FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1986004996> ?p ?o ?g. }

• W1986004996 endingPage "45842" @default.
• W1986004996 startingPage "45833" @default.
• W1986004996 abstract "Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional process that amplifies the repertoire of protein production. Recently, the induction of this process through up-regulation of the editing enzyme RNA-specific adenosine deaminase 1 (ADAR1) was documented during acute inflammation. Here we report that the inflammation-induced up-regulation of ADAR1 involves differential production and intracellular localization of several isoforms with distinct RNA-binding domains and localization signals. These include the full-length ADAR1 (p150) and two functionally active short isoforms (p80 and p110). ADAR1 p80 starts at a methionine 519 (M519) due to alternative splicing in exon 2, which deletes the putative nuclear localization signal, the Z-DNA binding domain, and the entire RNA binding domain I. ADAR1 p110 is the mouse homologue of the human ADAR1 110-kDa variant (M246), which retains the second half of the Z-DNA binding domain, all RNA binding domains, and the deaminase domain. Additional variations are found in the third RNA binding domain of ADAR1; they are differentially regulated during inflammation, generating isoforms with different levels of activities. Studies in several cell types transfected with ADAR1-EGFP chimeras demonstrated that the p150 and p80 variants are localized in the cytoplasm and nucleolus, respectively. In agreement with this observation, endogenous ADAR1 was identified in the cytoplasm and nucleolus of mouse splenocytes and HeLa cells. Since the ADAR1 variants are differentially regulated during acute inflammation, it suggests that the localization of these variants and of A-to-I RNA editing in the cytoplasm, nucleus, and nucleolus is intracellularly reorganized in response to inflammatory stimulation. Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional process that amplifies the repertoire of protein production. Recently, the induction of this process through up-regulation of the editing enzyme RNA-specific adenosine deaminase 1 (ADAR1) was documented during acute inflammation. Here we report that the inflammation-induced up-regulation of ADAR1 involves differential production and intracellular localization of several isoforms with distinct RNA-binding domains and localization signals. These include the full-length ADAR1 (p150) and two functionally active short isoforms (p80 and p110). ADAR1 p80 starts at a methionine 519 (M519) due to alternative splicing in exon 2, which deletes the putative nuclear localization signal, the Z-DNA binding domain, and the entire RNA binding domain I. ADAR1 p110 is the mouse homologue of the human ADAR1 110-kDa variant (M246), which retains the second half of the Z-DNA binding domain, all RNA binding domains, and the deaminase domain. Additional variations are found in the third RNA binding domain of ADAR1; they are differentially regulated during inflammation, generating isoforms with different levels of activities. Studies in several cell types transfected with ADAR1-EGFP chimeras demonstrated that the p150 and p80 variants are localized in the cytoplasm and nucleolus, respectively. In agreement with this observation, endogenous ADAR1 was identified in the cytoplasm and nucleolus of mouse splenocytes and HeLa cells. Since the ADAR1 variants are differentially regulated during acute inflammation, it suggests that the localization of these variants and of A-to-I RNA editing in the cytoplasm, nucleus, and nucleolus is intracellularly reorganized in response to inflammatory stimulation. A-to-I RNA editing is catalyzed by RNA-specific adenosine deaminase (ADAR), 1The abbreviations used are: ADAR, RNA-specific adenosine deaminase (ADAR1 isoforms were designated as ADAR1 followed by L or S for the long and short forms, and a, b, or c for the alternatively spliced forms; i.e. ADAR1Sa is a small isoform of ADAR1 with a-form alternative splicing in the dsRBDIII); ADAT, tRNA-specific adenosine deaminase; dsRNA, double-stranded RNA; dsRBD, dsRNA binding domain; gluR-B, glutamate receptor subunit B; NLS, nuclear localization signal; IFN, interferon; IL, interleukin; RT, reverse transcriptase; PBS, phosphate-buffered saline; dsRBDI, dsRBDII, and dsRBDIII, dsRNA-binding domain I, II, and III; TRITC, tetramethylrhodamine isothiocyanate. which converts adenosine to inosine and leads to the production of mRNA and protein variants. This process is ubiquitous and widely conserved; it has been identified in multiple species including mammals (1Chen C.X. Cho D.S. