Requirement of Dimerization for RNA Editing Activity of Adenosine Deaminases Acting on RNA
2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês
10.1074/jbc.m213127200
ISSN1083-351X
AutoresDan-Sung C. Cho, Weidong Yang, Joshua T. Lee, Ramin Shiekhattar, John M. Murray, Kazuko Nishikura,
Tópico(s)Viral Infections and Immunology Research
ResumoAdenosine deaminases acting on RNA (ADAR) convert adenosine residues into inosines in double-stranded RNA. Three vertebrate ADAR gene family members, ADAR1,ADAR2, and ADAR3, have been identified. The catalytic domain of all three ADAR gene family members is very similar to that of Escherichia coli cytidine deaminase and APOBEC-1. Homodimerization is essential for the enzyme activity of those cytidine deaminases. In this study, we investigated the formation of complexes between differentially epitope-tagged ADAR monomers by sequential affinity chromatography and size exclusion column chromatography. Both ADAR1 and ADAR2 form a stable enzymatically active homodimer complex, whereas ADAR3 remains as a monomeric, enzymatically inactive form. No heterodimer complex formation among different ADAR gene family members was detected. Analysis of HeLa and mouse brain nuclear extracts suggested that endogenous ADAR1 and ADAR2 both form a homodimer complex. Interestingly, endogenous ADAR3 also appears to form a homodimer complex, indicating the presence of a brain-specific mechanism for ADAR3 dimerization. Homodimer formation may be necessary for ADAR to act as active deaminases. Analysis of dimer complexes consisting of one wild-type and one mutant monomer suggests functional interactions between the two subunits during site-selective RNA editing. Adenosine deaminases acting on RNA (ADAR) convert adenosine residues into inosines in double-stranded RNA. Three vertebrate ADAR gene family members, ADAR1,ADAR2, and ADAR3, have been identified. The catalytic domain of all three ADAR gene family members is very similar to that of Escherichia coli cytidine deaminase and APOBEC-1. Homodimerization is essential for the enzyme activity of those cytidine deaminases. In this study, we investigated the formation of complexes between differentially epitope-tagged ADAR monomers by sequential affinity chromatography and size exclusion column chromatography. Both ADAR1 and ADAR2 form a stable enzymatically active homodimer complex, whereas ADAR3 remains as a monomeric, enzymatically inactive form. No heterodimer complex formation among different ADAR gene family members was detected. Analysis of HeLa and mouse brain nuclear extracts suggested that endogenous ADAR1 and ADAR2 both form a homodimer complex. Interestingly, endogenous ADAR3 also appears to form a homodimer complex, indicating the presence of a brain-specific mechanism for ADAR3 dimerization. Homodimer formation may be necessary for ADAR to act as active deaminases. Analysis of dimer complexes consisting of one wild-type and one mutant monomer suggests functional interactions between the two subunits during site-selective RNA editing. double-stranded RNA adenosine deaminase acting on RNA glutamate receptor 5-hydroxytryptamine or serotonin serotonin receptor subtype 2C single-stranded RNA monoclonal antibody dithiothreitol Caenorhabditis elegans adenosine deaminase acting on RNA One type of RNA editing converts adenosine residues into inosine within the double-stranded RNA (dsRNA)1 region of substrate RNAs (1Gerber A.P. Keller W. Trends Biochem. Sci. 2001; 26: 376-384Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 2Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (977) Google Scholar, 3Maas S. Rich A. Nishikura K. J. Biol. Chem. 2003; 278: 1391-1394Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Because inosine is treated as guanosine by the translational machinery, this A-to-I editing could lead to functional alterations of the affected genes. For instance, A-to-I RNA editing results in the expression of editing isoforms of glutamate receptor (GluR) ion channel subunits (4Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar, 5Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J.R.P. Kuner T. Monyer H. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 226: 1709-1713Crossref Scopus (642) Google Scholar) and serotonin 2C subtype receptors (5-HT2CR) (6Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar). Editing of the so-called "Q/R" site of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid GluR-B subunit dramatically decreases the Ca2+ permeability of the channel (7Köhler M. Burnashev N. Sakmann B. Seeburg P.H. Neuron. 1993; 10: 491-500Abstract Full Text PDF PubMed Scopus (367) Google Scholar). Substantial reduction in G-protein coupling efficiency is noted with A-to-I editing of 5-HT2CR RNA at five positions (A to E sites) located in the intracellular II loop region (6Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar, 8Niswender C.M. Copeland S.C. Herrick-Davis K. Emeson R.B. Sanders-Bush E. J. Biol. Chem. 1999; 274: 9472-9478Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 9Fitzgerald L.W. Iyer G. Conklin D.S. Krause C.M. Marshall A. Patterson J.P. Tran D.P. Jonak G.J. Hartig P.R. Neuropsychopharmacology. 1999; 21: 82S-90SCrossref PubMed Scopus (136) Google Scholar, 10Wang Q. Chen C-X. Cho D-S.C. O'Brien P. Murray J.M. Nishikura K. J. Neurochem. 2000; 74: 1290-1300Crossref PubMed Scopus (175) Google Scholar). A-to-I RNA editing also occurs in non-coding sequences. Editing of its own intron sequence by adenosine deaminase acting on RNA (ADAR) 2 creates an alternative splice acceptor site leading to synthesis of a truncated translation product, which may be a negative feedback mechanism to regulate the activity of ADAR2 (11Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 339: 75-80Crossref Scopus (508) Google Scholar). In all these examples, a dsRNA structure formed between the exonic sequences containing an editing site(s) and downstream or upstream intronic sequences has been proven to be essential for editing (4Higuchi M. Single F.N. Köhler M. Sommer B. Sprengel R. Seeburg P.H. Cell. 1993; 75: 1361-1370Abstract Full Text PDF PubMed Scopus (523) Google Scholar, 5Lomeli H. Mosbacher J. Melcher T. Höger T. Geiger J.R.P. Kuner T. Monyer H. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 226: 1709-1713Crossref Scopus (642) Google Scholar, 6Burns C.M. Chu H. Rueter S.M. Hutchinson L.K. Canton H. Sanders-Bush E. Emeson R.B. Nature. 1997; 387: 303-308Crossref PubMed Scopus (860) Google Scholar,12Herb A. Higuchi M. Sprengel R. Seeburg P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1875-1880Crossref PubMed Scopus (137) Google Scholar). Systematic search with a recently devised method for cloning of inosine-containing RNAs has led to identification of more than two dozen editing sites occurring in the intron and 3′-untranslated regions of new target genes. A-to-I RNA editing of these non-coding regions may affect the splicing rate, the translation efficacy, or stability of the edited mRNAs (13Morse D.P. Aruscavage P.J. Bass B.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7906-7911Crossref PubMed Scopus (182) Google Scholar). Furthermore, the intronic and untranslated region sequences subjected to A-to-I RNA editing often contain common repetitive elements such as Alu and LINE1 repeats forming a long dsRNA structure, raising the possibility that A-to-I RNA editing may be involved in a mechanism regulating the very abundant repetitive sequences in mammalian genomes (2Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (977) Google Scholar, 3Maas S. Rich A. Nishikura K. J. Biol. Chem. 2003; 278: 1391-1394Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 13Morse D.P. Aruscavage P.J. Bass B.