein and VIG associate with the RNA interference machinery. Genes Dev. 16, 2491–2496 (2002). 11.Caudy AA, Ketting RF, Hammond SM, Denli AM, Bathoorn AM, Tops BB, Silva JM, Myers MM, Hannon GJ, Plasterk RH: A micrococcal nuclease homologue in RNAi effector complexes. Nature 2003, 425:411-414. 12.Meister, G. et al. (2005) Identification of novel argonaute-associated proteins. Curr. Biol. 15, 2149–2155 13.Fabian MR, Mathonnet G, Sundermeier T, Mathys H, Zipprich JT,Svitkin YV, Rivas F, Jinek M, Wohlschlegel J, Doudna JA,Chen CY, Shyu AB, Yates JRr, Hannon GJ, Filipowicz W,Duchaine TF, Sonenberg N (2009) Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation. Mol Cell 35: 868–880 14.Ding, L. et al. (2005) The developmental timing regulator AIN-1 interacts with miRISCs and may target the Argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. Mol. Cell 19, 437–447 15.Reinhart, B.J., et al. The 21-nucleotide let-7 RNA regulates devel- opmental timing in Caenorhabditis elegans. Nature 2000;403(6772):901-6. 16.Pasquinelli, A.E., et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature 2000;408(6808) :86-9. 17.Ambros, V. A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans. Cell 1989;57(1):49-57 18.Ambros, V. and H.R. Horvitz. Heterochronic mutants of the nematode Caenorhabditis elegans. Science 1984;226(4673):409-16. 19.Slack, F.J., et al. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol Cell 2000;5(4):659-69. 20.Chan, S.P., Ramaswamy, G., Choi, E.Y., and Slack, F.J. (2008). Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans. RNA 14, 2104-2114. 21. Hutvagner, G., Simard, M.J., Mello, C.C., and Zamore, P.D. (2004) Sequence-specific inhibition of small RNA function. PLoS Biology, 2:465 22. Weir JR, Bonneau F, Hentschel J, Conti E. Structural analysis reveals the characteristic features of Mtr4, a DExH helicase involved in nuclear RNA processing and surveillance Proc Natl Acad Sci U S A. 2010 Jul 6;107(27):12139-44. Epub 2010 Jun 21. 23. Jia H, Wang X, Anderson JT, Jankowsky E.RNA unwinding by the Trf4/Air2 /Mtr4 polyadenylation (TRAMP) complex.Proc Natl Acad Sci U S A. 2012 May 8;109(19):7292-7. Epub 2012 Apr 24 24. Lubas M, Christensen MS, Kristiansen MS, Domanski M, Falkenby LG, Lykke-Andersen S, Andersen JS, Dziembowski A, Jensen TH. Interaction profiling identifies the human nuclear exosome targeting complex. Mol Cell. 2011 Aug 19;43(4):624-37. 25. Chekanova JA, Gregory BD, Reverdatto SV, Chen H, Kumar R, Hooker T, Yazaki J, Li P, Skiba N, Peng Q, Alonso J, Brukhin V, Grossniklaus U, Ecker JR, Belostotsky DA.Genome-wide high-resolution mapping of exosome substrates reveals hidden features in the Arabidopsis transcriptome. cell 2007 Dec 28;131(7):1340-53. 26. Van Wynsberghe PM, Kai ZS, Massirer KB, Burton VH, Yeo GW, Pasquinelli AE LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nat Struct Mol Biol. 2011 Mar;18(3):302-8. Epub 2011 Feb 6. 27. Monsalve GC, Van Buskirk C, Frand AR.LIN-42/PERIOD controls cyclical and developmental progression of C. elegans molts. Curr Biol. 2011 Dec 20;21(24):2033-45. Epub 2011 Dec 1. 28. Feinbaum RL and Ambros VR: 1999 . The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans. Dev Biol 210 : 87-95. 29. Clucas C et al.: 2002. Oncogenic potential of a C.elegans cdc25 gene is demonstrated by a gain-of-function allele. EMBO J 21: 665-74. 30. Ingo Bussing, Jr-Shiuan Yang,Eric C Lai,Helge Groshans The nuclear export receptor XPO-1 supports primary miRNA processing in C. elegans and Drosophila EMBO J. 2010 June 2; 29(11): 1830–1839 31. Pall and Hamilton. Improved northern blot method for enhanced detection of small RNA. Nat Protoc (2008) vol. 3 (6) pp. 1077-84 32. Hurteau et al. Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR. Cell Cycle (2006) vol. 5 (17) pp. 1951-6 33. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. 2000 Nov 16;408(6810):325-30. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65554 | - |
| dc.description.abstract | microRNAs在10幾年前被發現以來已經有相當多的研究在探討其調控細胞基因表現的機制，但是關於microRNAs本身是如何作用、有甚麼共同因子一起促進其作用或抑制其作用，至今還是有許多地方尚未釐清。在我的論文中主要是想要在C. elegans體內探討let-7上除了目前已知的一些RISC組成蛋白與其結合之外，是否還有其他蛋白質會與let-7專一性地結合，再進一步探討它們是如何參與在let-7調控基因表現的過程。我的論文策略主要是想利用2’-O-methyl oligo-
nucleotides pull-down assay來探討let-7上是否有專一性的結合蛋白。原理是利用人外加標定有biotin的let-7 antisense序列到線蟲細胞萃取液中去辨認內生性的let-7，再用帶有Streptavidin的基質將內生性的let-7及其結合蛋白分離出來並利用一維以及二維蛋白質電泳分析其蛋白質成分。 針對所有被antisense let-7 oligonucleotide純化出來的候選蛋白質，我們利用RNAi降低其基因表現並觀察其產生的性狀，rpa-1以及mtr-4在RNAi處理後會出現col-19表現量降低、vulva 發育缺失、seam cell分化異常、alae斷裂等典型的let-7功能不全時所會看到的型態學變化。進一步的研究發現抑制mtr-4的表現會造成mature let-7量下降、pri-let-7以及pre-let-7量增加的情形，代表著mtr-4可能參與在let-7生合成過程中。然而抑制rpa-1的表現對於let-7的生合成則沒有影響，是否rpa-1在let-7執行功能時一起參與其中則是還要再進一步去證實。 | zh_TW |
| dc.description.abstract | MicroRNAs are a class of small non-coding RNAs which regulate more than half of human genes by targeting to their complementary sites on mRNA 3’UTRs. MicroRNAs assemble with additional proteins into ribonucleopreotein complexes called miRNA-induced silencing complexes (miRISCs) and the composition of miRISCs is still not clear. Interestingly, in our previous study, we found that the let-7 miRNA and their family members, such as mir-84, can assemble into specific miRNPs in vitro. Here we aim to identify specific factors that associate with let-7. We use anti-sense oligonucleotides pull-down to isolate endogenous let-7 along with its associating proteins and then analyze the pull-down products by 1-D or 2-D protein electrophoresis. Further we use RNAi to reduce the gene expression of the candidate let-7-binding proteins and investigate their genetic interaction with let-7.
