RNA編輯被認為是分子生物學的中心教條中一個少數的例外，近五年來的研究揭露許多大量在人類基因體中的編輯點，雖然這些編輯點有何影響需要更多的研究，但是這些發現強烈地告訴我們RNA編輯是頻繁地並且影響大量的基因，藉由選擇性地修飾一個基因的一小片序列，RNA編輯可以使一個細胞中的單一基因製造出不同性質的蛋白質。本論文主要探討在生物的生理中，A-to-I編輯在適應環境壓力機制上所扮演的角色，並且以果蠅做為實驗的模式生物。在先前的研究中指出，hypnos-2突變的果蠅在缺氧耐受性以及熱耐受性都不如wild type果蠅，更進一步研究hypnos-2基因時，更發現hypnos-2基因就是dADAR基因，支持了我們假設A-to-I編輯可能為果蠅適應環境壓力的一種策略。我們利用兩個面向的實驗來探查果蠅如何用A-to-I編輯來適應“熱”環境壓力，其一是探查A-to-I編輯的執行者dADAR，實驗發現果蠅dADAR基因會受到熱休克處理的作用使得exon 7自我編輯點編輯頻率會下降，可能代表dADAR的活性經熱休克處理而提高，值得注意的是，在兩個promoter活化的實驗中發現，不含-1序列的轉錄物表現量有明顯地增加，含-1序列的轉錄物則相對地減少，但是dADAR mRNA的表現量及兩個promoter活化的程度都沒有明顯地增加或減少。其二則是探查已知的30個A-to-I編輯點，發現大多數編輯點的編輯頻率會因為熱休克處理而有所改變。兩個面向的實驗證實dADAR的活性、dADAR基因轉錄物的表現量差異與dADAR目標基因的A-to-I編輯頻率會因為熱休克處理而改變，推測可能因此是果蠅適應能力的來源，然而這些改變如何讓果蠅適應環境壓力更詳盡的分子機制，仍然需要更多的實驗來證明。

RNA editing had been considered as a rare exception to the central dogma of molecular biology in which the mRNA truthfully carries genetic code from nucleus to the ribosome for translation. However, researches in the last five years have revealed numerous, widespread RNA A-to-I editing sites in human genome. Although the effects of these editing events require further study, this finding strongly suggests RNA editing occurs frequently, and affects large number of genes. By selectively modifying a few sequences of a gene, RNA editing allows a cell to produce a population of proteins with different properties from a single gene. The major question of this thesis study is whether such editing event is actually dynamically regulated when the cellular physiological processes have to be adjusted in response to changing environment. A previous study screening for Drosophila mutants defected in hypoxia and heat tolerance discovered a hypnos-2 mutant strain which was later found to be defective in dADAR, the drosophila gene encoding the A-to-I editing enzyme, supporting the hypothesis that cells/organisms response to stressful environment by dADAR-mediated RNA editing. Two directions are used to approach how Drosophila uses A-to-I editing to adapt “heat” environment stress. First, whether the expression pattern of dADAR changes after heat shock was investegated. The result showed the dADAR gene exon 7 self-editing frequency was decreased by heat shock, thus possibly enhances dADAR activity after heat shock processing. Moreover it is worth noting that the isoform without -1 exon transcript were obviously up-regulated, and transcript with -1 sequence is relatively down-regulated. On the other hand, no significant changes in the dADAR mRNA expression levels and in the degrees of two dADAR promoters activity were observed. Second, the changes of editing frequency of 30 known A-to-I editing sites were investigated. Generally the editing frequency of majority editing sites changed after heat shock. Therefore, the dADAR activity, the dADAR gene transcript expression alternations, and A-to-I editing frequency of dADAR target genes did change after heat shock, supporting the notion that change of RNA editing pattern is a mechanism for organism to adapt to drastic environmental change. However, how the edited protein isoforms contribute to heat resistance requires further investigation.