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7906-7911Crossref PubMed Scopus (182) Google Scholar). Finally, A-to-I RNA editing of dsRNAs derived from transgenes appears to prevent silencing of the transgenes regulated by RNA interference, revealing the potential intersection of the two mechanisms, RNA editing and RNA interference both evolved to deal with dsRNA (14Knight S.W. Bass B.L. Mol. Cell. 2002; 10: 809-817Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Members of the ADAR gene family have been implicated as the enzymes responsible for A-to-I RNA editing. Three separate mammalian gene family members (ADAR1 to ADAR3) have been identified (15Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (372) Google Scholar, 16O'Connell M.A. Krause S. Higuchi M. Hsuan J.J. Totty N. Jenny A. Keller W. Mol. Cell. Biol. 1995; 15: 1389-1397Crossref PubMed Google Scholar, 17Patterson J.B. Samuel C.E. Mol. Cell. Biol. 1995; 15: 5376-5388Crossref PubMed Scopus (437) Google Scholar, 18Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (433) Google Scholar, 19Gerber A.P. O'Connell M.A. Keller W. RNA. 1997; 3: 453-463PubMed Google Scholar, 20Lai F. Chen C.-X. Carter K.C. Nishikura K. Mol. Cell. Biol. 1997; 17: 2413-2424Crossref PubMed Scopus (159) Google Scholar, 21Melcher T. Maas S. Herb A. Sprengel R. Higuchi M. Seeburg P.H. J. Biol. Chem. 1996; 271: 31795-31798Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 22Chen C-X. Cho D-S.C. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar). Data base search has identified corresponding fish ADARsrevealing the conservation of the complete set of ADAR gene family members in vertebrates through evolution (23Slavov D. Clark M. Gardiner K. Gene (Amst.). 2000; 250: 45-51Google Scholar, 24Slavov D. Crnogorac-Jurcevic T. Clark M. Gardiner K. Gene (Amst.). 2000; 250: 53-60Crossref PubMed Scopus (34) Google Scholar). In invertebrates, a single Drosophila dADAR, very similar to mammalian ADAR2 (25Palladino M.J. Keegan L.P. O'Connell M.A. Reenan R.A. Cell. 2000; 102: 437-449Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar), and two less conserved Caenorhabditis elegans c.e.ADAR1 and c.e.ADAR2 have been identified (15Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (372) Google Scholar, 26Hough R.F. Lingam A.T. Bass B.L. Nucleic Acids Res. 1999; 27: 3424-3432Crossref PubMed Scopus (28) Google Scholar). Mammalian ADAR1 and ADAR2 are detected ubiquitously (15Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (372) Google Scholar, 16O'Connell M.A. Krause S. Higuchi M. Hsuan J.J. Totty N. Jenny A. Keller W. Mol. Cell. Biol. 1995; 15: 1389-1397Crossref PubMed Google Scholar, 18Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (433) Google Scholar, 19Gerber A.P. O'Connell M.A. Keller W. RNA. 1997; 3: 453-463PubMed Google Scholar, 20Lai F. Chen C.-X. Carter K.C. Nishikura K. Mol. Cell. Biol. 1997; 17: 2413-2424Crossref PubMed Scopus (159) Google Scholar), whereas the expression of mammalian ADAR3, Drosophila dADAR, and C. elegans c.e.ADAR1 is restricted mainly to nervous systems (21Melcher T. Maas S. Herb A. Sprengel R. Higuchi M. Seeburg P.H. J. Biol. Chem. 1996; 271: 31795-31798Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 22Chen C-X. Cho D-S.C. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar, 25Palladino M.J. Keegan L.P. O'Connell M.A. Reenan R.A. Cell. 2000; 102: 437-449Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 27Tonkin L.A. Saccomanno L. Morse D.P. Brodigan T. Krause M. Bass B.L. EMBO J. 2002; 21: 6025-6035Crossref PubMed Scopus (171) Google Scholar). Analysis of ADAR null mutation phenotypes has revealed the importance of A-to-I RNA editing. Flies with a null mutation of dADAR, although viable, display defective locomotion and behavior accompanied by various neurological and anatomical changes in the brain. This phenotype is most likely because of the lack of editing in the transcripts of several known target genes such as cacCa2+ channel and para Na+ channel (25Palladino M.J. Keegan L.P. O'Connell M.A. Reenan R.A. Cell. 2000; 102: 437-449Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). C. elegans strains containing homozygous deletions of both c.e.ADAR1 and c.e.ADAR2 genes show defects in chemotaxis, whereas aberrant development of the vulva is occasionally detected with worms lacking c.e.ADAR1 (27Tonkin L.A. Saccomanno L. Morse D.P. Brodigan T. Krause M. Bass B.L. EMBO J. 2002; 21: 6025-6035Crossref