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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡素宜(Su-Yi Tsai) | |
| dc.contributor.author | Serena Huei-An Lu | en |
| dc.contributor.author | 盧薈安 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:09:02Z | - |
| dc.date.available | 2021-11-03 | |
| dc.date.available | 2022-11-24T03:09:02Z | - |
| dc.date.copyright | 2021-11-03 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-10-28 | |
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Developmental control of titin isoform expression and passive stiffness in fetal and neonatal myocardium. Circ Res 94, 505-513. 24. Li, S., Guo, W., Dewey, C.N., and Greaser, M.L. (2013). Rbm20 regulates titin alternative splicing as a splicing repressor. Nucleic Acids Res 41, 2659-2672. 25. Lian, X., Zhang, J., Azarin, S.M., Zhu, K., Hazeltine, L.B., Bao, X., Hsiao, C., Kamp, T.J., and Palecek, S.P. (2013). Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat Protoc 8, 162-175. 26. Liu, J., Kong, X., Zhang, M., Yang, X., and Xu, X. (2019). RNA binding protein 24 deletion disrupts global alternative splicing and causes dilated cardiomyopathy. Protein Cell 10, 405-416. 27. Luther, P.K. (2009). The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling. J Muscle Res Cell Motil 30, 171-185. 28. Mi, H., Muruganujan, A., Ebert, D., Huang, X., and Thomas, P.D. (2019). PANTHER version 14: more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools. Nucleic Acids Res 47, D419-D426. 29. Myhre, J.L., and Pilgrim, D.B. (2012). At the Start of the Sarcomere: A Previously Unrecognized Role for Myosin Chaperones and Associated Proteins during Early Myofibrillogenesis. Biochem Res Int 2012, 712315. 30. Ohtsuka, H., Yajima, H., Maruyama, K., and Kimura, S. (1997). The N-terminal Z repeat 5 of connectin/titin binds to the C-terminal region of alpha-actinin. Biochem Biophys Res Commun 235, 1-3. 31. Olson, E.N. (2006). Gene regulatory networks in the evolution and development of the heart. Science 313, 1922-1927. 32. Paz, I., Kosti, I., Ares, M., Jr., Cline, M., and Mandel-Gutfreund, Y. (2014). RBPmap: a web server for mapping binding sites of RNA-binding proteins. Nucleic Acids Res 42, W361-367. 33. Poon, K.L., Tan, K.T., Wei, Y.Y., Ng, C.P., Colman, A., Korzh, V., and Xu, X.Q. (2012). RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility. Cardiovasc Res 94, 418-427. 34. Rhee, D., Sanger, J.M., and Sanger, J.W. (1994). The premyofibril: evidence for its role in myofibrillogenesis. Cell Motil Cytoskeleton 28, 1-24. 35. Schiano, C., Costa, V., Aprile, M., Grimaldi, V., Maiello, C., Esposito, R., Soricelli, A., Colantuoni, V., Donatelli, F., Ciccodicola, A., et al. (2017). Heart failure: Pilot transcriptomic analysis of cardiac tissue by RNA-sequencing. Cardiol J 24, 539-553. 36. Schoenauer, R., Emmert, M.Y., Felley, A., Ehler, E., Brokopp, C., Weber, B., Nemir, M., Faggian, G.G., Pedrazzini, T., Falk, V., et al. (2011). EH-myomesin splice isoform is a novel marker for dilated cardiomyopathy. Basic Research in Cardiology 106, 233-247. 37. Tohyama, S., Hattori, F., Sano, M., Hishiki, T., Nagahata, Y., Matsuura, T., Hashimoto, H., Suzuki, T., Yamashita, H., Satoh, Y., et al. (2013). Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127-137. 38. Tsai, S.Y., Ghazizadeh, Z., Wang, H.J., Amin, S., Ortega, F.A., Badieyan, Z.S., Hsu, Z.T., Gordillo, M., Kumar, R., Christini, D.J., et al. (2020). A human embryonic stem cell reporter line for monitoring chemical-induced cardiotoxicity. Cardiovasc Res 116, 658-670. 39. Turnacioglu, K.K., Mittal, B., Dabiri, G.A., Sanger, J.M., and Sanger, J.W. (1997). An N-terminal fragment of titin coupled to green fluorescent protein localizes to the Z-bands in living muscle cells: overexpression leads to myofibril disassembly. Mol Biol Cell 8, 705-717. 