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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹世鵬 | |
| dc.contributor.author | Hsin-Kai Chen | en |
| dc.contributor.author | 陳昕楷 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:21:51Z | - |
| dc.date.available | 2016-10-09 | |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-15 | |
| dc.identifier.citation | 1 Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297 (2004).
2 Winter, J., Jung, S., Keller, S., Gregory, R. I. & Diederichs, S. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nature cell biology 11, 228-234, doi:10.1038/ncb0309-228 (2009). 3 Johnson, C. D. et al. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer research 67, 7713-7722, doi:10.1158/0008-5472.CAN-07-1083 (2007). 4 Tutar, L., Tutar, E. & Tutar, Y. MicroRNAs and Cancer; an Overview. Current pharmaceutical biotechnology (2014). 5 Esquela-Kerscher, A. & Slack, F. J. Oncomirs - microRNAs with a role in cancer. Nature reviews. Cancer 6, 259-269, doi:10.1038/nrc1840 (2006). 6 Slack, F. J. & Weidhaas, J. B. MicroRNAs as a potential magic bullet in cancer. Future oncology 2, 73-82, doi:10.2217/14796694.2.1.73 (2006). 7 Skalsky, R. L. & Cullen, B. R. Viruses, microRNAs, and host interactions. Annual review of microbiology 64, 123-141, doi:10.1146/annurev.micro.112408.134243 (2010). 8 Trang, P., Weidhaas, J. B. & Slack, F. J. MicroRNAs as potential cancer therapeutics. Oncogene 27 Suppl 2, S52-57, doi:10.1038/onc.2009.353 (2008). 9 Slack, F. J. & Weidhaas, J. B. MicroRNA in cancer prognosis. The New England journal of medicine 359, 2720-2722, doi:10.1056/NEJMe0808667 (2008). 10 Medina, P. P. & Slack, F. J. microRNAs and cancer: an overview. Cell cycle 7, 2485-2492 (2008). 11 Takamizawa, J. et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer research 64, 3753-3756, doi:10.1158/0008-5472.CAN-04-0637 (2004). 12 Reinhart, B. J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901-906, doi:10.1038/35002607 (2000). 13 Bracht, J., Hunter, S., Eachus, R., Weeks, P. & Pasquinelli, A. E. Trans-splicing and polyadenylation of let-7 microRNA primary transcripts. Rna 10, 1586-1594, doi:10.1261/rna.7122604 (2004). 14 Nimmo, R. A. & Slack, F. J. An elegant miRror: microRNAs in stem cells, developmental timing and cancer. Chromosoma 118, 405-418, doi:10.1007/s00412-009-0210-z (2009). 15 Roush, S. & Slack, F. J. The let-7 family of microRNAs. Trends in cell biology 18, 505-516, doi:10.1016/j.tcb.2008.07.007 (2008). 16 Vella, M. C., Choi, E. Y., Lin, S. Y., Reinert, K. & Slack, F. J. The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3'UTR. Genes & development 18, 132-137, doi:10.1101/gad.1165404 (2004). 17 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. Molecular cell 5, 659-669 (2000). 18 Fuller-Pace, F. V. RNA helicases: modulators of RNA structure. Trends in cell biology 4, 271-274 (1994). 19 Rocak, S. & Linder, P. DEAD-box proteins: the driving forces behind RNA metabolism. Nature reviews. Molecular cell biology 5, 232-241, doi:10.1038/nrm1335 (2004). 20 Linder, P. & Jankowsky, E. From unwinding to clamping - the DEAD box RNA helicase family. Nature reviews. Molecular cell biology 12, 505-516, doi:10.1038/nrm3154 (2011). 21 Fukuda, T. et al. DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs. Nature cell biology 9, 604-611, doi:10.1038/ncb1577 (2007). 22 Hammell, C. M., Lubin, I., Boag, P. R., Blackwell, T. K. & Ambros, V. nhl-2 Modulates microRNA activity in Caenorhabditis elegans. Cell 136, 926-938, doi:10.1016/j.cell.2009.01.053 (2009). 23 Curran, S. P. & Ruvkun, G. Lifespan regulation by evolutionarily conserved genes essential for viability. PLoS genetics 3, e56, doi:10.1371/journal.pgen.0030056 (2007). 