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar, 2Kim 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, 3Maas S. Gerber A.P. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8895-8900Crossref PubMed Scopus (72) Google Scholar, 4Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (433) Google Scholar, 5Melcher 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), Xenopus (6Bass B.L. Weintraub H. Cell. 1988; 55: 1089-1098Abstract Full Text PDF PubMed Scopus (533) Google Scholar), Drosophila (7Palladino M.J. Keegan L.P. O'Connell M.A. Reenan R.A. RNA. 2000; 6: 1004-1018Crossref PubMed Scopus (143) Google Scholar), and zebrafish (8Slavov D. Clark M. Gardiner K. Gene (Amst.). 2000; 250: 41-51Crossref PubMed Scopus (22) Google Scholar). Four A-to-I RNA editing enzymes, termed ADAR1, ADAR2, ADAR3, and ADAT1, have been cloned so far from mammals (1Chen C.X. Cho D.S. Wang Q. Lai F. Carter K.C. Nishikura K. RNA. 2000; 6: 755-767Crossref PubMed Scopus (378) Google Scholar, 2Kim 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, 3Maas S. Gerber A.P. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8895-8900Crossref PubMed Scopus (72) Google Scholar, 4Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (433) Google Scholar, 5Melcher 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). ADAR1 and ADAR2 are widely expressed in a variety of cells and tissues (2Kim 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, 4Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (433) Google Scholar), with the highest expression in the brain and spleen. ADAR3 was identified solely in the brain, and its deaminase activity has not yet been established. ADAT1 targets tRNA and has been cloned from humans (3Maas S. Gerber A.P. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8895-8900Crossref PubMed Scopus (72) Google Scholar), mice (9Maas S. Kim Y.G. Rich A. Gene (Amst.). 2000; 243: 59-66Crossref PubMed Scopus (22) Google Scholar), and yeast (10Gerber A. Grosjean H. Melcher T. Keller W. EMBO J. 1998; 17: 4780-4789Crossref PubMed Scopus (126) Google Scholar). ADARs are conserved in their adenosine deaminase domain but differ in their RNA binding domains. ADAR1 and ADAR2 contain two or three dsRNA binding domains (dsRBD) in addition to an adenosine deaminase domain. These editases are capable of both nonspecific editing of dsRNA and site-specific editing of glutamate receptor subunit B (gluR-B) mRNA and serotonin receptor mRNA (11Yang J.H. Sklar P. Axel R. Maniatis T. Nature. 1995; 374: 77-81Crossref PubMed Scopus (116) Google Scholar, 12Burns 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). ADAR2 selectively edits gluR-B at the Q/R site and serotonin at the D site; ADAR1 preferentially targets gluR-B at the hot spot and serotonin at the A and C sites (12Burns 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). Q/R site editing requires the formation of a base-pairing structure around the editing site for specific substrate recognition by ADAR2. The importance of the secondary structure in substrate recognition was confirmed in a study in which deletion of the stem-loop structures around the Q/R editing site abolished site-specific editing (11Yang J.H. Sklar P. Axel R. Maniatis T. Nature. 