Our results indicate that knock-down of rpa-1 or mtr-4, which encode candidate let-7-binding proteins found in our pull-down assay, resulted in let-7 defective phenotypes, like col-19 down- regulation, seam cell differentiation defects, alae formation defects and vulva protruding. We further found that knocking down mtr-4 caused accumulation of pri-let-7 and pre-let-7 but decrease of mature let-7 level. On the other hand, when we knocked down rpa-1, the level of let-7 was not altered. We hypothesize that MTR-4 may be involved in let-7 primary transcript processing or pre-let-7 exporting from the nucleus to the cytoplasm. The role of rpa-1 in the let-7 regulation pathway is still unknown and awaited to be further investigated. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:50:08Z (GMT). No. of bitstreams: 1 ntu-101-R99445126-1.pdf: 7215332 bytes, checksum: 6fc5b4accafb71dabf4a240ce8e6b4a1 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書 -------------------------------------------i
誌謝 ----------------------------------------------------ii 中文摘要 -=----------------------------------------------iii 英文摘要 ===--------------------------------------------iv-v 1.簡介 -------------------------------------------1-4 1.1 MicroRNAs --------------------------------------------1 1.2 RNA-induced silencing complex (RISC) ---------------1-2 1.3 let-7 ----------------------------------------------2-4 2.材料與方法 --------------------------------------5-13 2.1 線蟲品系與轉殖基因 ----------------------------------------5 2.2 液態培養基培養法 ---------------------------------5-6 2.3 線蟲細胞萃取液製備 -------------------------------6-7 2.4 2’-O-methyl oligonucleotide pull-down assay ---7-8 2.5 西方墨點法 ----------------------------------------8 2.6 二維蛋白質膠體電泳 -------------------------------8--9 2.7 北方墨點法 --------------------------------------9-11 2.8 RNAi ------------------------------------------12-13 2.9 Tailing qPCR ----------------------------------13-14 3. 實驗結果 ---------------------------------------15-19 3.1利用antisense let-7 oligonucleotide將內生性let-7專一性地純化出來 -15 3.2確認在pull down assay的系統可以純化出RISC的核心蛋白質-ALG -1、以及ALG-1的結合蛋白-AIN-1 --------------------------------15-16 3.3在antisense let-7 oligonucleotide pull-down產物的一維、二維蛋白質電泳的分析結果中發現RPA-1以及MTR-4可能參與在let-7所調控的路徑--------------------------------------------------------16-18 3.4 當抑制mtr-4時會造成pri-let-7以及pre-let-7的累積，進而使mature let-7的量下降 -----------------------------------------18-19 3.5 RPA-1確實能夠與let-7專一性的結合 -----------------19 4. 討論 ------------------------------------------20-22 5. 圖表-------------------------------------------23-46 圖一 --------------------------------------------------23 圖二 --------------------------------------------------24-25 圖三 --------------------------------------------------26-27 圖四---------------------------------------------------28-30 表一 --------------------------------------------------31 圖五 --------------------------------------------------32 圖六 --------------------------------------------------33-34 圖七 --------------------------------------------------35-36 圖八 --------------------------------------------------37-38 圖九 --------------------------------------------------39-40 圖十 --------------------------------------------------41-42 圖十一 ------------------------------------------------43 圖十二 ------------------------------------------------44 圖十三 ------------------------------------------------45 圖十四 ------------------------------------------------46-47 圖十五 -------------------------------------------------48 6. 參考文獻---------------------------------------49-53 | |
| dc.language.iso | zh-TW | |
| dc.subject | 二維蛋白質電泳 | zh_TW |
| dc.subject | 專一性的結合蛋白 | zh_TW |
| dc.subject | pull-down assay | en |
| dc.subject | let-7 | en |
| dc.subject | RISC | en |
| dc.title | 線蟲體內微小核醣核酸let-7之專一性結合蛋白鑑定 | zh_TW |
| dc.title | Identification of let-7-specific binding proteins in Caenorhabditis elegans | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧述諄,朱家瑩 | |
| dc.subject.keyword | 二維蛋白質電泳,專一性的結合蛋白, | zh_TW |
| dc.subject.keyword | let-7,RISC,pull-down assay, | en |
| dc.relation.page | 53 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-07-20 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-101-1.pdf 未授權公開取用 | 7.05 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。