24 Eki, T., Ishihara, T., Katsura, I. & Hanaoka, F. A genome-wide survey and systematic RNAi-based characterization of helicase-like genes in Caenorhabditis elegans. DNA research : an international journal for rapid publication of reports on genes and genomes 14, 183-199, doi:10.1093/dnares/dsm016 (2007). 25 Strauss, E. J. & Guthrie, C. PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro. Nucleic acids research 22, 3187-3193 (1994). 26 Konishi, T., Uodome, N. & Sugimoto, A. The Caenorhabditis elegans DDX-23, a homolog of yeast splicing factor PRP28, is required for the sperm-oocyte switch and differentiation of various cell types. Developmental dynamics : an official publication of the American Association of Anatomists 237, 2367-2377, doi:10.1002/dvdy.21649 (2008). 27 Kamath, R. Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313-321, doi:10.1016/s1046-2023(03)00050-1 (2003). 28 Van Wynsberghe, P. M. et al. LIN-28 co-transcriptionally binds primary let-7 to regulate miRNA maturation in Caenorhabditis elegans. Nature structural & molecular biology 18, 302-308, doi:10.1038/nsmb.1986 (2011). 29 Jeon, M., Gardner, H. F., Miller, E. A., Deshler, J. & Rougvie, A. E. Similarity of the C. elegans developmental timing protein LIN-42 to circadian rhythm proteins. Science 286, 1141-1146 (1999). 30 Bussing, I., Yang, J. S., Lai, E. C. & Grosshans, H. The nuclear export receptor XPO-1 supports primary miRNA processing in C. elegans and Drosophila. The EMBO journal 29, 1830-1839, doi:10.1038/emboj.2010.82 (2010). 31 Sarov, M. et al. A genome-scale resource for in vivo tag-based protein function exploration in C. elegans. Cell 150, 855-866, doi:10.1016/j.cell.2012.08.001 (2012). 32 Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes & development 18, 3016-3027, doi:10.1101/gad.1262504 (2004). 33 Chen, J. Y. et al. Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor. Molecular cell 7, 227-232 (2001). 34 Teigelkamp, S., Mundt, C., Achsel, T., Will, C. L. & Luhrmann, R. The human U5 snRNP-specific 100-kD protein is an RS domain-containing, putative RNA helicase with significant homology to the yeast splicing factor Prp28p. Rna 3, 1313-1326 (1997). 35 Kataoka, N., Fujita, M. & Ohno, M. Functional association of the Microprocessor complex with the spliceosome. Molecular and cellular biology 29, 3243-3254, doi:10.1128/MCB.00360-09 (2009). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56284 | - |
| dc.description.abstract | DEAD-box RNA 解旋酶參與在許多改變RNA 結構或改變RNA 和蛋白交互作用的生化機制，其可能也包含了微型核醣核酸生合成以及發揮功能的某些步驟，但目前相關的研究並不多。在我們先前的RNA干擾篩選實驗中，發現藉由RNA 干擾將線蟲DEAD-box RNA 解旋酶F01F1.7/DDX-23的表現減低會加劇 let-7微型核醣核酸突變種let-7(mg279)的性狀，原因可能是減低F01F1.7的表現導致了成熟let-7微型核醣核酸的減少，在本篇論文中我們試圖探討F01F1.7是否可能在微型核醣核酸生合成中扮演著某種角色。我們發現減少F01F1.7/DDX-23表現量後也會減低其他微型核醣核酸的表現量，包括線蟲成熟過程中調控異時基因路徑 (heterochronic gene pathway)中的lin-4, miR-48, miR-241等。我們觀察到這些微型核醣核酸的初始物 (primary miRNAs) 在F01F1.7/DDX-23表現減少時有累積的現象，顯示這些初始微型核醣核酸似乎無法順利被Drosha 切割成前驅微型核醣核酸 (precursor miRNAs)。由於F01F1.7/DDX-23是酵母菌中訊息核醣核酸的PRP-28剪接因子 (pre-mRNA splicing factor) 的同源蛋白，減少F01F1.7/DDX-23表現時也有可能影響到初始微型核醣核酸處理過程中所需的 Drosha與Pasha的基因表現，然而我們卻沒有觀察到Drosha與Pasha的基因表現有任何降低的現象，顯示前述初始微型核醣核酸的累積並非因為Drosha與Pasha的表現量有所降低，而有可能是因為F01F1.7/DDX-23量降低後，Drosha無法順利切割處理這些初始微型核醣核酸所致。另外，除了與異時基因路徑相關的微型核醣核酸，我們也觀察到減少F01F1.7/DDX-23表現也導致一個與神經分化有關的微型核醣核酸lsy-6對其目標基因的調控有所降低。我們在線蟲中表現帶有GFP-Flag的F01F1.7/DDX-23 蛋白，並發現它如同預期地主要表現在細胞核。我們也試圖表現有抗原標記的Drosha蛋白，並利用免疫沈澱法來觀察是否F01F1.7/DDX-23跟含有Drosha的蛋白複合體有交互作用，但在本篇論文中我們並沒有成功表現該蛋白。我們在老鼠HEK293細胞中利用免疫沈澱也沒有觀察到Drosha與DDX23有交互作用。無論如何，我們的實驗結果顯示，F01F1.7/DDX-23可能幫助Drosha去切割處理某些初始微型核醣核酸，但其詳細機制仍須進一步的研究來闡明。 | zh_TW |