1995; 374: 77-81Crossref PubMed Scopus (116) Google Scholar). ADAR3 and ADAT1 do not seem to edit these substrates. A nuclear localization signal (NLS) and a Z-DNA binding domain are present near the N-terminal region of ADAR1 and are conserved in all species. This NLS is also called a nuclear export signal, since the human ADAR1 was initially localized to the cytoplasm and was found in the nucleus after nuclear export was blocked (13Poulsen H. Nilsson J. Damgaard C.K. Egebjerg J. Kjems J. Mol. Cell Biol. 2001; 21: 7862-7871Crossref PubMed Scopus (124) Google Scholar). The human ADAR1 also has an atypical NLS within its dsRBDIII and thus displays the characteristics of a shuttling protein (14Eckmann C.R. Neunteufl A. Pfaffstetter L. Jantsch M.F. Mol. Biol. Cell. 2001; 12: 1911-1924Crossref PubMed Scopus (89) Google Scholar). In contrast, the Xenopus ADAR1 contains a distinct NLS, which leads this enzyme to the nascent ribonucleoprotein matrix on Xenopus lampbrush chromosomes, where it is specifically associated with active transcriptional sites. Thus, it is conceivable that the editing activity of Xenopus ADAR1 is coupled with transcriptional events or that it targets newly synthesized RNAs (15Eckmann C.R. Jantsch M.F. J. Cell Biol. 1999; 144: 603-615Crossref PubMed Scopus (31) Google Scholar). Functional consequences of A-to-I RNA editing have been observed in the central nervous system. In the mammalian brain, editing of gluR-B pre-mRNA by ADAR2 has been shown to alter the calcium permeability of excitatory neurons (16Lomeli H. Mosbacher J. Melcher T. Hoger T. Geiger J.R. Kuner T. Monyer H. Higuchi M. Bach A. Seeburg P.H. Science. 1994; 266: 1709-1713Crossref PubMed Scopus (642) Google Scholar, 17Feldmeyer D. Kask K. Brusa R. Kornau H.C. Kolhekar R. Rozov A. Burnashev N. Jensen V. Hvalby O. Sprengel R. Seeburg P.H. Nat. Neurosci. 1999; 2: 57-64Crossref PubMed Scopus (232) Google Scholar). The role of ADAR2 in the nervous system was further studied in mice homozygous for a targeted functional null allele. In ADAR2 -/- mice, A-to-I RNA editing of diverse mRNAs is substantially reduced; seizure activity and early death occur (18Higuchi 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). In the Drosophila brain, disruption of the dADAR gene (a homologue of ADAR2) abolishes sodium, calcium, and chloride channels (19Hanrahan C.J. Palladino M.J. Ganetzky B. Reenan R.A. Genetics. 2000; 155: 1149-1160Crossref PubMed Google Scholar, 20Ma E. Gu X.Q. Wu X. Xu T. Haddad G.G. J. Clin. Invest. 2001; 107: 685-693Crossref PubMed Scopus (57) Google Scholar, 21Palladino 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). Mutants lacking dADAR exhibit extreme behavioral deficits and neurodegeneration (21Palladino 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). Furthermore, an oxygen-sensitive dADAR mutant suffers prolonged recovery from anoxic stupor, vulnerability to heat shock, and increased O2 demands (20Ma E. Gu X.Q. Wu X. Xu T. Haddad G.G. J. Clin. Invest. 2001; 107: 685-693Crossref PubMed Scopus (57) Google Scholar). Thus, editing of ion channel pre-mRNAs by dADAR appears to be critical for the integrity and function of the central nervous system. Studies in ADAR1 chimeric mouse embryos demonstrated that this editing enzyme affects embryonic erythropoiesis (22Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar). In these studies, most embryos died before the 14th embryonic day due to hematopoietic defects, suggesting that editing in this developmental stage is critical for normal erythrocyte proliferation and/or differentiation. Several observations suggest that A-to-I RNA editing plays a role in the immune system. First, ADAR1 can be induced by interferon (IFN) in human amnion U cells (23Patterson J.B. Thomis D.C. Hans S.L. Samuel C.E. Virology. 1995; 210: 508-511Crossref PubMed Scopus (135) Google Scholar, 24Weier H.U. George C.X. Greulich K.M. Samuel C.E. Genomics. 1995; 30: 372-375Crossref PubMed Scopus (61) Google Scholar) and pulmonary macrophages (25Rabinovici R. Kabir K. Chen M. Su Y. Zhang D. Luo X. Yang J.H. Circ. Res. 2001; 88: 1066-1071Crossref PubMed Scopus (43) Google Scholar). Second, IFN-induced enzymes such as dsRNA-dependent protein kinase (PKR), 2′,5′-oligo(A) nuclease, and ADAR1 all interact with dsRNA (23Patterson J.B. Thomis D.C. Hans S.L. Samuel C.E. Virology. 1995; 210: 508-511Crossref PubMed Scopus (135) Google Scholar, 26Samuel C.E. Curr. Top. Microbiol. Immunol. 