致謝 i
摘要 ii
ABSTRACT iii
縮寫表 v
目錄 vii
圖目錄 x
表目錄 xiii
1. 前言 - 1 -
1.1 RNA A-to-I編輯 - 1 -
1.1.1 Editing site Complementary Sequence (ECS) - 2 -
1.1.2 A-to-I編輯對RNA的影響 - 3 -
1.2 Mammalian ADAR - 4 -
1.2.1 Mammalian ADAR家族的成員與domain結構 - 5 -
1.2.2 ADAR1 - 5 -
1.2.3 ADAR2 - 6 -
1.2.4 ADAR3 - 7 -
1.3 哺乳動物中神經傳導相關基因的A-to-I編輯 - 7 -
1.4 在Drosophila的A-to-I編輯 - 8 -
1.4.1 dADAR - 9 -
1.4.2 果蠅目前發現有被A-to-I編輯的基因及其編輯點可能的影響 - 10 -
1.4.3 環境壓力(stress)與果蠅A-to-I編輯的關係 - 12 -
2. HYPOTHESIS AND SPECIFIC AIMS - 14 -
3. 實驗設計 - 16 -
4. 材料與方法 - 18 -
4.1 果蠅的培養(Fly Stock) - 18 -
4.1.1 果蠅培養基(medium)的製作 - 18 -
4.1.2 果蠅培養瓶的製作 - 18 -
4.1.3 準備實驗的果蠅 - 18 -
4.2 熱休克處理(Heat Shock) - 19 -
4.3 果蠅total RNA的純化 - 19 -
4.4 Reverse Transcription – cDNA Synthesis - 20 -
4.5 Polymerase Chain Reaction (PCR) - 21 -
4.6 One-step RT-PCR - 21 -
4.7 純化PCR產物和定序 - 22 -
4.8 測定實驗組和對照組編輯頻率的差異 - 22 -
5. 結果 - 23 -
5.1 dADAR基因mRNA表現量的變化 - 23 -
5.2 dADAR 3a序列表現量的變化 - 23 -
5.3 dADAR 基因exon 7自我編輯點其編輯頻率的變化 - 24 -
5.4 dADAR 基因兩種promoter活化的變化與其mRNA isoform表現量的變化 - 25 -
5.5 在神經系統的dADAR目標基因編輯點其編輯頻率的變化 - 26 -
6. 討論 - 27 -
7. 結論 - 32 -
8. 參考文獻 - 33 -
附錄 - 40 -
A. 圖 - 41 -
B. 表 - 78 -

Athanasiadis A, Rich A, Maas S (2004) Widespread A-to-I RNA editing of Alu-containing mRNAs in the human transcriptome. PLoS biology 2(12): e391

Bass BL (2002) RNA editing by adenosine deaminases that act on RNA. Annual Review of Biochemistry 71: 817-846

Bass BL, Weintraub H (1987) A developmentally regulated activity that unwinds RNA duplexes. Cell 48(4): 607-613

Bass BL, Weintraub H (1988) An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 55(6): 1089-1098

Benne R, Van den Burg J, Brakenhoff JP, Sloof P, Van Boom JH, Tromp MC (1986) Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell 46(6): 819-826

Blow M, Futreal PA, Wooster R, Stratton MR (2004) A survey of RNA editing in human brain. Genome Research 14(12): 2379-2387

Burns CM, Chu H, Rueter SM, Hutchinson LK, Canton H, Sanders-Bush E, Emeson RB (1997) Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 387(6630): 303-308

Chapman ER, Desai RC, Davis AF, Tornehl CK (1998) Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin. The Journal of Biological Chemistry 273(49): 32966-32972

Chen CX, Cho DS, Wang Q, Lai F, Carter KC, Nishikura K (2000) A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains. RNA (New York, NY) 6(5): 755-767

Clutterbuck DR, Leroy A, O'Connell MA, Semple CA (2005) A bioinformatic screen for novel A-I RNA editing sites reveals recoding editing in BC10. Bioinformatics (Oxford, England) 21(11): 2590-2595

Davis AF, Bai J, Fasshauer D, Wolowick MJ, Lewis JL, Chapman ER (1999) Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes. Neuron 24(2): 363-376

Dawson TR, Sansam CL, Emeson RB (2004) Structure and sequence determinants required for the RNA editing of ADAR2 substrates. The Journal of Biological Chemistry 279(6): 4941-4951

Eaholtz G, Scheuer T, Catterall WA (1994) Restoration of inactivation and block of open sodium channels by an inactivation gate peptide. Neuron 12(5): 1041-1048

Egebjerg J, Heinemann SF (1993) Ca2+ permeability of unedited and edited versions of the kainate selective glutamate receptor GluR6. Proceedings of the National Academy of Sciences of the United States of America 90(2): 755-759

Egebjerg J, Kukekov V, Heinemann SF (1994) Intron sequence directs RNA editing of the glutamate receptor subunit GluR2 coding sequence. Proceedings of the National Academy of Sciences of the United States of America 91(22): 10270-10274

Fitzgerald LW, Iyer G, Conklin DS, Krause CM, Marshall A, Patterson JP, Tran DP, Jonak GJ, Hartig PR (1999) Messenger RNA editing of the human serotonin 5-HT2C receptor. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 21(2 Suppl): 82S-90S

