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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 朱家瑩(Chia-Ying Chu) | |
| dc.contributor.author | Ying-Ying Chiang | en |
| dc.contributor.author | 江盈盈 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:37:40Z | - |
| dc.date.available | 2021-07-10T21:37:40Z | - |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-18 | |
| dc.identifier.citation | Adams, D.K., Sewell, M.A., Angerer, R.C., and Angerer, L.M. (2011). Rapid adaptation to food availability by a dopamine-mediated morphogenetic response. Nat Commun 2, 592. Alon, S., Garrett, S.C., Levanon, E.Y., Olson, S., Graveley, B.R., Rosenthal, J.J., and Eisenberg, E. (2015). The majority of transcripts in the squid nervous system are extensively recoded by A-to-I RNA editing. Elife 4. Angerer, L.M., Yaguchi, S., Angerer, R.C., and Burke, R.D. (2011). The evolution of nervous system patterning: insights from sea urchin development. Development 138, 3613-3623. Bass, B.L. (2002). RNA editing by adenosine deaminases that act on RNA. Annual Review of Biochemistry 71, 817-846. Behm, M., and Öhman, M. (2016). RNA Editing: A Contributor to Neuronal Dynamics in the Mammalian Brain. Trends in genetics : TIG 32, 165-175. Behm, M., Wahlstedt, H., Widmark, A., Eriksson, M., and Ohman, M. (2017). Accumulation of nuclear ADAR2 regulates adenosine-to-inosine RNA editing during neuronal development. J Cell Sci 130, 745-753. Bhalla, T., Rosenthal, J.J., Holmgren, M., and Reenan, R. (2004). Control of human potassium channel inactivation by editing of a small mRNA hairpin. Nat Struct Mol Biol 11, 950-956. Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120. Burke, R.D., Moller, D.J., Krupke, O.A., and Taylor, V.J. (2014). Sea urchin neural development and the metazoan paradigm of neurogenesis. Genesis 52, 208-221. Burke, R.D., Osborne, L., Wang, D., Murabe, N., Yaguchi, S., and Nakajima, Y. (2006). Neuron-specific expression of a synaptotagmin gene in the sea urchin Strongylocentrotus purpuratus. J Comp Neurol 496, 244-251. Burns, C.M., Chu, H., Rueter, S.M., Hutchinson, L.K., Canton, H., SandersBush, E., and Emeson, R.B. (1997). Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 387, 303-308. Cai, Y.Y., Cronin, C.N., Engel, A.G., Ohno, K., Hersh, L.B., and Rodgers, D.W. (2004). Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders. Embo Journal 23, 2047-2058. Choksi, S.P., Southall, T.D., Bossing, T., Edoff, K., de Wit, E., Fischer, B.E., van Steensel, B., Micklem, G., and Brand, A.H. (2006). Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells. Dev Cell 11, 775-789. Cole, A.J., Saffen, D.W., Baraban, J.M., and Worley, P.F. (1989). Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340, 474-476. Dawson, T.R., Sansam, C.L., and Emeson, R.B. (2004). Structure and sequence determinants required for the RNA editing of ADAR2 substrates. J Biol Chem 279, 4941-4951. Deffit, S.N., Yee, B.A., Manning, A.C., Rajendren, S., Vadlamani, P., Wheeler, E.C., Domissy, A., Washburn, M.C., Yeo, G.W., and Hundley, H.A. (2017). The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis. Elife 6. Deng, P., Khan, A., Jacobson, D., Sambrani, N., McGurk, L., Li, X., Jayasree, A., Hejatko, J., Shohat-Ophir, G., O'Connell, M.A., et al. (2020). Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila. Nat Commun 11, 1580. Desterro, J.M., Keegan, L.P., Lafarga, M., Berciano, M.T., O'Connell, M., and Carmo-Fonseca, M. (2003). Dynamic association of RNA-editing enzymes with the nucleolus. J Cell Sci 116, 1805-1818. Duclot, F., and Kabbaj, M. (2017). The Role of Early Growth Response 1 (EGR1) in Brain Plasticity and Neuropsychiatric Disorders. Front Behav Neurosci 11, 35. Duclot, F., Perez-Taboada, I., Wright, K.N., and Kabbaj, M. (2016). Prediction of individual differences in fear response by novelty seeking, and disruption of contextual fear memory reconsolidation by ketamine. Neuropharmacology 109, 293-305. Dymowska, A.K., Schultz, A.G., Blair, S.D., Chamot, D., and Goss, G.G. (2014). Acid-sensing ion channels are involved in epithelial Na+ uptake in the rainbow trout Oncorhynchus mykiss. Am J Physiol Cell Physiol 307, C255-265. Garner, S., Zysk, I., Byrne, G., Kramer, M., Moller, D., Taylor, V., and Burke, R.D. (2016). Neurogenesis in sea urchin embryos and the diversity of deuterostome neurogenic mechanisms. Development 143, 286-297. Han, Y., Ren, J., Lee, E., Xu, X., Yu, W., and Muegge, K. (2017). Lsh/HELLS regulates self-renewal/proliferation of neural stem/progenitor cells. Scientific reports 7, 1136. Higuchi, M., Stefan, M., Single, F.N., Hartner, J., Rozov, A., Burnashev, N., Feldmeyer, D., Sprengel, R., and Seeburg, P.H. (2000). Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406, 78-81. Hinman, V.F., and Burke, R.D. (2018). Embryonic neurogenesis in echinoderms. Wiley Interdiscip Rev Dev Biol 7, e316. Hollis, F., Gaval-Cruz, M., Carrier, N., Dietz, D.M., and Kabbaj, M. (2012). Juvenile and adult rats differ in cocaine reward and expression of zif268 in the forebrain. Neuroscience 200, 91-98. Hoopengardner, B., Bhalla, T., Staber, C., and Reenan, R. (2003). Nervous system targets of RNA editing identified by comparative genomics. Science 301, 832-836. Hsu, T., and Schulz, R.A. (2000). Sequence and functional properties of Ets genes in the model organism Drosophila. Oncogene 19, 6409-6416. Hung, L.Y., Chen, Y.J., Mai, T.L., Chen, C.Y., Yang, M.Y., Chiang, T.W., Wang, Y.D., and Chuang, T.J. (2018). An Evolutionary Landscape of A-to-I RNA Editome across Metazoan Species. Genome Biol Evol 10, 521-537. Jacobs, M.M., Fogg, R.L., Emeson, R.B., and Stanwood, G.D. (2009). ADAR1 and ADAR2 expression and editing activity during forebrain development. Dev Neurosci 31, 223-237. Jékely, G., Colombelli, J., Hausen, H., Guy, K., Stelzer, E., Nédélec, F., and Arendt, D. (2008). Mechanism of phototaxis in marine zooplankton. Nature 456, 395-399. Jin, B., Tao, Q., Peng, J., Soo, H.M., Wu, W., Ying, J., Fields, C.R., Delmas, A.L., Liu, X., Qiu, J., et al. (2008). DNA methyltransferase 3B (DNMT3B) mutations in ICF syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function. Hum Mol Genet 17, 690-709. Jin, Y., Zhang, W., and Li, Q. (2009). Origins and evolution of ADAR-mediated RNA editing. IUBMB Life 61, 572-578. Jung, C.G., Kim, H.J., Kawaguchi, M., Khanna, K.K., Hida, H., Asai, K., Nishino, H., and Miura, Y. (2005). Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation. Development 132, 5137-5145. Kent, W.J. (2002). BLAT - The BLAST-like alignment tool. Genome Research 12, 656-664. Kosugi, S., Hasebe, M., Tomita, M., and Yanagawa, H. (2009). Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc Natl Acad Sci U S A 106, 10171-10176. Lai Wing Sun, K., Correia, J.P., and Kennedy, T.E. (2011). Netrins: versatile extracellular cues with diverse functions. Development 138, 2153-2169. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., and Genome Project Data, P. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079. Li, I.C., Chen, Y.C., Wang, Y.Y., Tzeng, B.W., Ou, C.W., Lau, Y.Y., Wu, K.M., Chan, T.M., Lin, W.H., Hwang, S.P., et al. (2014). Zebrafish Adar2 Edits the Q/R site of AMPA receptor Subunit gria2α transcript to ensure normal development of nervous system and cranial neural crest cells. PLoS One 9, e97133. Lin, C.Y., and Su, Y.H. (2016). Genome editing in sea urchin embryos by using a CRISPR/Cas9 system. Dev Biol 409, 420-428. Lundell, M.J., and Hirsh, J. (1992). The zfh-2 gene product is a potential regulator fo neuron-specific dopa decarboxylase gene expression in Drosophila. Developmental Biology 154, 84-94. Luo, T., Lee, Y.H., Saint-Jeannet, J.P., and Sargent, T.D. (2003). Induction of neural crest in Xenopus by transcription factor AP2alpha. Proc Natl Acad Sci U S A 100, 532-537. Lynagh, T., Mikhaleva, Y., Colding, J.M., Glover, J.C., and Pless, S.A. (2018). Acid-sensing ion channels emerged over 600 Mya and are conserved throughout the deuterostomes. Proc Natl Acad Sci U S A 115, 8430-8435. McClay, D.R. (2011). Evolutionary crossroads in developmental biology: sea urchins. Development 138, 2639-2648. Metzstein, M.M., and Horvitz, H.R. (1999). The C-elegans cell death. specification gene ces-1 encodes a snail family zinc finger protein. Molecular Cell 4, 309-319. Monge, I., Krishnamurthy, R., Sims, D., Hirth, F., Spengler, M., Kammermeier, L., Reichert, H., and Mitchell, P.J. (2001). Drosophila transcription factor AP-2 in proboscis, leg and brain central complex development. Development 128, 1239-1252. Monge, I., and Mitchell, P.J. (1998). DAP-2, the Drosophila homolog of transcription factor AP-2. Mechanisms of Development 76, 191-195. Nishikura, K. (2016). A-to-I editing of coding and non-coding RNAs by ADARs. Nat Rev Mol Cell Biol 17, 83-96. Oakes, E., Anderson, A., Cohen-Gadol, A., and Hundley, H.A. (2017). Adenosine Deaminase That Acts on RNA 3 (ADAR3) Binding to Glutamate Receptor Subunit B Pre-mRNA Inhibits RNA Editing in Glioblastoma. J Biol Chem 292, 4326-4335. Okuno, H. (2011). Regulation and function of immediate-early genes in the brain: beyond neuronal activity markers. Neurosci Res 69, 175-186. Palladino, M.J., Keegan, L.P., O'Connell, M.A., and Reenan, R.A. (2000a). dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing. Rna 6, 1004-1018. Palladino, M.J., Keegan, L.P., O'Connell, M.A., and Reenan, R.A. (2000b). A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity. Cell 102, 437-449. Picardi, E., and Pesole, G. (2013). REDItools: high-throughput RNA editing detection made easy. Bioinformatics 29, 1813-1814. Porath, H.T., Carmi, S., and Levanon, E.Y. (2014). A genome-wide map of hyper-edited RNA reveals numerous new sites. Nat Commun 5, 4726. Porath, H.T., Hazan, E., Shpigler, H., Cohen, M., Band, M., Ben-Shahar, Y., Levanon, E.Y., Eisenberg, E., and Bloch, G. (2019). RNA editing is abundant and correlates with task performance in a social bumblebee. Nat Commun 10, 1605. Porath, H.T., Knisbacher, B.A., Eisenberg, E., and Levanon, E.Y. (2017). Massive A-to-I RNA editing is common across the Metazoa and correlates with dsRNA abundance. Genome Biol 18, 185. Prado, V.F., Roy, A., Kolisnyk, B., Gros, R., and Prado, M.A. (2013). Regulation of cholinergic activity by the vesicular acetylcholine transporter. Biochem J 450, 265-274. Purves, D. (2018). Neuroscience (New York, United States of America: Oxford University Press, 113-120). Reece-Hoyes, J.S., Deplancke, B., Barrasa, M.I., Hatzold, J., Smit, R.B., Arda, H.E., Pope, P.A., Gaudet, J., Conradt, B., and Walhout, A.J. (2009). The C. elegans Snail homolog CES-1 can activate gene expression in vivo and share targets with bHLH transcription factors. Nucleic Acids Res 37, 3689-3698. Rieder, L.E., and Reenan, R.A. (2012). The intricate relationship between RNA structure, editing, and splicing. Semin Cell Dev Biol 23, 281-288. Rizzo, F., Fernandez-Serra, M., Squarzoni, P., Archimandritis, A., and Arnone, M.I. (2006). Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus). Dev Biol 300, 35-48. Roark, M., Sturtevant, M.A., Emery, J., Vaessin, H., Grell, E., and Bier, E. (1995). scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development. Genes Dev 9, 2384-2398. Sapiro, A.L., Shmueli, A., Henry, G.L., Li, Q., Shalit, T., Yaron, O., Paas, Y., Billy Li, J., and Shohat-Ophir, G. (2019). Illuminating spatial A-to-I RNA editing signatures within the Drosophila brain. Proc Natl Acad Sci U S A 116, 2318-2327. Sea Urchin Genome Sequencing, C., Sodergren, E., Weinstock, G.M., Davidson, E.H., Cameron, R.A., Gibbs, R.A., Angerer, R.C., Angerer, L.M., Arnone, M.I., Burgess, D.R., et al. (2006). The genome of the sea urchin Strongylocentrotus purpuratus. Science 314, 941-952. Shamay-Ramot, A., Khermesh, K., Porath, H.T., Barak, M., Pinto, Y., Wachtel, C., Zilberberg, A., Lerer-Goldshtein, T., Efroni, S., Levanon, E.Y., et al. (2015). Fmrp Interacts with Adar and Regulates RNA Editing, Synaptic Density and Locomotor Activity in Zebrafish. Plos Genetics 11. Slota, L.A., and McClay, D.R. (2018). Identification of neural transcription factors required for the differentiation of three neuronal subtypes in the sea urchin embryo. Dev Biol 435, 138-149. Slota, L.A., Miranda, E., Peskin, B., and McClay, D.R. (2020). Developmental origin of peripheral ciliary band neurons in the sea urchin embryo. Dev Biol 459, 72-78. Slota, L.A., Miranda, E.M., and McClay, D.R. (2019). Spatial and temporal patterns of gene expression during neurogenesis in the sea urchin Lytechinus variegatus. EvoDevo 10, 2. Strathmann, R.R. (2007). Time and extent of ciliary response to particles in a non-filtering feeding mechanism. The Biological bulletin 212, 93-103. Tavano, S., Taverna, E., Kalebic, N., Haffner, C., Namba, T., Dahl, A., Wilsch-Brauninger, M., Paridaen, J., and Huttner, W.B. (2018). Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7. Neuron 97, 1299-1314 e1298. Tonkin, L.A., Saccomanno, L., Morse, D.P., Brodigan, T., Krause, M., and Bass, B.L. (2002). RNA editing by ADARs is important for normal behavior in Caenorhabditis elegans. Embo Journal 21, 6025-6035. Vallecillo-Viejo, I.C., Liscovitch-Brauer, N., Diaz Quiroz, J.F., Montiel-Gonzalez, M.F., Nemes, S.E., Rangan, K.J., Levinson, S.R., Eisenberg, E., and Rosenthal, J.J.C. (2020). Spatially regulated editing of genetic information within a neuron. Nucleic Acids Res 48, 3999-4012. Wang, Z., Lian, J., Li, Q., Zhang, P., Zhou, Y., Zhan, X., and Zhang, G. (2016). RES-Scanner: a software package for genome-wide identification of RNA-editing sites. Gigascience 5, 37. Washburn, M.C., Kakaradov, B., Sundararaman, B., Wheeler, E., Hoon, S., Yeo, G.W., and Hundley, H.A. (2014). The dsRBP and inactive editor ADR-1 utilizes dsRNA binding to regulate A-to-I RNA editing across the C. elegans transcriptome. Cell Rep 6, 599-607. Wei, Z., Angerer, R.C., and Angerer, L.M. (2011). Direct development of neurons within foregut endoderm of sea urchin embryos. Proc Natl Acad Sci U S A 108, 9143-9147. Wu, T.D., and Nacu, S. (2010). Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26, 873-881. Yaguchi, S., Yaguchi, J., Angerer, R.C., Angerer, L.M., and Burke, R.D. (2010). TGFbeta signaling positions the ciliary band and patterns neurons in the sea urchin embryo. Dev Biol 347, 71-81. Yazdani, U., and Terman, J.R. (2006). The semaphorins. Genome Biol 7, 211. Yu, H.L., Wang, L.Z., Zhang, L.L., Chen, B.L., Zhang, H.J., Li, Y.P., Xiao, G.D., and Chen, Y.Z. (2019). ESE1 expression correlates with neuronal apoptosis in the hippocampus after cerebral ischemia/reperfusion injury. Neural regeneration research 14, 841-849. Zhang, F., Lu, Y., Yan, S., Xing, Q., and Tian, W. (2017). SPRINT: an SNP-free toolkit for identifying RNA editing sites. Bioinformatics 33, 3538-3548. Zuker, M. (2003). Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research 31, 3406-3415. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76816 | - |