1998; 233: 125-145PubMed Google Scholar) either as a substrate or an activator. Third, ADAR1 and ADAR2 can destroy dsRNA or mRNA with dsRNA stretches, indicating that they may indirectly regulate the activity of dsRNA-binding proteins such as PKR. Fourth, a C-to-U editing enzyme has been reported to play a role in the specific immune response by modulating class switch recombination and somatic hypermutation in B-lymphocytes (27Longacre A. Storb U. Cell. 2000; 102: 541-544Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 28Neuberger M.S. Scott J. Science. 2000; 289: 1705-1706Crossref PubMed Scopus (12) Google Scholar, 29Muramatsu M. Sankaranand V.S. Anant S. Sugai M. Kinoshita K. Davidson N.O. Honjo T. J. Biol. Chem. 1999; 274: 18470-18476Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar, 30Muramatsu M. Kinoshita K. Fagarasan S. Yamada S. Shinkai Y. Honjo T. Cell. 2000; 102: 553-563Abstract Full Text Full Text PDF PubMed Scopus (2726) Google Scholar, 31Revy P. Muto T. Levy Y. Geissmann F. Plebani A. Sanal O. Catalan N. Forveille M. Dufourcq-Labelouse R. Gennery A. Tezcan I. Ersoy F. Kayserili H. Ugazio A.G. Brousse N. Muramatsu M. Notarangelo L.D. Kinoshita K. Honjo T. Fischer A. Durandy A. Cell. 2000; 102: 565-575Abstract Full Text Full Text PDF PubMed Scopus (1351) Google Scholar). Introduction of the C-to-U editing enzyme triggers hybridoma cells to generate somatic hypermutations in immunoglobulins (32Martin A. Bardwell P.D. Woo C.J. Fan M. Shulman M.J. Scharff M.D. Nature. 2002; 415: 802-806Crossref PubMed Scopus (229) Google Scholar). We have recently reported that A-to-I RNA editing by ADAR1 is also involved in both local (25Rabinovici R. Kabir K. Chen M. Su Y. Zhang D. Luo X. Yang J.H. Circ. Res. 2001; 88: 1066-1071Crossref PubMed Scopus (43) Google Scholar) and systemic (33Yang J.H. Luo X.X. Nie Y.Z. Su Y.J. Zhao Q.C. Kabir K. Zhang D.X. Rabinovici R. Immunology. 2003; 109: 15-23Crossref PubMed Scopus (105) Google Scholar) acute inflammation. In the lung, A-to-I RNA editing is up-regulated following endotoxin stimulation due to up-regulation of ADAR1, which precedes the development of pulmonary edema and leukocyte accumulation. Similarly, editing activity and ADAR1 expression are induced in cultured alveolar macrophages (MH-S cells) stimulated with endotoxin or IFN. In systemic inflammation produced by endotoxin in mice, inosine-containing mRNA is markedly induced to ∼5% of adenosine in total mRNA (33Yang J.H. Luo X.X. Nie Y.Z. Su Y.J. Zhao Q.C. Kabir K. Zhang D.X. Rabinovici R. Immunology. 2003; 109: 15-23Crossref PubMed Scopus (105) Google Scholar). This induction results from up-regulation of A-to-I RNA editing, since both dsRNA editing activity and ADAR1 expression are increased in the spleen, thymus, and peripheral lymphocytes of endotoxin-treated mice. Up-regulation of ADAR1 has been confirmed in vitro in T lymphocytes and macrophages stimulated with a variety of inflammatory mediators including tumor necrosis factor-α and IFN-γ. Late induction of RNA editing occurs in ConA-activated splenocytes stimulated with IL-2 in vitro. Taken together, these data suggest that during local and systemic inflammation, ADAR1-mediated RNA editing is increased, which may affect the inflammatory response through modulation of protein production. Acute inflammation is the underlying process of many critical illnesses, including the systemic inflammatory response syndrome, multiple organ failure, sepsis, adult respiratory distress syndrome, and ischemia/reperfusion injury (34Hudson L.D. Steinberg K.P. Chest. 1999; 116: 74-82Abstract Full Text Full Text PDF PubMed Google Scholar). One fundamental event that occurs in all of these stress situations is the intense production of multiple pro- and anti-inflammatory proteins. Thus, additional insight into the regulation of protein production during inflammation could shed light on the pathogenesis of this condition. It is within this context that we aimed to further examine the role of A-to-I RNA editing in acute inflammation. Specifically, following the identification of ADAR1 as a key player in local and systemic inflammation (25Rabinovici R. Kabir K. Chen M. Su Y. Zhang D. Luo X. Yang J.H. Circ. Res. 2001; 88: 1066-1071Crossref PubMed Scopus (43) Google Scholar, 33Yang J.H. Luo X.X. Nie Y.Z. Su Y.J. Zhao Q.C. Kabir K. Zhang D.X. Rabinovici R. Immunology. 