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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡欣祐(Hsin-Yue Tsai) | |
| dc.contributor.author | Hsian-Tang Cheng | en |
| dc.contributor.author | 鄭獻堂 | zh_TW |
| dc.date.accessioned | 2021-06-17T00:14:59Z | - |
| dc.date.available | 2020-03-12 | |
| dc.date.copyright | 2020-03-12 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-02-13 | |
| dc.identifier.citation | 1.Ward, S., Y. Argon, and G.A. Nelson, Sperm morphogenesis in wild-type and fertilization-defective mutants of Caenorhabditis elegans. J Cell Biol, 1981. 91(1): p. 26-44.
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Fire, Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science, 2007. 315(5809): p. 241-4. 8.Sijen, T., et al., Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science, 2007. 315(5809): p. 244-7. 9.Claycomb, J.M., et al., The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation. Cell, 2009. 139(1): p. 123-34. 10.Conine, C.C., et al., Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans. Proc Natl Acad Sci U S A, 2010. 107(8): p. 3588-93. 11.Lee, H.C., et al., C. elegans piRNAs mediate the genome-wide surveillance of germline transcripts. Cell, 2012. 150(1): p. 78-87. 12.Shirayama, M., et al., piRNAs initiate an epigenetic memory of nonself RNA in the C. elegans germline. Cell, 2012. 150(1): p. 65-77. 13.Vasale, J.J., et al., Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway. Proc Natl Acad Sci U S A, 2010. 107(8): p. 3582-7. 14.Yigit, E., et al., Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell, 2006. 127(4): p. 747-57. 15.Youngman, E.M. and J.M. Claycomb, From early lessons to new frontiers: the worm as a treasure trove of small RNA biology. Front Genet, 2014. 5: p. 416. 16.Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-11. 17.Lee, R., R. Feinbaum, and V. Ambros, A short history of a short RNA. Cell, 2004. 116(2 Suppl): p. S89-92, 1 p following S96. 18.Conine, C.C., et al., Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans. Cell, 2013. 155(7): p. 1532-44. 19.Gent, J.I., et al., Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma. Mol Cell, 2010. 37(5): p. 679-89. 20.Duchaine, T.F., et al., Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways. Cell, 2006. 124(2): p. 343-54. 21.Pavelec, D.M., et al., Requirement for the ERI/DICER complex in endogenous RNA interference and sperm development in Caenorhabditis elegans. Genetics, 2009. 183(4): p. 1283-95. 22.Matsushita, K., et al., Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay. Nature, 2009. 458(7242): p. 1185-90. 23.Xu, J., et al., Structural study of MCPIP1 N-terminal conserved domain reveals a PIN-like RNase. Nucleic Acids Res, 2012. 40(14): p. 6957-65. 24.Wawro, M., et al., Intact NYN/PIN-Like Domain is Crucial for the Degradation of Inflammation-Related Transcripts by ZC3H12D. J Cell Biochem, 2017. 118(3): p. 487-498. 25.Habacher, C., et al., Ribonuclease-Mediated Control of Body Fat. Dev Cell, 2016. 39(3): p. 359-369. 26.Tsai, H.Y., et al., A ribonuclease coordinates siRNA amplification and mRNA cleavage during RNAi. Cell, 2015. 160(3): p. 407-19. 27.Wang, L., J. Kimble, and M. Wickens, Tissue-specific modification of gld-2 mRNA in C. elegans: likely C-to-U editing. Rna, 2004. 10(9): p. 1444-8. 28.Thompson, F.J., et al., Biochemical and molecular characterization of two cytidine deaminases in the nematode Caenorhabditis elegans. Biochem J, 2002. 365(Pt 1): p. 99-107. 29.Brenner, S., The genetics of Caenorhabditis elegans. Genetics, 1974. 77(1): p. 71-94. 30.Shakes, D.C. and S. Ward, Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis. Dev Biol, 1989. 134(1): p. 189-200. 31.Nelson, G.A. and S. Ward, Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin. Cell, 1980. 19(2): p. 457-64. 32.Phillips, C.M., K.L. McDonald, and A.F. Dernburg, Cytological analysis of meiosis in Caenorhabditis elegans. Methods Mol Biol, 2009. 558: p. 171-95. 33.Doitsidou, M., et al., C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy. PLoS One, 2010. 5(11): p. e15435. 34.L'Hernault, S.W., Spermatogenesis. WormBook, 2006: p. 1-14. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/65905 | - |