PubMed Scopus (171) Google Scholar). Mice with a homozygous ADAR2 null mutation die several weeks after birth with repeated episodes of epileptic seizures because of underediting of GluR-B RNA at the Q/R site, a major target of ADAR2 (28Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar). Chimeric mouse embryos derived fromADAR1+/− ES cells die at the midgestation stage with a phenotype indicative of dyserythropoietic defects (29Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar). It has not yet been ruled out that antisense effects generated by transcripts derived from the ADAR1-targeted allele may contribute to the observed embryonic lethal phenotype (29Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar). Purified recombinant ADAR1 and ADAR2 proteins displayed in vitro their distinctive editing site selectivity with known RNA substrates (18Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (433) Google Scholar, 19Gerber A.P. O'Connell M.A. Keller W. RNA. 1997; 3: 453-463PubMed Google Scholar, 20Lai F. Chen C.-X. Carter K.C. Nishikura K. Mol. Cell. Biol. 1997; 17: 2413-2424Crossref PubMed Scopus (159) Google Scholar, 30Dabiri G.A. Lai F. Drakas R.A. Nishikura K. EMBO J. 1996; 15: 34-45Crossref PubMed Scopus (73) Google Scholar, 31Maas S. Melcher T. Herb A. Seeburg P.H. Keller W. Krause S. Higuchi M. O'Connell M.A. J. Biol. Chem. 1996; 271: 12221-12226Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). For instance, ADAR2 edits almost exclusively the D site of 5-HT2CR and the Q/R site of GluR-B RNA, whereas ADAR1 barely edits these sites. However, ADAR1 selectively edits the A and B sites of 5-HT2CR RNA and the intronic hot spot (+60 site) of GluR-B RNA. The result of in vitro editing studies indicate a significant difference among ADAR gene family members in their interaction with substrate RNA. Specific structural features of the dsRNA binding domains and their N-terminal regions may form the molecular basis of this editing site selectivity. There are only two dsRNA binding motif repeats in the RNA binding domain of ADAR2 and ADAR3, in contrast to three dsRNA binding motifs in ADAR1. ADAR2 lacks a part of the N terminus region of ADAR1, just upstream of its RNA binding domain, where ADAR1 contains two repeats of a Z-DNA binding motif (32Herbert A. Alfken J. Kim Y.-G. Mian I.S. Nishikura K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8421-8426Crossref PubMed Scopus (264) Google Scholar). ADAR3 has a unique N-terminal region containing the arginine-rich R domain (21Melcher T. Maas S. Herb A. Sprengel R. Higuchi M. Seeburg P.H. J. Biol. Chem. 1996; 271: 31795-31798Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 22Chen C-X. Cho D-S.C. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar). Alternatively, the deaminase domains and relatively divergent C-terminal regions of ADAR gene family members may also contribute to the difference observed in their RNA editing site selectivity as indicated by the studies of domain exchange between ADAR1 and ADAR2 (21Melcher T. Maas S. Herb A. Sprengel R. Higuchi M. Seeburg P.H. J. Biol. Chem. 1996; 271: 31795-31798Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 33Wong S.K. Sato S. Lazinski D.W. RNA. 2001; 7: 846-858Crossref PubMed Scopus (170) Google Scholar). A longstanding question with regard to the enzymatic activities of ADARs is whether they act as monomeric or oligomeric forms and whether oligomerization plays a role in the site-selective editing mechanism. The catalytic domain of ADAR is very similar to that of the cytidine deaminase gene family (1Gerber A.P. Keller W. Trends Biochem. Sci. 2001; 26: 376-384Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 2Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (977) Google Scholar, 15Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (372) Google Scholar). E. coli cytidine deaminase forms a homodimer (34Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar), as does APOBEC-1, another cytidine deaminase involved in C-to-U RNA editing of apolipoprotein B mRNAs (35Lau P.P. Zhu H-J. Baldini A. Charnsanavej C. Chan L. Proc. Natl. Acad. Sci U. S. A. 1994; 91: 8522-8526Crossref PubMed Scopus (136) Google Scholar, 36MacGinnitie A.J. Anant S. Davidson N.O. J. Biol. Chem. 1995; 270: 14768-14775Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 37Navaratnam N. Fujino T. Bayliss J. Jarmuz A. How A. Richardson N. Somasekaram A. Bhattacharya S. Carter C. Scott J. J. Mol. Biol. 1998; 275: 695-714Crossref PubMed Scopus (126) Google Scholar). In both cases homodimerization is required for enzymatic activity (34Betts L. Xiang S. Short S.A. Wolfenden R. Carter Jr., C.W. J. Mol. Biol. 1994; 235: 635-656Crossref PubMed Scopus (342) Google Scholar, 35Lau P.P. Zhu H-J. Baldini A. Charnsanavej C. Chan L. Proc. Natl. Acad. Sci U. S. A. 1994; 91: 8522-8526Crossref PubMed Scopus (136) Google Scholar, 36MacGinnitie A.J. Anant S. Davidson N.O. J. Biol. Chem. 1995; 270: 14768-14775Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 37Navaratnam N. Fujino T. Bayliss J. Jarmuz A. How A. Richardson N. Somasekaram A. Bhattacharya S. Carter C. Scott J. J. Mol. Biol. 1998; 275: 695-714Crossref PubMed Scopus (126) Google Scholar). It is possible that the RNA editing site selectivity observed with ADAR1 and ADAR2 is dependent on their state of oligomerization. Curiously, the third member of the ADAR gene family, ADAR3, is incapable of editing all known sites of GluR-B and 5-HT2CR RNAs. The lack of enzymatic activity may be related to its oligomerization state. In the present studies, we have investigated whether ADAR gene family members undergo oligomerization. In addition, we have examined the possible formation of heteromeric oligomers among different ADAR gene family members. The following oligonucleotides used for construction of 6His-tagged ADAR baculovirus constructs were synthesized at the University of Pennsylvania, Cancer Center Nucleic Acid Facility. All ADAR oligonucleotides correspond to the human sequence. The nucleotide positions indicated in parentheses are relative to the initiation codon ATG of ADAR1, ADAR2, and ADAR3 (GenBankTM accession numbers U10439, U76420, and AF034837, respectively), in which A was assigned as position +1. The 6His epitope tag sequence is underlined, and all restriction sites within the oligonucleotides are shown in bold. Not-H-ADAR1UP, 5′-AAGGAAAAAAGCGGCCGCAGAATAAAAATGAATCATCACCATCACCATCACAATCCGCGGCAGGGGTATTCCCTC-3′ (+4 to +27); H-ADAR1DW, 5′-GTGGCAGTGACGGTGTCTAG-3 (+196 to +177); Not-H-ADAR2UP, 5′-AAGGAAAAAAGCGGCCGCAGAATAAAAATGAATCATCACCATCACCATCACGATATAGAAGATGAAGAAACATG-3′ (+4 to +27); H-ADAR2DW, 5′-GTTGACAGACAGGGTCCTC-3′ (+486 to +468); Bam-H-ADAR3UP, 5′-CGCGGATCCAGAATAAAAATGAATCATCACCATCACCATCACGCCTCGGTCCTGGGGAGCGGC-3′ (+4 to +24); H-ADAR3DW, 5′-AGACCAGCTGCAGTTTGCACA-3′ (+349 to +329). A 6His epitope tag sequence was introduced into the N termini of the existing FLAG epitope-tagged expression constructs, pBac-F-ADAR1, pBac-F-ADAR2a, and pBac-F-ADAR3 (20Lai F. Chen C.-X. Carter K.C. Nishikura K. Mol. Cell. Biol. 1997; 17: 2413-2424Crossref PubMed Scopus (159) Google Scholar, 22Chen C-X. Cho D-S.C. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar, 38Lai F. Drakas R. Nishikura K. J. Biol. Chem. 1995; 270: 17098-17105Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Different regions of human ADAR1 (amino acids 2–72), ADAR2a (amino acids 2–35), or ADAR3 (amino acids 2–100) were prepared by PCR amplification of human ADAR1 (15Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (372) Google Scholar), ADAR2a (20Lai F. Chen C.-X. Carter K.C. Nishikura K. Mol. Cell. Biol. 1997; 17: 2413-2424Crossref PubMed Scopus (159) Google Scholar), and ADAR3 cDNA plasmids (22Chen C-X. Cho D-S.C. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar) using a set of oligonucleotide primers designed to create NotI and BamHI restriction sites. Not-H-ADAR1UP and H-ADAR1DW primers were used for PCR amplification of the ADAR1 sequence, Not-H-ADAR2UP and H-ADAR2DW primers for ADAR2a, and Bam-H-ADAR3UP and H-ADAR3DW primers for ADAR3. Restriction sitesAflII, StuI, and NotI were utilized for ligation of the PCR products at their 3′ ends into pBac-F-ADAR1, pBac-F-ADAR2a, and pBac-F-ADAR3, respectively. The resultant constructs, termed pBac-H-ADAR1, pBac-H-ADAR2a, and pBac-H-ADAR3, contain a Kozak sequence that is strongly preferred by baculovirus for protein translation initiation at the N terminus region (39Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2810) Google Scholar). The region amplified by PCR was confirmed by sequencing. Baculovirus expression constructs were then transformed in DH10Bac for transposition into the bacmid and subjected to blue/white screening for identification of recombinant baculoviruses. Sf9 cells were grown to a density of 2 × 106 cells/ml and infected with either a single or a combination of two ADAR recombinant viruses (1:1 ratio) at a multiplicity of infection of 10–20. At 48 h post-infection, ∼1 × 109 cells were collected. All procedures were carried out at 4 °C. HeLa cell extract was prepared as described previously (40Wagner R.W. Nishikura K. Mol. Cell. Biol. 1988; 8: 770-777Crossref PubMed Scopus (96) Google Scholar). Mouse brain nuclear extract was prepared by the Dignam method (41Dignam J.D. Lebovits R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar) with a minor modification as follows. Fresh mouse brains were minced using a pair of scissors, and further homogenized by a motor-driven Potter homogenizer in 3 times the packed cell volume of phosphate-buffered saline. The cell pellet was suspended in a buffer containing 10 mm Hepes (pH 7.9), 10 mm KCl, 1.5 mm MgCl2, 0.5 mm DTT, 1× complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN), and 0.5 mm phenylmethylsulfonyl fluoride, and kept on ice for 20 min. Cells were lysed by 10–20 strokes with a glass Dounce homogenizer followed by centrifugation at 10,000 rpm for 15 min in a Type 65 Ti Beckman rotor. The nuclear pellet was suspended in 3 pellet volumes of a buffer containing 20 mm Hepes (pH 7.9), 0.42m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 257 glycerol, 1.0 mm DTT, 1× Complete protease inhibitor mixture, and 0.5 mmphenylmethylsulfonyl fluoride. After five gentle strokes in a glass Dounce homogenizer, the protein extract was cleared of debris by centrifugation at 30,000 rpm for 30 min. All column chromatography procedures were carried out at 4 °C. Total cell extract was prepared from Sf9 cells infected with a single or combination of two recombinant baculoviruses (38Lai F. Drakas R. Nishikura K. J. Biol. Chem. 1995; 270: 17098-17105Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The cell extract, dialyzed against buffer A (0.05 mTris, pH 7.0, 0.15 m NaCl, 5 mm EDTA, 1.0 mm DTT, 207 glycerol, 0.25 mmphenylmethylsulfonyl fluoride, 0.057 Nonidet P-40) was first passed through a 1.0-ml (1.0 × 1.3 cm) anti-FLAG M2-monoclonal antibody (mAb)-agarose gel (Sigma) affinity column equilibrated with buffer A containing 0.15 m NaCl and 1 mmऔ-mercaptoethanol instead of 1 mm DTT. After washing the column with 10 ml each of buffer A containing 0.15 m NaCl, 0.75 m NaCl, and again 0.15 m NaCl, the complex was eluted with 5 ml of buffer A containing 0.15 m NaCl and 200 ॖg/ml FLAG peptide. The pooled peak fractions were dialyzed against buffer B (10 mm Tris, pH 7.5, 0.3 mNaCl, 207 glycerol, 0.057 Nonidet P-40, 1 mmऔ-mercaptoethanol) and then applied to a TALON metal resin (BD Biosciences, Palo Alto, CA) affinity column. Following extensive washing with buffer B containing 10 mm imidazole, proteins were eluted with 150 mm imidazole. The yield of recombinant proteins during the sequential affinity chromatography was followed by Western blotting analysis using an anti-FLAG M2 mAb (Sigma) or anti-6His 6XHN mAb (BD Biosciences). The purity of recombinant proteins purified by the first ("1× purified") and second ("2× purified") affinity column chromatography were determined by electrophoresis on a 107 SDS-PAGE gel followed by silver staining. In some experiments, the ADAR complex purified on a M2 mAb-agarose column was treated with RNases prior to its application to