非脊椎动物Kv2基因A至I RNA编辑位点的鉴定和形成机制的初步研究
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摘要
RNA编辑的分子机制和生理功能的研究已经非常广泛,但其在生物进化过程中的意义仍然是一个谜团。本研究从约占动物界总种数85%的节肢动物门出发,通过基因组序列与RT-PCR序列的比对,鉴定Kv2基因第二外显子A至I RNA编辑靶位点。结果发现,尽管各物种所在纲和目不尽相同,进化年代上也相差甚远,但是却有三个编辑位点Y/C,S/G,I/V是非常保守的,表明这种修饰对蛋白质的功能是有重要作用的。不仅如此,同一个目或同一个纲亲缘关系相近的物种,往往会共享一些编辑位点,同时保留一些自己特有的编辑位点。
A至I RNA编辑酶ADAR作用的底物一般认为是由外显子与其下游内含子的一小段序列回折形成的双链RNA,而根据本研究对节肢动物门中模式生物家蚕(Bombyx mori)、果蝇(Drosophila melanogaster)、意大利蜜蜂(Apis mellifera)二级结构的预测,发现这些物种Kv2基因的exon2这段序列自身就可以形成完美的RNA编辑结构。由此运用细胞转染的方法建立了一套体内编辑系统来验证这一预测。结果显示果蝇的Kv2基因片段发生编辑,但家蚕和意大利蜜蜂的不能被编辑。继而我们推断果蝇的Kv2基因编辑是由exon2自身形成二级结构介导的,而家蚕和蜜蜂的Kv2基因编辑需要除exon2以外的片段辅助形成二级结构介导。同时,利用生物信息学手段重新预测出了家蚕和蜜蜂ECS序列(编辑互补序列)。
The molecular mechanism and physiological function of recoding by A-to-I RNA editing is well known, but its evolutionary significance remains a mystery. The RNA editing of the Kv2 K~+channel from different classes of Arthropoda are extensively analyzed. By comparing the RNA RT-PCR sequence with the genomic sequence, we verify several editing sites that change codon usage within exon 2. It is notable that three of these editing sites were conserved in most species, suggesting that a crucial role of function during hundreds of millions of years' evolution. We also found that in the same classis, different species share identical editing sites, meantime own their specific editing sites. We all know that the occurrence of RNA editing is catalyzed by the ADAR enzyme family. Their editing substrates are RNA stem-loop structures that are occasionally formed by an exon sequence and an inverted repeat located in the downstream intron. Surprisingly, bioinformatical analysis of B.mori, D. melanogaster and A.mellifera kv2 RNA secondary structures show that the entire exon 2 sequence form a perfect double-stranded RNA structure that could be the editing substrate. Although it has been found in mammals Kv1.1 and Gabra-3 before, it is still rare that the editing sites and the editing complementary sequences are located in the same exon. Based on these results, experiments are undertaken to demonstrate our predictions. However, the results show that only D. melanogaster can be edited.