2003; 109: 15-23Crossref PubMed Scopus (105) Google Scholar), we examined and characterized ADAR1 production, localization, and regulation during acute systemic inflammation in vitro and in vivo. The present study aims to further delineate the A-to-I RNA editing response to acute inflammation, with special emphasis on the generation through alternative splicing of ADAR1 variants with distinct functional domains. In addition, this study investigates the regulation and translocation of the inflammation-induced ADAR1 variants in the cytoplasm, nucleus, and nucleolus. Animal Model of Acute Inflammation—The model used in the present study was described previously in detail (35Kabir K. Gelinas J.P. Chen M. Chen D. Zhang D. Luo X. Yang J.H. Carter D. Rabinovici R. Shock. 2002; 17: 300-303Crossref PubMed Scopus (132) Google Scholar). In brief, endotoxin at a dose of 15 mg/kg (LD60) was injected into the peritoneal cavity of conscious adult (6-week-old, 25-g) male C57Bl/6 mice (Charles River Laboratories). The mice were anesthetized (pentobarbital; 30 mg/kg) at various time points, and tissues were harvested and processed for analysis, as described below. The Yale Animal Care and Use Committee approved all animal protocols. Five mice were used per group, and a mixture of five similar organs from these animals was used for each analysis. RNA and Protein Isolation—Total RNA and mRNA at each time point (n = 5) were isolated using Trizol (Trizol, Inc.) and OligoTex (Qiagen) separately for Northern blotting, RT-PCR, and ADAR1 cloning. Total protein was isolated by homogenizing mouse splenic cells in four volumes of an editing buffer (containing 20 mm Hepes, pH 7.9, 100 mm KCl, 5 mm EDTA, 0.5% Nonidet P-40, and 10% glycerol). The lysate was sonicated for 30 s and centrifuged for 30 s at 4,000 × g. Protein concentration in the supernatant was determined using a Bio-Rad protein kit and adjusted to 10 mg/ml for Western blotting or editing analyses. Northern Blotting, RT-PCR, and Western Blot Analysis—Equal amounts (∼2 μg) of mRNA from mouse tissues (n = 5) were used for Northern blotting. ADAR1 was detected by hybridization of the blots with 32P-labeled antisense probes (positions 1305-1265 and 3004-2966; GenBank accession number AF291050). Membranes were hybridized at 65 °C overnight and then subjected to a final wash was with 0.1× SSC at 55 °C for 10 min. For RT-PCR, 2 μg of total RNA were used for reverse transcription primed with poly(dT)12-18. ADAR1 mRNA was determined by RT-PCR using primers that flank exons 6-8 (positions 1975-2003 and 2436-2408; GenBank number AF291050) or the entire coding region (positions 1-24 and 3459-3435; GenBank number AF291050). The relative expression of ADAR1 mRNA was estimated in comparison with GAPDH. For Western blotting, 60 μg of total protein from splenic cells was resolved on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The blots were detected using rabbit antiserum against the C-terminal of recombinant mouse ADAR1 expressed in E. coli (positions 2765-3459; GenBank number AF291050) or the N terminus of a synthetic mouse ADAR1 peptide (Santa Cruz Biotechnology). Cloning and Sequencing of ADAR1 Variants—Full-length ADAR1 from mouse splenic cells harvested 24 h after endotoxin stimulation (n = 5) was amplified by RT-PCR. The PCR products were cloned into pCRII (Invitrogen), and the diversity of ADAR1 cDNAs was analyzed by EcoRI digestion. Alternative splice junction sites were mapped by sequencing and analyzed by sequence alignment. Four typical ADAR1 cDNAs with open reading frames, ADAR1Sa (GenBank number AF291875), ADAR1Sb (AF291877), ADAR1La (AF291050), and ADAR1Lb (AF291876), were subcloned for further analysis. In Vitro Translation and Expression of ADAR1 Isoforms in a Baculovirus System—The cDNAs of ADAR1Sa, ADAR1Sb, ADAR1La, or ADAR1Lb in the pCRII vector were translated in vitro using a TNT T7 quick translation system following the manufacturer's protocol (Promega). For recombinant proteins, these variants were subcloned into pFastBac (Invitrogen) in frame with a His tag at the N termini to generate recombinant ADAR1-Bacmids. After transfection of Bacmids into Sf9 insect cells, recombinant ADAR1 isoforms were expressed, and proteins were isolated using nickel columns following the manufacturer's protocol (Amersham Biosciences). RNA Editing Assay Using dsRNA—RNA editing activity was evaluated by measuring A-to-I conversion of synthetic dsRNA (36Yang J.H. Sklar P. Axel R. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4354-4359Crossref PubMed Scopus (42) Google Scholar). In a typical dsRNA editing assay, 10 μl of cell extracts (100 μg of total protein) or 0.098 μg of recombinant ADAR1 isoforms were mixed with 32P-labeled dsRNA (∼5 × 103 cpm) and incubated at 37 °C for 1 h. The mixture was treated with an equal volume of a proteinase K buffer (containing 10 mm Tris-HCl, pH 8.0, 300 mm NaCl, 0.2% SDS, and 0.5 mg/ml proteinase K). The edited dsRNA was extracted with phenol, precipitated in ethanol, resuspended in 10 μl of RNase P1 mixture (containing 5 mm Tris-Cl, pH 7.5, 10 mm NaCl, and 1 unit of RNase P1), and incubated at 37 °C for 2 h. After being developed in saturated (NH4)2SO4-isopropyl alcohol (95:5) solution, the converted inosine was analyzed by TLC and visualized by autoradiography. The radioactivity of each spot was measured by scintillation, and the intensity of each nucleotide on x-ray film was quantitated with ScionImage (available on the World Wide Web at www.scioncorp.com). The specific activity was calculated by dividing the percentage of A-to-I conversion by the amount of protein (μg) used in the reaction, which was normalized to the specific activity of ADARLb. Construction of ADAR1-EGFP Chimera and Localization Analysis—A restriction enzyme site (BamHI) was added at each end of ADAR1 cDNAs (ADAR1Sa, ADAR1Sb, ADAR1La, and ADAR1Lb) by PCR using the pCRII-ADAR1 plasmids as templates. The PCR product was cleaved with BamHI and directly subcloned into the pEGFP-N1 vector (Clontech) in frame with the N-terminal of EGFP. Different cells, including mouse fibroblasts (3T3), neuroblastoma cells (N18), monocytes (RAW2.17), or human HeLa and 293 cells, were transfected (n = 3) with pEGFP-ADAR1 DNAs; fluorescence was observed under a microscope 6 h after transfection. Editing activity of the transiently expressed ADAR1-EGFP chimeras was tested in 293 cells and proved able to convert adenosine to inosine on synthetic dsRNA. Electron Microscopy—HeLa cells were fixed in 4 and 8% paraformaldehyde solutions in 0.25 m Hepes (pH 7.4) at 4 °C for 1 h and overnight, respectively. Cells were washed in PBS, scraped, and pelleted in a 10% solution of gelatin in PBS. Pieces of the pellet were infiltrated overnight at 4 °Cina2.3 m sucrose solution in PBS and then mounted on aluminum studs and frozen in liquid nitrogen. Frozen sections were cut on a Leica ultramicrotome at -108 °C. They were collected with a 1:1 mixture of 2.3 m sucrose and 2% methylcellulose solutions, thawed, and transferred onto Formvar- and carbon-coated nickel grids. For immunolabeling, grids were incubated with a solution of 0.1 m NH4Cl in PBS for 10 min, followed by 0.5% fish skin gelatin solution in PBS for 20 min at 22 °C. They were then incubated with anti-C-terminal ADAR1 antibody (1:50) for 30 min, followed by 10-nm protein A-gold complex (Department of Cell Biology, Utrecht University, The Netherlands) also for 30 min at 22 °C. After several washes in PBS, the sections were fixed in 1% glutaraldehyde in PBS, washed in water, and incubated with 1.8% methylcellulose and 0.5% uranyl acetate solution before being air-dried. Sections were examined in a Tecnai 12 Biotwin electron microscope (FEI) at 80 kV. For quantification, 20 random micrographs of nuclei were taken and printed at a final magnification of ×34,000. Labeling densities over the nucleus or nucleolus were calculated by dividing the number of gold particles over these structures by the surface of profiles estimated by point counting using a lattice grid with 20 mm between lines. Two ADAR1 mRNA Transcripts Are Expressed in a Tissue-specific Manner—To determine whether ADAR1 has splice variants and whether its expression is tissue-specific, ADAR1 transcripts in a variety of mouse tissues were analyzed by Northern blotting. An antisense sequence complementary to the dsRNA-binding domain of ADAR1 cDNA from positions 1305-1265 (GenBank number AF291050) was selected as the hybridization probe to reduce the nonspecific signal from mouse ribosomal RNA. Two ADAR1 transcripts