Flomen R, Knight J, Sham P, Kerwin R, Makoff A (2004) Evidence that RNA editing modulates splice site selection in the 5-HT2C receptor gene. Nucleic Acids Research 32(7): 2113-2122

Gott JM, Emeson RB (2000) Functions and mechanisms of RNA editing. Annual Review of Genetics 34: 499-531

Greger IH, Khatri L, Kong X, Ziff EB (2003) AMPA receptor tetramerization is mediated by Q/R editing. Neuron 40(4): 763-774

Greger IH, Khatri L, Ziff EB (2002) RNA editing at arg607 controls AMPA receptor exit from the endoplasmic reticulum. Neuron 34(5): 759-772

Haddad GG, Sun Y, Wyman RJ, Xu T (1997) Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster. Proceedings of the National Academy of Sciences of the United States of America 94(20): 10809-10812

Hanrahan CJ, Palladino MJ, Ganetzky B, Reenan RA (2000) RNA editing of the Drosophila para Na(+) channel transcript. Evolutionary conservation and developmental regulation. Genetics 155(3): 1149-1160

Herb A, Higuchi M, Sprengel R, Seeburg PH (1996) Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences. Proceedings of the National Academy of Sciences of the United States of America 93(5): 1875-1880

Herbert A, Alfken J, Kim YG, Mian IS, Nishikura K, Rich A (1997) A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase. Proceedings of the National Academy of Sciences of the United States of America 94(16): 8421-8426

Herrick-Davis K, Grinde E, Niswender CM (1999) Serotonin 5-HT2C receptor RNA editing alters receptor basal activity: implications for serotonergic signal transduction. Journal of Neurochemistry 73(4): 1711-1717

Higuchi M, Maas S, Single FN, Hartner J, Rozov A, Burnashev N, Feldmeyer D, Sprengel R, Seeburg PH (2000) Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature 406(6791): 78-81

Higuchi M, Single FN, Kohler M, Sommer B, Sprengel R, Seeburg PH (1993) RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency. Cell 75(7): 1361-1370

Hoopengardner B, Bhalla T, Staber C, Reenan R (2003) Nervous system targets of RNA editing identified by comparative genomics. Science (New York, NY) 301(5634): 832-836

Imoto K, Konno T, Nakai J, Wang F, Mishina M, Numa S (1991) A ring of uncharged polar amino acids as a component of channel constriction in the nicotinic acetylcholine receptor. FEBS Letters 289(2): 193-200

Keegan LP, Brindle J, Gallo A, Leroy A, Reenan RA, O'Connell MA (2005) Tuning of RNA editing by ADAR is required in Drosophila. The EMBO Journal 24(12): 2183-2193

Kim DD, Kim TT, Walsh T, Kobayashi Y, Matise TC, Buyske S, Gabriel A (2004) Widespread RNA editing of embedded alu elements in the human transcriptome. Genome Research 14(9): 1719-1725

Kim U, Wang Y, Sanford T, Zeng Y, Nishikura K (1994) Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing. Proceedings of the National Academy of Sciences of the United States of America 91(24): 11457-11461

Knight SW, Bass BL (2002) The role of RNA editing by ADARs in RNAi. Molecular Cell 10(4): 809-817

Konno T, Busch C, Von Kitzing E, Imoto K, Wang F, Nakai J, Mishina M, Numa S, Sakmann B (1991) Rings of anionic amino acids as structural determinants of ion selectivity in the acetylcholine receptor channel. ProceedingsBiological Sciences / The Royal Society 244(1310): 69-79

Kung SS, Chen YC, Lin WH, Chen CC, Chow WY (2001) Q/R RNA editing of the AMPA receptor subunit 2 (GRIA2) transcript evolves no later than the appearance of cartilaginous fishes. FEBS Letters 509(2): 277-281

Lai F, Drakas R, Nishikura K (1995) Mutagenic analysis of double-stranded RNA adenosine deaminase, a candidate enzyme for RNA editing of glutamate-gated ion channel transcripts. The Journal of Biological Chemistry 270(29): 17098-17105

Lehmann KA, Bass BL (1999) The importance of internal loops within RNA substrates of ADAR1. Journal of Molecular Biology 291(1): 1-13

Levanon EY, Eisenberg E, Yelin R, Nemzer S, Hallegger M, Shemesh R, Fligelman ZY, Shoshan A, Pollock SR, Sztybel D, Olshansky M, Rechavi G, Jantsch MF (2004) Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nature Biotechnology 22(8): 1001-1005