A至I RNA编辑酶ADAR作用的底物一般认为是由外显子与其下游内含子的一小段序列回折形成的双链RNA,而根据本研究对节肢动物门中模式生物家蚕(Bombyx mori)、果蝇(Drosophila melanogaster)、意大利蜜蜂(Apis mellifera)二级结构的预测,发现这些物种Kv2基因的exon2这段序列自身就可以形成完美的RNA编辑结构。由此运用细胞转染的方法建立了一套体内编辑系统来验证这一预测。结果显示果蝇的Kv2基因片段发生编辑,但家蚕和意大利蜜蜂的不能被编辑。继而我们推断果蝇的Kv2基因编辑是由exon2自身形成二级结构介导的,而家蚕和蜜蜂的Kv2基因编辑需要除exon2以外的片段辅助形成二级结构介导。同时,利用生物信息学手段重新预测出了家蚕和蜜蜂ECS序列(编辑互补序列)。
The molecular mechanism and physiological function of recoding by A-to-I RNA editing is well known, but its evolutionary significance remains a mystery. The RNA editing of the Kv2 K~+channel from different classes of Arthropoda are extensively analyzed. By comparing the RNA RT-PCR sequence with the genomic sequence, we verify several editing sites that change codon usage within exon 2. It is notable that three of these editing sites were conserved in most species, suggesting that a crucial role of function during hundreds of millions of years' evolution. We also found that in the same classis, different species share identical editing sites, meantime own their specific editing sites. We all know that the occurrence of RNA editing is catalyzed by the ADAR enzyme family. Their editing substrates are RNA stem-loop structures that are occasionally formed by an exon sequence and an inverted repeat located in the downstream intron. Surprisingly, bioinformatical analysis of B.mori, D. melanogaster and A.mellifera kv2 RNA secondary structures show that the entire exon 2 sequence form a perfect double-stranded RNA structure that could be the editing substrate. Although it has been found in mammals Kv1.1 and Gabra-3 before, it is still rare that the editing sites and the editing complementary sequences are located in the same exon. Based on these results, experiments are undertaken to demonstrate our predictions. However, the results show that only D. melanogaster can be edited.
引文
1.王海芳,罗小星.A至IRNA编辑:遗传信息修饰的新机制.科学通报,2003,48(12):1251-1252
2.尚观胜,李晓辉.RNA编辑:受体表达的信息调节新机制.国外医学·生理、病理科学与临床分册,2000,20(6):445-446
3.孙芳,江红,郑晓飞.RNA编辑研究进展.国际遗传学杂志,2006,29(1):27-28
4.Benne R,Van den Burg J,Brakenhoff JP,et al.Major transcript of the frameshifted coxⅡ gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA.Cell.1986,46(6):819-826
5.Bass BL,Weintraub H.An unwinding activity that covalently modifies its double-stranded RNA substrate.Cell.1988,55:1089-1098
6.Bass BL.Double-stranded RNA as a template for gene silencing.Cell.2000,101(3):235-238
7.Bass BL.RNA editing by adenosine deaminases that act on RNA.Annu Rev Biochem.2002,71:817-846
8.Bhalla T,Rosenthal JJ,Holmgren M,Reenan R.Control of human potassium channel inactivation by editing of a small mRNA hairpin.Nat Struet Mol Biol.2004,11(10):950-956
9.Cattaneo R.Different types of messager RNA editing.Annu Rev Genet.1991,25:71-88
10.Chan,L.Apolipopretein B,the major protein component of triglyceride-rich and low density lipoproteins.J.Biol.Chem.1992,267:25621-25624
11.Chen CX,Cho DS,Wang Q,et al.A third member of the RNA-specific adenosine deaminase gene family.ADAR3 contains both single- and double-stranded RNA domains.RNA.2000,6(5):755-67
12.Gott JM,Emeson RB.Functions and mechanisms of RNA editing.Annu Rev Genet.2000,34:499-531
13. Gutman GA, Chandy KG, Grissmer S et al. International Union of Pharmacology. LIII. Nomenclature and Molecular Relationships of Voltage-Gated Potassium Channels. Pharmacol Rev. 2005 ,57(4):473-508
14. Hwang PM, Glatt CE, Bredt DS, et al. A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization. Neuron. 1992 ,8(3):473-481
15. Hiesel R, Comgettes B, Brennicke A. Evidence for RNA editing in mitochondria of all major groups of land plants except the bryothyta. Proc Natl Acad Sci USA. 1994, 91:629-633