measuring ∼7 and 5 kb were detected in all tested tissues (Fig. 1A). The total ADAR1 mRNA level showed tissue-specific differences, with the highest signal in the brain and spleen and the lowest in the liver. The ratio between the 7- and 5-kb transcripts also varied in a tissue-specific manner, with the highest value observed in the spleen and the lowest in the brain. Similar results were noted when antisense probes complementary to the deaminase domain or to full-length ADAR1 were used (data not shown). Multiple ADAR1 Variants Are Induced in Response to Inflammatory Stimulation—Northern blot analysis of splenic tissue from endotoxin-challenged mice demonstrated up-regulation of both the 7- and 5-kb ADAR1 transcripts (Fig. 1B). To characterize the sequence variations, the coding region of ADAR1 cDNA in spleens from sham-injected or endotoxin-stimulated mice was amplified using RT-PCR (Fig. 1C). In sham-injected animals, a dominant (∼20-fold excess) long form (ADAR1L) measuring 3.4 kb and a short form (ADAR1S) of 2.0 kb were identified. In contrast, spleens from endotoxin-challenged mice demonstrated equal levels of ADAR1L and ADAR1S due to a selective induction of ADAR1S. It should be noted that the 1.4-kb difference between ADAR1L and ADAR1S may not reflect the difference between the 7- and 5-kb ADAR1 transcripts observed in Northern blotting (Fig. 1, B and C). In addition, the PCR products of ADAR1L and ADAR1S displayed diverse variants, which slightly differed in size. To identify these variants, RT-PCR products of the ADAR1L fragments from a mixture of spleen and thymus tissues of normal or endotoxin-stimulated mice were recovered and cloned. Diversity of the selected individual clones was analyzed by restriction enzyme mapping (Fig. 1D). In agreement with the sequencing analysis, three fragments (I, II, and III) and a vector were identified. In sham-injected mice, fragment III had three major variations in 14 clones, whereas only a single size was identified for fragments I and II. In stimulated mice, fragments II and III were found to have three and four variants in 16 clones, respectively, whereas fragment I remained unchanged, with a similar size in all clones. As described in the legends to Figs. 1C and 2, variations in fragment I could be detected only when the mapping of ADAR1L and ADAR1S was compared. In conclusion, diverse ADAR1L and ADAR1S variants were generated in response to inflammatory stimulation. ADAR1 cDNA Variants Are Generated through Inflammation-induced Alternative Splicing—ADAR1 cDNA variants were sequenced. Alignment analysis confirmed that all variants were identical at their 5′- or 3′-ends. The difference between ADAR1L and ADAR1S resulted from alternative selection of the 3′ splice site of intron 1, which deleted the entire exon 2, comprising ∼1.4 kb of ADAR1 mRNA (Fig. 2A). Variations in fragment III resulted from alternative splicing of exon 7 and selective usage of a few cryptic 5′ splice sites (Fig. 2B). Three ADAR1 variants with slightly different sizes, termed a-, b-, and c-forms, were generated. The b-form matched the previously identified mouse cDNA sequence (25Rabinovici R. Kabir K. Chen M. Su Y. Zhang D. Luo X. Yang J.H. Circ. Res. 2001; 88: 1066-1071Crossref PubMed Scopus (43) Google Scholar) and was dominant in sham-injected mice (9 of 14 clones). The a-form, which had an additional 78 bp in exon 7 due to a cryptic 5′ splice site within intron 7, occurred less frequently (3 of 14 clones). A similar insertio" @default.
• W1986004996 created "2016-06-24" @default.
• W1986004996 creator A5017787702 @default.
• W1986004996 creator A5033173435 @default.
• W1986004996 creator A5056499636 @default.
• W1986004996 creator A5060279706 @default.
• W1986004996 creator A5065971181 @default.
• W1986004996 creator A5075777094 @default.
• W1986004996 creator A5080177287 @default.
• W1986004996 date "2003-11-01" @default.
• W1986004996 modified "2023-10-16" @default.
• W1986004996 title "Intracellular Localization of Differentially Regulated RNA-specific Adenosine Deaminase Isoforms in Inflammation" @default.
• W1986004996 cites W1533516721 @default.
• W1986004996 cites W1547213195 @default.
• W1986004996 cites W1575039917 @default.