| dc.description.abstract | RNA編輯可以藉由微調RNA的序列來影響RNA二級結構、microRNA結合位置、RNA穩定度以及氨基酸序列來達到增加蛋白質多樣性的功能。大多數的RNA編輯是由adenosine deaminases acting on RNA (ADARs)來執行，ADARs能夠將雙股RNA上的adenosine藉由水解脫氨的作用修飾成inosine。在許多不同物種的研究上顯示ADAR經常作用於神經傳導物質受體以及離子通道的mRNA上。此外，ADAR適當的RNA編輯程度對於腦部正常功能扮演重要角色。然而現在我們仍然不了解ADAR如何影響神經系統的發育。海膽胚胎擁有相較簡單的神經系統並且可以容易的用於研究早期胚胎發育，因此我們使用海膽胚胎來研究ADAR在神經系統發育過程當中所扮演的角色。海膽胚胎的基因體包含兩個ADAR基因，adar1以及adar2，我們發現adar1專一的表現在神經細胞當中。adar1與一些神經標記物的雙重染色證實了adar1表現的細胞是會分泌乙醯膽鹼的神經細胞。抑制adar1蛋白質轉譯的胚胎，其形態以及軸突的分佈與正常胚胎沒有差異。為了要了解抑制adar1是否會影響神經活化的程度，我們利用鈣離子偵測物GCaMP6紀錄海膽胚胎活體中的神經活性。為了進一步尋找海膽胚胎當中可能的RNA編輯位點，我們利用次世代定序發現大約一萬一千個RNA編輯位點，其中96%都是adenosine修飾成inosine的RNA編輯並且落在大約100個基因上。我們已經利用一代定序確定其中一個基因choline acetyltransferase (chat)的mRNA會被ADAR1編輯。此外，我們已經成功的在Hela細胞當中過度表現海膽胚胎的ChAT蛋白，未來我們將利用體外ChAT分析來了解adenosine修飾成inosine的RNA編輯作用是否會影響到ChAT的酵素活性，這可能會導致海膽胚胎當中神經活性的改變。這個研究首次發現在神經系統發育的過程當中ADAR1可能會藉由微調ChAT的氨基酸序列，進而影響到分泌乙醯膽鹼的神經細胞活性。 | zh_TW |
| dc.description.abstract | RNA editing provides proteomic complexity by fine-tuning RNA sequences, which may result in a wide range of functional changes by affecting RNA secondary structures, microRNA binding sites, RNA stability and amino acid sequences. The majority of RNA editing is A-to-I RNA editing by adenosine deaminases acting on RNA (ADARs) that modify double-stranded RNA to alter adenosine into inosine by deamination. Numerous studies on different species have demonstrated that ADARs frequently act on mRNAs encoding neurotransmitter receptors and ion channels. Furthermore, proper RNA editing levels mediated by ADARs are important for normal brain functions. However, how ADARs affect nervous system development remains unknown. Sea urchin embryos have a relatively simple nervous system and are easily accessible for early developmental studies. Therefore, I set to investigate the roles of ADARs during nervous system development in sea urchin embryos. The sea urchin genome contains two adar genes, adar1 and adar2. I discovered that adar1 is specifically expressed in neuronal cells during embryogenesis. Double staining of adar1 and several neuronal markers confirmed that adar1-positive cells are cholinergic neurons. Adar1-knockdown embryos displayed no difference in morphology and axonal organizations. In order to examine whether knockdown of adar1 affects neuronal activity, I utilized the calcium indicator GCaMP6 to record neuronal activity in live sea urchin embryos. To further identify possible RNA editing sites in sea urchin embryos, I performed next generation sequencing and uncovered 11,000 RNA editing sites, and 96% of which were A-to-I RNA editing sites located in ~100 gene products. One of such gene products, which encodes choline acetyltransferase (ChAT), was confirmed to be a direct target of ADAR1 by Sanger sequencing. Furthermore, I have successfully expressed the sea urchin ChAT in HeLa cells. This in vitro system will allow us to investigate whether A-to-I RNA editing affects the enzymatic activity of ChAT. This study will provide insights into whether ADAR1 regulates neuronal activity during nervous system development by fine-tuning the activity of cholinergic neurons via modulating chat. | en |