16. Hoopengardner, B. O'Connell, M.A. Reenan R, et al. Adenosine to inosine RNA editing in animal cells. In: Topics in Current Genetics. 2005, 12: 341-364
17. Johnson DF, Poksay KS, Innerarity TL.The mechanism for apo-B mRNA editing is deamination. Biochem.Biophy.Res.Commun. 1993,195: 1204-1210
18. Keegan LP, Leroy A, Sproul D, O'Connell MA. Adenosine deaminases acting on RNA (ADARs): RNA-editing enzymes. Genome Biol. 2004,5(2):209
19. Lai F, Chen C X, Carter K C, et al. Edting of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2 double-stranded RNA adenosine deaminases. Mol Cell Biol. 1997,17: 2413-2424
20. Lim ST, Antonucci DE, Scannevin RH,et al. A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons. Neuron. 2000 ,25(2):385-397
21. Long X,Hansen EH, Miro M. Determination of trace metal ions via on-line separation and preconcentration by means of chelating Sepharose beads in a sequential injection lab-on-valve (SI-LOV) system coupled to electrothermal atomic absorption spectrometric detection. Talanta. 2005 ,66(5): 1326-1332
22. Miyoko Higuchi, Stefan Maas, Frank N. Single,et al. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature. 2000,406:78-81
23. Misonou H, Mohapatra DP, Trimmer JS. Kv2.1: a voltage-gated k+ channel critical to dynamic control of neuronal excitability. Neurotoxicology. 2005 ,26(5):743-752.
24. Malysz J, Farrugia G, Ou Y, et al. The Kv2.2 alpha subunit contributes to delayed rectifier K(+) currents in myocytes from rabbit corpus cavernosum. J Androl. 2002 Nov-Dec;23(6):899-910.
25. Maas S, Kawahara Y, Tamburro KM, Nishikura K. A-to-I RNA editing and human disease. RNA Biol. 2006,3(1): 1-9.
26. Misonou H, Thompson SM, Cai X. Dynamic regulation of the Kv2.1 voltage-gated potassium channel during brain ischemia through neuroglial interaction. J Neurosci. 2008 ,28(34):8529-8538.
27. Nishikura K. Editor meets silencer: crosstalk between RNA editing and RNA interference. Nat Rev Mol Cell Biol, 2006 ,7(12):919-931.
28. O'Connell M A, Krause S, Highuchi M, et al. Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase. Mol Cell Biol. 1995,15: 1389-1397.
29. Ohlson J, Pedersen JS, Haussler D,et al. Editing modifies the GABA(A) receptor subunit alpha3. RNA. 2007 ,13(5):698-703.
30. Powell L M, Wallis S C, Pease R J, et al. Anovel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine. Cell. 1987 ,50(6):831-40.
31. Patton DE, Silva T, Bezanilla F.RNA Editing Generates a Diverse Array of Transcripts Encoding Squid Kv2 K+ Channels with Altered Functional Properties. Neuron. 1997,19(3):711-722.
32. Paul M S, Bass B L. Inosine exists in mRNA at tissue-specific levels and is most abundant in brain mRNA.EMBOJ. 1998,17: 1120-1127.
33. Pongs O. Voltage-gated potassium channels: from hyperexcitability to excitement. FEBS Lett. 1999,452(1-2):31-35.
34. Papazian DM.Potassium Channels: Some Assembly Required. Neuron. 1999,23(1):7-10.
35. Palladino M J, Keegan L P, O'Connell M A, et al. A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity. Cell. 2000,102:437-449.
36. Park KS, Mohapatra DP, Misonou H, et al. Graded regulation of the Kv2.1 potassium channel by variable phosphorylation. Science. 2006, 313(5789):976-979.
37. Rudy B. Diversity and ubiquity of K channels. Neuroscience. 1988 ,25(3):729-749.
38. Schuster W, Hiesel R, Brennicke A. RNA editing in plant mitochondria. Semin Cell Biol. 1993,4:279-284.
39. Rosenthal JJ, Bezanilla F. Extensive Editing of mRNAs for the Squid Delayed Rectifier K~+ Channel Regulates Subunit Tetramerization. Neuron. 2002, 34(5):743-757.