40. van den Hoogenhof, M.M.G., Beqqali, A., Amin, A.S., van der Made, I., Aufiero, S., Khan, M.A.F., Schumacher, C.A., Jansweijer, J.A., van Spaendonck-Zwarts, K.Y., Remme, C.A., et al. (2018). RBM20 Mutations Induce an Arrhythmogenic Dilated Cardiomyopathy Related to Disturbed Calcium Handling. Circulation 138, 1330-1342. 41. van den Hoogenhof, M.M.G., van der Made, I., Beqqali, A., de Groot, N.E., Damanafshan, A., van Oort, R.J., Pinto, Y.M., and Creemers, E.E. (2017). The RNA-binding protein Rbm38 is dispensable during pressure overload-induced cardiac remodeling in mice. PLOS ONE 12, e0184093. 42. Van der Ven, P.F., Ehler, E., Perriard, J.C., and Furst, D.O. (1999). Thick filament assembly occurs after the formation of a cytoskeletal scaffold. J Muscle Res Cell Motil 20, 569-579. 43. Wang, H., Chen, Y., Li, X., Chen, G., Zhong, L., Chen, G., Liao, Y., Liao, W., and Bin, J. (2016). Genome-wide analysis of alternative splicing during human heart development. Scientific Reports 6, 35520. 44. Wei, C., Qiu, J., Zhou, Y., Xue, Y., Hu, J., Ouyang, K., Banerjee, I., Zhang, C., Chen, B., Li, H., et al. (2015). Repression of the Central Splicing Regulator RBFox2 Is Functionally Linked to Pressure Overload-Induced Heart Failure. Cell Reports 10, 1521-1533. 45. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80546 | - |
| dc.description.abstract | 肌節是構成肌收縮的基本單位。肌節蛋白的基因缺陷可能導致肌節無法組成，進而引發心臟相關疾病。藉由選擇性剪接調控，肌節蛋白可於不同的肌纖維生成時期表現不同的剪接體，正確的剪接體調控對於肌節結構的穩定性十分重要。心臟剪接因子RBM24是一個組織專一的選擇性剪接調控分子，已知其在心臟生成中扮演重要的角色。然而，目前尚未了解RBM24如何透過調控發育過程中階段性的選擇性剪接，進而影響肌節的組裝與心生成。為了瞭解RBM24如何階段性調控肌節組裝及心臟生成，我們以人類胚胎幹細胞作為模型，利用CRISPR/Cas9剔除RBM24蛋白。RBM24剔除的胚胎幹細胞衍生心肌細胞呈現了肌節的混亂及點狀的Z線，我發現這與肌纖維生成早期的肌凝蛋白替換缺陷有關。轉錄體分析顯示RBM24調控了超過4000個以上的基因。在這些基因之中，RBM24缺失造成核心的肌纖維生成蛋白（包含ACTN2、TTN、MYH10等）不正常的調控，導致肌纖維生成停滯在前肌纖維時期，並引起了肌節的混亂。有趣的是，我發現RBM24調控了Z線蛋白ACTN2第六外顯子（exon 6）的剪接，而這不含exon 6的ACTN2會在心肌分化早期表現，到成人時心臟則表現含有exon 6的ACTN2。在人類胚胎幹細胞衍生之心肌細胞中，不論是藉由CRISPR/Cas9去除ACTN2基因的exon 6，或是在RBM24剔除的細胞中過表現全長的ACTN2，都證明了ACTN2轉錄子的exon6包含與否對於肌節的組裝十分重要。整體而言，我發現RBM24在特定的心肌細胞分化時期促進了ACTN2exon6的內含，進而調控了肌節的組裝及穩定。我的研究顯示RBM24可以時間性調控核心肌纖維生成蛋白的基因表現，進而影響肌節組成，可被視為一個主要調控者。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:09:02Z (GMT). No. of bitstreams: 1 U0001-2510202122471700.pdf: 5542560 bytes, checksum: 78fe90351d752eba4c44ba69a9cdb5e7 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "致謝 i 中文摘要 ii Abstract iii Contents v List of Figures viii List of Table ix Introduction 1 Alternative splicing and heart development 1 RNA-binding proteins involved in heart development and diseases 2 RBM24 and its known roles in heart development 3 Sarcomere assembly and the premyofibril model 5 Aims 7 Materials and Methods 9 hESC culture and directed cardiac differentiation 9 Generation of RBM24 and ACTN2 exon6 knockout hESC lines using the CRISPR/Cas9 technique 10 Overexpression of ACTN2 and doxycycline-inducible RBM24 in RBM24-knockout or WT hESC lines 11 Immunofluorescence analysis 12 Western blotting 13 Fluorescence-activated cell sorting (FACS) and flow cytometry 14 RNA isolation, RNA-sequencing and analyses 14 RNA immunoprecipitation (RNA IP) 16 Calcium imaging 17 Reverse Transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) 17 Transmission electron microscopy (TEM) 18 Construction of the ACTN2 splicing reporter and splicing assay 19 Statistics 20 Results 22 RBM24 is highly expressed during hESC-CM differentiation process 22 RBM24-/- hESCs differentiate into cardiomyocytes with lower efficiency 23 RBM24-/--CMs exhibit disorganized sarcomere 23 Calcium handling ability is impaired upon RBM24 ablation 24 Transcriptome analysis reveals the regulatory role of RBM24 