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| dc.description.tableofcontents | 口試委員會審定書 i 摘要 ii Abstract iii 1. Introduction 1 1.1 A-to-I RNA editing mediated by adenosine deaminases acting on RNA (ADARs) 1 1.2 ADARs are highly expressed in the central nervous system and they frequently act on mRNAs encoding neurotransmitter receptors and ion channels 1 1.3 Dysfunctions of ADARs in Drosophila and C. elegans resulted in abnormal behaviors 3 1.4 Sea urchin embryos are a good model for investigating roles of A-to-I RNA editing during nervous system development 5 2. Materials and Methods 7 2.1 Culture of sea urchin embryos 7 2.2 Cell culture and transfection 7 2.3 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 7 2.4 Molecular cloning 8 2.5 Fixation of sea urchin embryos for in situ hybridization 8 2.6 In situ hybridization 9 2.7 Generation of ADAR1 antibodies 11 2.8 Fixation of sea urchin embryos for immunostaining 11 2.9 Immunostaining 12 2.10 Protein extraction 13 2.11 Western blot 13 2.12 Gene knockdown by microinjections of morpholinos into the sea urchin eggs 14 2.13 Preparation of RNA for microinjection 15 2.14 Imaging of GCaMP6 signal by confocal microscopy 15 2.15 DNA and RNA extraction for next generation sequencing (NGS) 16 2.16 NGS analysis by REDItools 16 2.17 NGS analysis by RES-Scanner 17 2.18 Validation of A-to-I RNA editing sites by Sanger sequencing 18 2.19 RNA secondary structure prediction 18 2.20 Site-directed mutagenesis 18 3. Results 20 3.1 Sea urchin adar1 is specifically expressed in the ciliary band 20 3.2 ADAR1 antibody specifically recognizes ADAR1 protein 21 3.3 ADAR1-positive cells are neural progenitors at 48 hpf and extend axons along the ciliary band at 96 hpf 22 3.4 ADAR1 is specifically expressed in the ciliary band cholinergic neurons 23 3.5 adar1 translation-blocking morpholino efficiently knocks down ADAR1 in the sea urchin embryo 24 3.6 Knockdown of adar1 does not affect morphology and axonal organization 25 3.7 Utilizing the calcium indicator GCaMP6 to examine whether knockdown of adar1 affects the neural activity in sea urchin embryos 26 3.8 Identification of putative ADAR1 target genes using candidate gene approach 27 3.9 Utilizing REDItools pipeline to identify A-to-I RNA editing targets in sea urchin embryos 36 3.10 Utilizing RES-Scanner pipeline to identify A-to-I RNA editing targets in sea urchin embryos 38 3.11 Transcript of choline acetyltransferase (chat) is a target of the sea urchin ADAR1 41 3.12 ADAR2 may play a role in editing ADAR1 in sea urchin embryos 44 3.13 Transcript of vesicular acetylcholine transporter (vacht) was not a direct target of ADAR1 46 4. Discussions 47 4.1 The cytoplasmic location of sea urchin ADAR1 47 4.2 The acetylcholine pathway may be an important target of ADAR1 in sea urchin embryos 48 4.3 Both candidate gene approach and next generation sequencing data are necessary for identifying ADAR1 targets in sea urchin embryos 49 4.4 A-to-I RNA editing frequencies are highly variable among individuals 50 References 51 List of Figures Figure 1. Domain structures of the sea urchin ADARs. 58 Figure 2. Temporal expression profiles of sea urchin adar1 and adar2. 59 Figure 3. Spatial expression of sea urchin adar1 and adar2 throughout embryogenesis. 60 Figure 4. ADAR1 antibodies specifically recognized ADAR1 protein in the sea urchin embryos. 61 Figure 5. ADAR1-positive cells are neural progenitors at 48 hpf and extend axons along the ciliary band at 96 hpf. 63 Figure 6. ADAR1-positive cells are cholinergic neurons located in the ciliary band. 64 Figure 7. Adar1 was knockdowned by both adar1 translation-blocking morpholino (adar1 tMO) and adar1 splicing-blocking morpholino (adar1 sMO). 65 Figure 8. Knockdown of adar1 does not affect morphology and axonal organization in sea urchin embryos. 67 Figure 9. Neuronal activity in 72 hpf sea urchin embryos could be recorded by using the calcium indicator GCaMP6. 68 Figure 10. Spatial expression of (A) neutrin1 and (B) Semaphorin A (SemaA) from the blastula stage to pluteus stages. 