| dc.description.abstract | 小核糖核酸在精子生成的過程中扮演著重要的角色。先前的研究揭示了一組小核糖核酸與其相關的argonautes蛋白和秀麗隱桿線蟲的精子生成有關。我們發現當特定的小核糖核酸-argonautes複合物的缺失後，會導致精子在高溫的環境下（25℃）發生缺陷。而ALG-3/4即是這一組argonautes，而與其結合的26G小核糖核酸對高溫下精子的功能非常重要。過去的研究表明，小核糖核酸-ALG-3/4複合物能夠藉由觸發另一種argonaute，CSR-1，所介導的小核糖核酸路徑，確保在25°C時能大量生成精子相關的特異性蛋白。而除了ALG-3/4外，ERI/DICER蛋白複合物是另一群參與在ALG-3/4 26G小核糖核酸生成的成員。因此，這些複合物的成員一旦缺失，它們也會導致線蟲精子在25°C下有所缺陷。然而，這些ALG-3/4 26G小核糖核酸的產生機制為何尚不清楚。
在我的研究中，我們研究一個尚未被報導的基因，c29f5.3。並使用兩個c29f5.3的突變種：核糖核酸酶缺陷型突變種c29f5.3（hyt044）和一個假定的胞苷脫氨酶缺失突變種c29f5.3（tm2499）。發現兩者皆會在高溫下（25° C）不孕。進一步研究發現更這個不孕的現象是精子方面出現了問題，並且可能是發生在精子活化的過程中。相似的情況同樣發生在alg-3/4突變種中。此外，我們也發現c29f5.3突變體會導致ALG-3/4的26G小核糖核酸減少。最後，使用免疫沉澱法後能得到HA-C29F5.3的蛋白質相互作用物。在進行後續的質譜分析後發現，ALG-3確實是C29F5.3的蛋白質相互作用物之一。這些資訊表明C29F5.3參與在ALG-3/4調控的小RNA路徑中，並藉此去調控精子生成的機制。 | zh_TW |
| dc.description.abstract | Small RNAs participate in spermatogenesis is a conserved regulatory pathway from C. elegans to human. Previous studies have revealed a sub-group of small RNAs, 26G small RNA, and their associated argonautes, ALG-3 and ALG-4 (abbreviate as ALG-3/4), are essential for sperm function at elevated temperature (25°C) in C. elegans. The ALG-3/4 26G small RNA protein complex is known triggers RNA dependent RNA polymerase (RdRP), EGO-1, to synthesize a subset of 22G small RNAs and these 22G small RNAs specifically associate with CSR-1, which is another argonaute. The CSR-1 mediated gene regulatory pathway is known results in up-regulation in the transcription level of the targeted genes, which has been known as licensing processes. Previous studies have also shown that the ALG-3/4 26G small RNA targeted genes are majority sperm specific genes. Though several proteins have been known participates in biogenesis of ALG-3/4 26G small RNAs, such as, the class I ERIs in the ERI/DICER complex, the detail mechanism of the biogenesis of ALG-3/4 26G small RNAs are still unknown. Here, we have identified a novel ZC3H12A-like NYN ribonuclease domain protein, C29F5.3. Our data show that the two c29f5.3 mutant strains, including one ribonuclease defective mutant, c29f5.3(hyt044), and a putative cytidine deaminase deletion mutant, c29f5.3(tm2499), result in sterile phenomenon at elevated temperature (25°C). Our further characterization shows that the sterility is solely contributed from sperm failure and likely during sperm activation processes, similar to what is reported in alg-3/4 mutant strain. Furthermore, we have also shown that c29f5.3 mutants not only result in decreases in one of the ALG-3/4 related small RNA but also alg-3/4 targeted mRNAs. Moreover, our mass spectrometry analysis of the protein interactors of immunoprecipitated HA-C29F5.3 has revealed that ALG-3 is one of the protein interactors of HA-C29F5.3. Our data suggest that C29F5.3 participates in ALG-3/4 26G small RNA regulatory pathway and is likely via interaction between C29F5.3 and ALG-3. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T00:14:59Z (GMT). No. of bitstreams: 1 ntu-109-R04448014-1.pdf: 2972945 bytes, checksum: 4c7d7e00a8ac492a008fc366418485b7 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書 Page.ii
謝辭 Page.iii 摘要 Page.iv Abstract Page.v 目錄 Page.vii Chapter1:Introduction Page.1 1.1 The spermatogenesis and spermiogenesis of C. elegans Page.1 1.2 Small RNA pathways in C. elegans Page.2 1.3 Overview of c29f5.3 Page.6 Chapter2: Materials and methods Page.8 2.1 Worm strains Page.8 2.2 Brood size counting assay Page.8 2.3 Fertility assay Page.9 2.4 In vitro activation assay Page.9 2.5 Immunofluorescence Page.10 2.6 RNA extraction Page.11 2.7 RT-PCR and RT-qPCR Page.11 2.8 Protein extraction Page.12 2.9 Western blot Page.12 2.10 Immunoprecipitation Page.13 2.11 Mutagenesis of C. elegans Page.13 Chapter3. Results Page.14 3.1 Generating knock-in strains by CRISPR Page.14 3.1.1 ha-c29f5.3(tm2499) Page.14 