the TALON affinity column. The recombinant ADAR proteins (1× purified) were treated with single-stranded RNA (ssRNA) specific RNases A (0.5 units/ml) and T1 (10 unit/ml) obtained from Roche Diagnostics or with dsRNA-specific RNase V1 (1 unit/ml) obtained from Pierce. RNase-digested ADAR proteins were dialyzed against buffer B, and then subjected to TALON affinity column chromatography. The RNase digestion conditions were tested separately with uniformly [α-32P]ATP-labeled c-myc antisense ssRNA or dsRNA (40Wagner R.W. Nishikura K. Mol. Cell. Biol. 1988; 8: 770-777Crossref PubMed Scopus (96) Google Scholar), confirming their complete digestion with the relevant RNase(s). Purified ADAR proteins (1 ॖg) or crude nuclear extract (2 mg) was applied to a 24-ml (1 × 30 cm) column of Superose 12 HR 10/30 (Amersham Biosciences) for size exclusion chromatography. The buffer system used was 0.05 m Tris (pH 7.0), 0.5 m NaCl, 5 mm EDTA, 1 mm DTT, 207 glycerol, and 0.17 Nonidet P-40. Purified recombinant ADAR proteins were concentrated to 100 ॖl using Centricon (Amicon) before applying to the column. Fractions (0.5 ml) were collected at a flow rate of 0.4 ml/min using a fast protein liquid chromatography system. The molecular weight of ADAR (monomer or oligomer) was estimated by comparison with molecular weight standards obtained from Sigma; bovine thyroglobulin (669,000), horse spleen apoferritin (443,000), sweet potato औ-amylase (200,000), yeast alcohol dehydrogenase (150,000), bovine serum albumin (66,000), and bovine carbonic anhydrase (29,000). The peak for the ADAR complex was confirmed by Western blotting analysis, and the peak position of the marker proteins was determined by measuring the optical absorption at 280 nm. Editing of a synthetic 5-HT2C RNA C5 was assayed in vitro as described previously (22Chen C-X. Cho D-S.C. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar), using 1× or 2× purified recombinant homodimer complexes as well as 2× purified heterodimer complexes consisting of one wild-type and another non-functional mutant ADAR monomer. The standard editing reaction contained 20 fmol of a synthetic C5 RNA substrate, 10 ng of recombinant ADAR proteins, 0.02 m Hepes (pH 7.0), 0.1 m NaCl, 107 glycerol, 5 mm EDTA, 1 mm DTT, and 250 units/ml RNasin (Promega). The reactions were incubated at 30 °C for various times. Quantitation of editing efficiency at five sites of 5-HT2CR RNA was carried out by dideoxyoligonucleotide/primer extension assay as described previously (10Wang Q. Chen C-X. Cho D-S.C. O'Brien P. Murray J.M. Nishikura K. J. Neurochem. 2000; 74: 1290-1300Crossref PubMed Scopus (175) Google Scholar, 22Chen C-X. Cho D-S.C. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar). The ratio of the edited and unedited RNAs was estimated by quantifying the radioactivity of the primer-extended products with a phosphorimaging system (Amersham Biosciences). Proteins were fractionated on an SDS-87 polyacrylamide gel and transferred to ImmobilonTM-P nylon membrane (Millipore, Bedford, MA). Blots were blocked in a buffer containing phosphate-buffered saline and 37 nonfat dry milk. MAbs 15.8.6, 1.3.1, and 3.591 for detection of native and recombinant ADAR1, ADAR2, and ADAR3 proteins, respectively (22Chen C-X. Cho D-S.C. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar, 42Raizkin O. Cho D-S. Sperling J. Nishikura K. Sperling R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6571-6576Crossref PubMed Scopus (80) Google Scholar), and mAbs M2 and 6XHN for FLAG- and 6His epitope-tagged recombinant ADAR proteins, respectively, were used. ADAR-specific protein bands were detected by peroxidase-conjugated goat antibodies directed against mouse immunoglobulins (Kirkegaard and Perry Lab., Gaithersburg, MD) and chemiluminescense staining using RenaissanceTM (PerkinElmer Life Sciences). A set of baculovirus constructs for ectopic expression of ADAR1, ADAR2, and ADAR3 with either a FLAG or a 6His epitope tag at the N terminus were prepared. Two different s
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