69 Figure 11. Spatial expression of (A) neurogenin (Ngn), (B) prospero (prox1), and (C) synaptotagmin B (Syt1-1) from the blastula stage to the pluteus stages. 71 Figure 12. Spatial expression of (A) lymphoid-specific helicase isoform (hells), (B) acid sensing ion channel 4 (asicl4), and (C) AT-binding transcription factor (atbf1) from the blastula stage to the pluteus stage. 74 Figure 13. Spatial expression of transcription factor genes: (A) AP2, (B) scratch, (C) epithelium-specific ets factor (ese), (D) insulinoma-associated protein (insm), and (E) early growth response (egr) from the blastula stage to the pluteus stage. 77 Figure 14. Spatial expression of (A) vesicular acetylcholine transporter (vacht), and (B) dopamine receptor D1 (drd1) from the blastula stage to the pluteus stage. 78 Figure 15. Schematic summary of the single in situ hybridization of candidate genes in sea urchin embryos at 72 hpf. 79 Figure 16. ADAR1 is co-expressed with prox1 and vacht at 72 hpf. 80 Figure 17. The pipeline of REDItools and the schematic of the clustering strategy for filtering. 81 Figure 18. Under filtering condition of cluster>3, the REDItools pipeline identified ~10,000 A-to-G mismatches in 48 hpf and 72 hpf samples. 84 Figure 19. Distributions of the identified ~850 A-to-G sites within the annotated gene regions analyzed by the REDItools pipeline. 85 Figure 20. Distributions of ADARs’ A-to-I RNA editing frequencies and comparison of A-to-I RNA editing sites identified using REDItools from samples collected at two different developmental stages. 87 Figure 21. Analysis using the RES-Scanner pipeline. 90 Figure 22. Distributions of the identified ~450 A-to-G sites within the annotated gene regions analyzed by the RES-Scanner pipeline. 91 Figure 23. Distributions of ADARs’ A-to-I RNA editing frequencies identified using RES-Scanner from samples collected at two different developmental stages. 94 Figure 24. Distributions of amino acid changes caused by A-to-I RNA editing and comparison of the A-to-I RNA editing sites identified by RES-Scanner at two different stages of the embryos. 95 Figure 25. Summary and comparison of the A-to-I RNA editing sites identified using REDItools and RES-Scanner pipelines. 96 Figure 26. Transcript of choline acetyltransferase (chat) was edited by ADAR1. 100 Figure 27. ADAR1 was not self-edited in the sea urchin embryos. 103 Figure 28. Transcript of vesicular acetylcholine transporter (vacht) is not a direct target of ADAR1. 104 List of Tables Table 1. RT-qPCR primer list 106 Table 2. gene-specific primers for in situ hybridization 107 Table 3. recoding sites-specific primer list 108 Table 4. A-to-I RNA editing frequencies of candidate genes 109 | |
| dc.language.iso | en | |
| dc.subject | RNA 編輯作用 | zh_TW |
| dc.subject | 海膽神經發育 | zh_TW |
| dc.subject | ADARs | zh_TW |
| dc.subject | sea urchin neural development | en |
| dc.subject | RNA editing | en |
| dc.subject | ADARs | en |
| dc.title | 探討 A-to-I RNA 編輯作用在海膽神經發育扮演的功能 | zh_TW |
| dc.title | Deciphering the functions of A-to-I RNA editing in sea urchin neural development | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 蘇怡璇(Yi-Hsien Su) | |
| dc.contributor.oralexamcommittee | 陳俊安(Jun-An Chen),莊樹諄(Trees-Juen Chuang),周銘翊(Ming-Yi Chou) | |
| dc.subject.keyword | ADARs,RNA 編輯作用,海膽神經發育, | zh_TW |
| dc.subject.keyword | ADARs,RNA editing,sea urchin neural development, | en |
| dc.relation.page | 110 | |
| dc.identifier.doi | 10.6342/NTU202003627 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-19 | |
| 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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