3.1.2 c29f5.3(tm2499) rescue strain line Page.15 3.2 Phenotype of c29f5.3 mutants Page.16 3.2.1 Two c29f5.3 mutants are sterile at 25ºC Page.16 3.2.2 The sterility of c29f5.3 mutants are caused by spermatogenesis defect Page.18 3.3 The temperature-sensitive defect of c29f5.3 mutants are due to spermiogenesis (sperm activation) defect in C. elegans. Page.19 3.4 Examination the presence of truncated protein produced by c29f5.3 (tm2499) Page.21 3.4.1 C29F5.3 potentially promotes the transcription of c29f5.3 and target gene Page.21 3.4.2 The antibody of C29F5.3 was unable to detect the specific C29F5.3 Page.23 3.4.3 Truncated C29F5.3 protein is presented in ha-c29f5.3(tm2499); fog-2(q71) Page.23 3.5 C29F5.3 play a role in modulating small RNA biogenesis Page.24 3.6 C29F5.3 is expressed in the region of the male’s germline undergoing spermatogenesis Page.25 3.7 Identify C29F5.3 protein interactor by Mass spectrometry (MS) Page.26 3.7.1 Optimize the immunoprecipitated condition of C29F5.3 Page.26 3.7.2 ALG-3 are identified as C29F5.3 interactor in mass spectrometry Page.27 3.8 Identify novel factors suppressing small RNA mediated temperature-sensitive sperm defect phenotype in C. elegans by forward genetics screening Page.28 Chapter4. Discussion Page.30 4.1 C29F5.3 is involved in different small RNA pathway Page.30 4.2 Several possibilities of novel factors suppressing small RNA mediated temperature-sensitive sperm defect by forward genetics screening Page.31 Figures and Tables Page.33 Figure I. Schematic model of spermatogenesis Page.2 Figure II. Small RNA pathways in C. elegans Page.5 Figure 1. Established two gene knock-in strain by CRISPR Page.33 Figure 2. Schematic of c29f5.3 gene in C. elegans Page.36 Figure 3. c29f5.3(tm2499) and c29f5.3(hyt044) show the temperature-sensitive defects in fertility Page.38 Figure 4. Two c29f5.3 mutants show the sperm dependent temperature-sensitive sterility Page.41 Figure 5. c29f5.3(tm2499) mutants showed the spermiogenesis defect at elevated temperature Page.45 Figure 6. The level of mRNA from c29f5.3 and ALG-3/4 26G small RNAs targets Page.47 Figure 7. The antibody of C29F5.3 was failed to detect the specific C29F5.3 Page.49 Figure 8. Detecting presence of C29F5.3(tm2499) Page.50 Figure 9. TaqMan qPCR validations of the ALG3/4 small RNA (f36h12.4) and ERGO-1 26G small RNA (siR26-1) Page.52 Figure 10. HA::C29F5.3 is expressed during spermatogenesis Page.54 Figure 11. Optimize the immunoprecipitated condition of C29F5.3 for Mass spectrometry Page.56 Figure 12. The current model in this study Page.58 Table 1. Statistical data of microinjection of ha-c29f5.3(tm2499) Page.59 Table 2. Statistical data of microinjection of c29f5.3(tm2499) rescue strain Page.59 Table 3. The percentage of the number of larvae of c29f5.3(tm2499) and c29f5.3(hyt044) relative to wild-type at three temperatures Page.60 Table 4. The percentage of the number of larvae in different cross at three temperatures Page.60 Table 5. Candidate C29F5.3 interact protein Page.61 Appendix Page.62 Strains used in this study Page.62 Primers used in this study Page.63 References Page.65 | |
| dc.language.iso | en | |
| dc.subject | 成精作用 | zh_TW |
| dc.subject | C29F5.3 | zh_TW |
| dc.subject | 小核醣核酸 | zh_TW |
| dc.subject | 秀麗隱桿線蟲 | zh_TW |
| dc.subject | Spermatogenesis | en |
| dc.subject | Small RNA | en |
| dc.subject | C. elegans | en |
| dc.subject | C29F5.3 | en |
| dc.title | 核糖核酸酶C29F5.3於秀麗隱桿線蟲中調控精子生成機制 | zh_TW |
| dc.title | The ribonuclease C29F5.3 regulates spermatogenesis in C. elegans | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳瑞菁(Jui-Ching Wu),詹世鵬(Shih-Peng Chan) | |
| dc.subject.keyword | C29F5.3,成精作用,小核醣核酸,秀麗隱桿線蟲, | zh_TW |
| dc.subject.keyword | C29F5.3,Spermatogenesis,Small RNA,C. elegans, | en |
| dc.relation.page | 69 | |
| dc.identifier.doi | 10.6342/NTU202000460 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-02-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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