40. Sommer B,Kohler M, Sprengen R, et al. RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell. 1991, 69:11-19.
41. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 2002, 84: 791-803.
42. Tempel BL, Papazian DM, Schwarz TL,et al. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. Science. 1987, 237(4816):770-775.
43. Vega-Saenz de Miera EC. Modification of Kv2.1 K+ currents by the silent Kv10 subunits. Brain Res Mol Brain Res. 2004, 123(1-2):91-103.
44. Yang Y, Lv J, Gui B, et al. A-to-I RNA editing alters less-conserved residues of highly conserved coding regions: Implications for its dual functions in evolution. RNA. 2008,14(8):1516-1525.
2.尚观胜,李晓辉.RNA编辑:受体表达的信息调节新机制.国外医学·生理、病理科学与临床分册,2000,20(6):445-446
3.孙芳,江红,郑晓飞.RNA编辑研究进展.国际遗传学杂志,2006,29(1):27-28
4.Benne R,Van den Burg J,Brakenhoff JP,et al.Major transcript of the frameshifted coxⅡ gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA.Cell.1986,46(6):819-826
5.Bass BL,Weintraub H.An unwinding activity that covalently modifies its double-stranded RNA substrate.Cell.1988,55:1089-1098
6.Bass BL.Double-stranded RNA as a template for gene silencing.Cell.2000,101(3):235-238
7.Bass BL.RNA editing by adenosine deaminases that act on RNA.Annu Rev Biochem.2002,71:817-846
8.Bhalla T,Rosenthal JJ,Holmgren M,Reenan R.Control of human potassium channel inactivation by editing of a small mRNA hairpin.Nat Struet Mol Biol.2004,11(10):950-956
9.Cattaneo R.Different types of messager RNA editing.Annu Rev Genet.1991,25:71-88
10.Chan,L.Apolipopretein B,the major protein component of triglyceride-rich and low density lipoproteins.J.Biol.Chem.1992,267:25621-25624
11.Chen CX,Cho DS,Wang Q,et al.A third member of the RNA-specific adenosine deaminase gene family.ADAR3 contains both single- and double-stranded RNA domains.RNA.2000,6(5):755-67
12.Gott JM,Emeson RB.Functions and mechanisms of RNA editing.Annu Rev Genet.2000,34:499-531
13. Gutman GA, Chandy KG, Grissmer S et al. International Union of Pharmacology. LIII. Nomenclature and Molecular Relationships of Voltage-Gated Potassium Channels. Pharmacol Rev. 2005 ,57(4):473-508
14. Hwang PM, Glatt CE, Bredt DS, et al. A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization. Neuron. 1992 ,8(3):473-481
15. Hiesel R, Comgettes B, Brennicke A. Evidence for RNA editing in mitochondria of all major groups of land plants except the bryothyta. Proc Natl Acad Sci USA. 1994, 91:629-633
16. Hoopengardner, B. O'Connell, M.A. Reenan R, et al. Adenosine to inosine RNA editing in animal cells. In: Topics in Current Genetics. 2005, 12: 341-364
17. Johnson DF, Poksay KS, Innerarity TL.The mechanism for apo-B mRNA editing is deamination. Biochem.Biophy.Res.Commun. 1993,195: 1204-1210
18. Keegan LP, Leroy A, Sproul D, O'Connell MA. Adenosine deaminases acting on RNA (ADARs): RNA-editing enzymes. Genome Biol. 2004,5(2):209
19. Lai F, Chen C X, Carter K C, et al. Edting of glutamate receptor B subunit ion channel RNAs by four alternatively spliced DRADA2 double-stranded RNA adenosine deaminases. Mol Cell Biol. 1997,17: 2413-2424
20. Lim ST, Antonucci DE, Scannevin RH,et al. A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons. Neuron. 2000 ,25(2):385-397
21. Long X,Hansen EH, Miro M. Determination of trace metal ions via on-line separation and preconcentration by means of chelating Sepharose beads in a sequential injection lab-on-valve (SI-LOV) system coupled to electrothermal atomic absorption spectrometric detection. Talanta. 2005 ,66(5): 1326-1332
22. Miyoko Higuchi, Stefan Maas, Frank N. Single,et al. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature. 2000,406:78-81
23. Misonou H, Mohapatra DP, Trimmer JS. Kv2.1: a voltage-gated k+ channel critical to dynamic control of neuronal excitability. Neurotoxicology. 2005 ,26(5):743-752.