on both structural and functional genes in hESC-CMs 26 RBM24 regulates myofibrillogenesis genes through alternative splicing 27 Exon6-excluded ACTN2 is a fetal form and is abnormally induced in RBM24-/--CMs 30 Sarcomere assembly is arrested in premyofibril stage in RBM24-/--CMs 32 Exclusion of exon 6 in ACTN2 causes sarcomere disorganization and reduced expression of ACTN2 in hESC-CMs 34 Ectopic expression of exon6-included ACTN2 reduces sarcomere stagnation and disruption in RBM24-/--CMs 36 RBM24 directly mediate the inclusion of exon 6 in ACTN2 in cooperation with splicing repressors 37 Discussion 41 Figures 47 Figure 1. RBM24 is a highly express splicing factor in hESC-CM. 48 Figure 2. Disrupted sarcomeric structures with punctate Z-lines in RBM24-/--CMs. 49 Figure 3. RBM24-/--CMs exhibit abnormal calcium transients and impaired responses to β-adrenergic stimulation. 52 Figure 4. Transcriptome analysis reveals the regulatory role of RBM24. 53 Figure 5. RBM24 regulates the expression of sarcomere-related genes, especially Z-line. 55 Figure 6. Conserved and unique roles of RBM24 in gene regulation through alternative splicing. 57 Figure 7. RBM24 regulates the expression of core myofibril genes through alternative splicing. 59 Figure 8. An exon 6 exclusion form of ACTN2 is induced in RBM24-/- -CMs and human fetal. 62 Figure 9. Sarcomere assembly is arrested at the premyofibril stage. 63 Figure 10. RBM24 knockout leads to accumulation of TTN and MYBPC3, causing defects in the interaction between TTN and ACTN2. 65 Figure 11. Generation of ACTN2 exon6-knockout lines in hESCs. 67 Figure 12. ACTN2 exon 6 inclusion is critical for Z-line integrity. 69 Figure 13. Ectopic expression of full-length ACTN2 rescues RBM24 knockout phenotypes. 71 Figure 14. Ectopic expression of full-length ACTN2 in WT hESC-CM do not affect Z-line length. 73 Figure 15. RBM24 alone is sufficient to regulate ACTN2 exon 6 splicing. 75 Figure 16. RBM24 directly regulates core myofibril genes and potentially orchestrates alternative splicing together with splicing repressors. 78 Figure 17. A working model of RBM24 function in human sarcomere assembly and integrity. 80 Table 81 Table 1. Primers 81 Reference 86 Appendix 95 Appendix. Knockout of RBM24 using CRISPR/Cas9 resulted in reduced differentiation efficiency. 95 " | |
| dc.language.iso | en | |
| dc.subject | 心肌纖維生成 | zh_TW |
| dc.subject | 擴張型心肌病 | zh_TW |
| dc.subject | 人類胚胎幹細胞 | zh_TW |
| dc.subject | 選擇性剪接 | zh_TW |
| dc.subject | 心臟剪接因子 | zh_TW |
| dc.subject | CRIPR/Cas9技術 | zh_TW |
| dc.subject | CRISPR/Cas9 technique | en |
| dc.subject | RNA binding protein | en |
| dc.subject | alternative splicing | en |
| dc.subject | cardiac myofibrillogenesis | en |
| dc.subject | human embryonic stem cells | en |
| dc.subject | dilated cardiomyopathy | en |
| dc.title | 心臟剪接因子RBM24透過選擇性剪接調控人類胚胎幹細胞衍生心肌細胞的肌節組成 | zh_TW |
| dc.title | Alternative Splicing Mediated by RNA-binding Protein RBM24 Modulates Sarcomere Organization in hESC-derived Cardiomyocytes | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.author-orcid | 0000-0003-0434-8840 | |
| dc.contributor.oralexamcommittee | 郭紘志(Hsin-Tsai Liu),李士傑(Chih-Yang Tseng),夏國強,楊鎧鍵 | |
| dc.subject.keyword | 心臟剪接因子,選擇性剪接,心肌纖維生成,人類胚胎幹細胞,擴張型心肌病,CRIPR/Cas9技術, | zh_TW |
| dc.subject.keyword | RNA binding protein,alternative splicing,cardiac myofibrillogenesis,human embryonic stem cells,dilated cardiomyopathy,CRISPR/Cas9 technique, | en |
| dc.relation.page | 95 | |
| dc.identifier.doi | 10.6342/NTU202104188 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-10-29 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生命科學系 | zh_TW |
| 顯示於系所單位： | 生命科學系 | |
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