| dc.description.abstract | The DEAD-box RNA helicases are required for biological mechanisms with rearrangements of inter- or intra-molecular RNA structures and/or conformational changes of ribonucleoprotein complexes, which include microRNA biogenesis and function. However, little is known about the roles of DEAD-box RNA helicases in these miRNA processes. In our previous studies, we found that knockdown of a DEAD-box RNA helicase F01F1.7/DDX-23 in C. elegans enhanced let-7-related phenotypes in let-7(mg279) mutants, perhaps due to the reduced let-7 levels when F01F1.7/DDX-23 was depleted. Here, we seek further investigation in how F01F1.7/DDX-23 affects miRNA biogenesis. We found that depletion of F01F1.7/DDX-23 also reduced the levels of several miRNAs, in addition to let-7, involved in the C. elegans heterochronic gene pathway, including lin-4, miR-48 and miR-241. Depletion of F01F1.7/DDX-23 caused accumulation of the primary form of these miRNAs, suggesting reduced pri-miRNA processing by Drosha. Since F01F1.7/DDX-23 is an ortholog of yeast pre-mRNA splicing factor PRP-28, it is possible that depletion of F01F1.7/DDX-23 affected the expression of Drosha and its partner, Pasha. However, we found no decrease in their expression upon F01F1.7/DDX-23 knockdown. This suggests that depletion of F01F1.7/DDX-23 reduced the efficacy of pri-miRNA processing but not the levels of machinery. We also observed that depletion of F01F1.7/DDX-23 inhibited target regulation of the lsy-6 miRNA, which is involved in neuronal differentiation. We expressed GFP-Flag tagged F01F1.7/DDX-23 in C. elegans and found a predominately nuclear expression that was expected. We attempted to express tagged Drosha in C. elegans and examine possible protein interaction between Drosha and F01F1.7/DDX-23 but failed to establish stable strains. Instead, we examined this interaction in mouse HEK293 cells by co-immunoprecipitation and no evidence of interaction has been observed. Nevertheless, our results suggest that F01F1.7/DDX-23 facilitates Drosha function in the processing of some pri-miRNAs. The detailed mechanism will be a focus of our future studies. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:21:51Z (GMT). No. of bitstreams: 1 ntu-103-R01445118-1.pdf: 3077610 bytes, checksum: ce03098a3fa0d016626242259dffc31a (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 口試委員會審定書 #
致謝 i 中文摘要 ii ABSTRACT iv CONTENTS vi Chapter 1 緒論 1 Chapter 2 材料與方法 8 2.1 線蟲品系(Strains)與轉殖基因(Transgenic genes) 8 2.2 固態培養基培養法 8 2.3 線蟲培養方式 8 2.4 RNAi 9 2.5 線蟲細胞萃取液製備 9 2.6 西方墨點法 10 2.7 北方墨點法 11 2.8 Random priming qRT-PCR 13 2.9 RT-PCR 15 2.10 Microinjection 16 2.11 Integration 18 2.12 細胞培養 19 2.13 Transfection 19 2.14 免疫共沉澱實驗 20 Chapter 3 實驗結果 21 3.1 F01F1.7(RNAi)造成mature let-7以及某些microRNA量下降 21 3.2 F01F1.7(RNAi)造成pri-let-7 SL-1 form累積 21 3.3 F01F1.7(RNAi)造成primary lin-4、mir-241和mir-48累積 22 3.4 F01F1.7(RNAi)並不會降低Drosha、Pasha以及Argonaute ALG-1的表現量 22 3.5 F01F1.7(RNAi)後lsy-6 miRNA調控cog-1 mRNA功能下降 23 3.6 F01F1.7在細胞核內表現 23 3.7 DDX23無法跟Drosha一起被免疫共沉澱下來 24 Chapter 4 討論 25 Chapter 5 圖表 28 5.1 圖一 28 5.2 圖二 29 5.3 圖三 33 5.4 圖四 34 5.5 圖五 36 5.6 圖六 38 5.7 圖七 39 Chapter 6 參考文獻 41 附錄 44 附圖一 44 附圖二 45 附圖三 46 附圖四 47 附圖五 48 附圖六 49 附圖七 50 附圖八 51 附圖九 52 附圖十 53 附圖十一 54 附表一 55 附表二 57 附表三 59 | |
| dc.language.iso | zh-TW | |
| dc.subject | 線蟲 | zh_TW |
| dc.subject | F01F1.7 | zh_TW |
| dc.subject | DDX-23 | zh_TW |
| dc.subject | let-7 | zh_TW |
| dc.subject | 微小核醣核酸 | zh_TW |
| dc.subject | 核糖核酸解旋? | zh_TW |
| dc.subject | F01F1.7 | en |
| dc.subject | let-7 | en |
| dc.subject | miRNA | en |
| dc.subject | c.elegans | en |
| dc.subject | DDX-23 | en |
| dc.subject | RNA helicase | en |
| dc.title | 線蟲體內DEAD-box核糖核酸解旋酶F01F1.7 /DDX-23
在let-7生合成中所扮演的角色 | zh_TW |
| dc.title | The role of C. elegans DEAD-box RNA helicase F01F1.7/DDX-23 in let-7 microRNA biogenesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 譚婉玉,陳俊宏 | |
| dc.subject.keyword | DDX-23,F01F1.7,核糖核酸解旋?,線蟲,微小核醣核酸,let-7, | zh_TW |
| dc.subject.keyword | DDX-23,F01F1.7,RNA helicase,c.elegans,miRNA,let-7, | en |
| dc.relation.page | 63 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-15 | |
| 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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