24. Malysz J, Farrugia G, Ou Y, et al. The Kv2.2 alpha subunit contributes to delayed rectifier K(+) currents in myocytes from rabbit corpus cavernosum. J Androl. 2002 Nov-Dec;23(6):899-910.
25. Maas S, Kawahara Y, Tamburro KM, Nishikura K. A-to-I RNA editing and human disease. RNA Biol. 2006,3(1): 1-9.
26. Misonou H, Thompson SM, Cai X. Dynamic regulation of the Kv2.1 voltage-gated potassium channel during brain ischemia through neuroglial interaction. J Neurosci. 2008 ,28(34):8529-8538.
27. Nishikura K. Editor meets silencer: crosstalk between RNA editing and RNA interference. Nat Rev Mol Cell Biol, 2006 ,7(12):919-931.
28. O'Connell M A, Krause S, Highuchi M, et al. Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase. Mol Cell Biol. 1995,15: 1389-1397.
29. Ohlson J, Pedersen JS, Haussler D,et al. Editing modifies the GABA(A) receptor subunit alpha3. RNA. 2007 ,13(5):698-703.
30. Powell L M, Wallis S C, Pease R J, et al. Anovel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine. Cell. 1987 ,50(6):831-40.
31. Patton DE, Silva T, Bezanilla F.RNA Editing Generates a Diverse Array of Transcripts Encoding Squid Kv2 K+ Channels with Altered Functional Properties. Neuron. 1997,19(3):711-722.
32. Paul M S, Bass B L. Inosine exists in mRNA at tissue-specific levels and is most abundant in brain mRNA.EMBOJ. 1998,17: 1120-1127.
33. Pongs O. Voltage-gated potassium channels: from hyperexcitability to excitement. FEBS Lett. 1999,452(1-2):31-35.
34. Papazian DM.Potassium Channels: Some Assembly Required. Neuron. 1999,23(1):7-10.
35. Palladino M J, Keegan L P, O'Connell M A, et al. A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity. Cell. 2000,102:437-449.
36. Park KS, Mohapatra DP, Misonou H, et al. Graded regulation of the Kv2.1 potassium channel by variable phosphorylation. Science. 2006, 313(5789):976-979.
37. Rudy B. Diversity and ubiquity of K channels. Neuroscience. 1988 ,25(3):729-749.
38. Schuster W, Hiesel R, Brennicke A. RNA editing in plant mitochondria. Semin Cell Biol. 1993,4:279-284.
39. Rosenthal JJ, Bezanilla F. Extensive Editing of mRNAs for the Squid Delayed Rectifier K~+ Channel Regulates Subunit Tetramerization. Neuron. 2002, 34(5):743-757.
40. Sommer B,Kohler M, Sprengen R, et al. RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell. 1991, 69:11-19.
41. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 2002, 84: 791-803.
42. Tempel BL, Papazian DM, Schwarz TL,et al. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. Science. 1987, 237(4816):770-775.
43. Vega-Saenz de Miera EC. Modification of Kv2.1 K+ currents by the silent Kv10 subunits. Brain Res Mol Brain Res. 2004, 123(1-2):91-103.
44. Yang Y, Lv J, Gui B, et al. A-to-I RNA editing alters less-conserved residues of highly conserved coding regions: Implications for its dual functions in evolution. RNA. 2008,14(8):1516-1525.