Lomeli H, Mosbacher J, Melcher T, Hoger T, Geiger JR, Kuner T, Monyer H, Higuchi M, Bach A, Seeburg PH (1994) Control of kinetic properties of AMPA receptor channels by nuclear RNA editing. Science (New York, NY) 266(5191): 1709-1713

Ma E, Gu XQ, Wu X, Xu T, Haddad GG (2001) Mutation in pre-mRNA adenosine deaminase markedly attenuates neuronal tolerance to O2 deprivation in Drosophila melanogaster. The Journal of Clinical Investigation 107(6): 685-693

Maas S, Patt S, Schrey M, Rich A (2001) Underediting of glutamate receptor GluR-B mRNA in malignant gliomas. Proceedings of the National Academy of Sciences of the United States of America 98(25): 14687-14692

Maas S, Rich A, Nishikura K (2003) A-to-I RNA editing: recent news and residual mysteries. The Journal of Biological Chemistry 278(3): 1391-1394

Melcher T, Maas S, Herb A, Sprengel R, Higuchi M, Seeburg PH (1996) RED2, a brain-specific member of the RNA-specific adenosine deaminase family. The Journal of Biological Chemistry 271(50): 31795-31798

Morse DP, Aruscavage PJ, Bass BL (2002) RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA. Proceedings of the National Academy of Sciences of the United States of America 99(12): 7906-7911

Nishikura K (2006) Editor meets silencer: crosstalk between RNA editing and RNA interference. Nature reviewsMolecular cell biology 7(12): 919-931

Niswender CM, Copeland SC, Herrick-Davis K, Emeson RB, Sanders-Bush E (1999) RNA editing of the human serotonin 5-hydroxytryptamine 2C receptor silences constitutive activity. The Journal of Biological Chemistry 274(14): 9472-9478

Palladino MJ, Keegan LP, O'Connell MA, Reenan RA (2000a) dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing. RNA (New York, NY) 6(7): 1004-1018

Palladino MJ, Keegan LP, O'Connell MA, Reenan RA (2000b) A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity. Cell 102(4): 437-449

Rebagliati MR, Melton DA (1987) Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity. Cell 48(4): 599-605

Reenan RA, Hanrahan CJ, Barry G (2000) The mle(napts) RNA helicase mutation in drosophila results in a splicing catastrophe of the para Na+ channel transcript in a region of RNA editing. Neuron 25(1): 139-149

Rueter SM, Dawson TR, Emeson RB (1999) Regulation of alternative splicing by RNA editing. Nature 399(6731): 75-80

Ryter JM, Schultz SC (1998) Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA. The EMBO Journal 17(24): 7505-7513

Seeburg PH, Single F, Kuner T, Higuchi M, Sprengel R (2001) Genetic manipulation of key determinants of ion flow in glutamate receptor channels in the mouse. Brain Research 907(1-2): 233-243

Smith LA, Wang X, Peixoto AA, Neumann EK, Hall LM, Hall JC (1996) A Drosophila calcium channel alpha1 subunit gene maps to a genetic locus associated with behavioral and visual defects. The Journal of Neuroscience : the official journal of the Society for Neuroscience 16(24): 7868-7879

Sommer B, Kohler M, Sprengel R, Seeburg PH (1991) RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67(1): 11-19

Teng B, Burant CF, Davidson NO (1993) Molecular cloning of an apolipoprotein B messenger RNA editing protein. Science 260(5115): 1816-1819

Valente L, Nishikura K (2005) ADAR gene family and A-to-I RNA editing: diverse roles in posttranscriptional gene regulation. Prog Nucleic Acid Res Mol Biol 79: 299-338

Wagner RW, Smith JE, Cooperman BS, Nishikura K (1989) A double-stranded RNA unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and Xenopus eggs. Proceedings of the National Academy of Sciences of the United States of America 86(8): 2647-2651

Wang Q, Khillan J, Gadue P, Nishikura K (2000a) Requirement of the RNA editing deaminase ADAR1 gene for embryonic erythropoiesis. Science (New York, NY) 290(5497): 1765-1768

Wang Q, Miyakoda M, Yang W, Khillan J, Stachura DL, Weiss MJ, Nishikura K (2004) Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene. The Journal of Biological Chemistry 279(6): 4952-4961

Wang Q, O'Brien PJ, Chen CX, Cho DS, Murray JM, Nishikura K (2000b) Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors. Journal of Neurochemistry 74(3): 1290-1300

Yoshihara M, Littleton JT (2002) Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release. Neuron 36(5): 897-908