• W1986004996 cites W1580101590 @default.
• W1986004996 cites W1833905419 @default.
• W1986004996 cites W1967155911 @default.
• W1986004996 cites W1979845442 @default.
• W1986004996 cites W1984407095 @default.
• W1986004996 cites W1986923087 @default.
• W1986004996 cites W1989022221 @default.
• W1986004996 cites W1991278351 @default.
• W1986004996 cites W1995956654 @default.
• W1986004996 cites W1996063915 @default.
• W1986004996 cites W2002043709 @default.
• W1986004996 cites W2008476211 @default.
• W1986004996 cites W2011536289 @default.
• W1986004996 cites W2011700223 @default.
• W1986004996 cites W2012463423 @default.
• W1986004996 cites W2014127990 @default.
• W1986004996 cites W2017472306 @default.
• W1986004996 cites W2018793883 @default.
• W1986004996 cites W2021261491 @default.
• W1986004996 cites W2021956465 @default.
• W1986004996 cites W2026169253 @default.
• W1986004996 cites W2029526949 @default.
• W1986004996 cites W2029991642 @default.
• W1986004996 cites W2036566192 @default.
• W1986004996 cites W2046641567 @default.
• W1986004996 cites W2047398077 @default.
• W1986004996 cites W2055465738 @default.
• W1986004996 cites W2061556445 @default.
• W1986004996 cites W2061680089 @default.
• W1986004996 cites W2062355463 @default.
• W1986004996 cites W2063954657 @default.
• W1986004996 cites W2066987191 @default.
• W1986004996 cites W2074562954 @default.
• W1986004996 cites W2083443360 @default.
• W1986004996 cites W2083787533 @default.
• W1986004996 cites W2101543755 @default.
• W1986004996 cites W2109759870 @default.
• W1986004996 cites W2120276739 @default.
• W1986004996 cites W2132002570 @default.
• W1986004996 cites W2133435396 @default.
• W1986004996 cites W2139387072 @default.
• W1986004996 cites W214526914 @default.
• W1986004996 cites W2155103924 @default.
• W1986004996 cites W2161048703 @default.
• W1986004996 cites W2163879429 @default.
• W1986004996 cites W2165380953 @default.
• W1986004996 cites W2165608602 @default.
• W1986004996 cites W64310402 @default.
• W1986004996 doi "https://doi.org/10.1074/jbc.m308612200" @default.
• W1986004996 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12954622" @default.
• W1986004996 hasPublicationYear "2003" @default.
• W1986004996 type Work @default.
• W1986004996 sameAs 1986004996 @default.
• W1986004996 citedByCount "55" @default.
• W1986004996 countsByYear W19860049962012 @default.
• W1986004996 countsByYear W19860049962013 @default.
• W1986004996 countsByYear W19860049962015 @default.
• W1986004996 countsByYear W19860049962016 @default.
• W1986004996 countsByYear W19860049962017 @default.
• W1986004996 countsByYear W19860049962018 @default.
• W1986004996 countsByYear W19860049962019 @default.
• W1986004996 countsByYear W19860049962020 @default.
• W1986004996 countsByYear W19860049962022 @default.
• W1986004996 countsByYear W19860049962023 @default.
• W1986004996 crossrefType "journal-article" @default.
• W1986004996 hasAuthorship W1986004996A5017787702 @default.
• W1986004996 hasAuthorship W1986004996A5033173435 @default.
• W1986004996 hasAuthorship W1986004996A5056499636 @default.
• W1986004996 hasAuthorship W1986004996A5060279706 @default.
• W1986004996 hasAuthorship W1986004996A5065971181 @default.
• W1986004996 hasAuthorship W1986004996A5075777094 @default.
• W1986004996 hasAuthorship W1986004996A5080177287 @default.
• W1986004996 hasBestOaLocation W19860049961 @default.
• W1986004996 hasConcept C104317684 @default.
• W1986004996 hasConcept C165912504 @default.
• W1986004996 hasConcept C185592680 @default.
• W1986004996 hasConcept C203014093 @default.
• W1986004996 hasConcept C2776914184 @default.
• W1986004996 hasConcept C2776991684 @default.
• W1986004996 hasConcept C34628245 @default.
• W1986004996 hasConcept C53345823 @default.
• W1986004996 hasConcept C55493867 @default.
• W1986004996 hasConcept C67705224 @default.

• next