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• W1989656471 abstract "Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo- and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent. Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo- and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent. Double-stranded RNAs (dsRNAs) 2The abbreviations used are: dsRNA, double-stranded RNA; ADAR, adenosine deaminase that acts on RNA; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; GFP, green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole.2The abbreviations used are: dsRNA, double-stranded RNA; ADAR, adenosine deaminase that acts on RNA; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; GFP, green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole. in eukaryotes are subject to a variety of processing reactions, including cleavage by the RNase III family members Drosha and Dicer in the micro RNA and small interfering RNA gene-silencing pathways and editing by members of the ADAR (adenosone deaminase that acts on RNA) enzyme family (1Carmell M.A. Hannon G.J. Nat. Struct. Mol. Biol. 2004; 11: 214-218Crossref PubMed Scopus (311) Google Scholar, 2Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (941) Google Scholar). This latter reaction involves the hydrolytic deamination of adenosine (A) to inosine (I) within the context of dsRNA. Editing events of this type have been demonstrated in both cellular and viral transcripts and have been shown to function in altering the coding properties of the edited RNAs. For example, the life cycle of the Hepatitis δ virus is regulated by an editing event in the anti-genome in which a UAG stop codon is converted to a UIG tryptophan codon (3Polson A.G. Bass B.L. Casey J.L. Nature. 1996; 380: 454-456Crossref PubMed Scopus (265) Google Scholar). An A to I modification is involved in the functional regulation of a growing number of cellular factors. These include the tissue-specific editing of the serotonin 5-HT2C receptor, which results in a reduction in response to serotonin agonists (4Burns 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 (852) Google Scholar). Transcripts for subunits of the neural-specific AMPA class of glutamate-gated (GluR) ion channels undergo A to I modification at two positions, the Q/R and R/G editing sites, that affect the properties of the resulting channels (5Sommer B. Kohler M. Sprengel R. Seeburg P.H. Cell. 1991; 67: 11-19Abstract Full Text PDF PubMed Scopus (1172) Google Scholar, 6Lomeli 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 (636) Google Scholar). In addition to the editing of these and other neuronal transcripts to effect codon changes, one deaminase family member, ADAR2, has been shown to autoregulate its own expression by the creation of a 3′-splice site (CAA to CAI) (7Rueter S.M. Dawson T.R. Emeson R.B. Nature. 1999; 399: 75-80Crossref PubMed Scopus (495) Google Scholar). Despite the identification of these editing substrates, the global role of A to I modification in higher eukaryotes remains unclear. Measurement of inosine levels in RNA isolated from rat tissue suggests a greater level of editing than indicated by known RNA substrates (8Paul M.S. Bass B.L. EMBO J. 1998; 17: 1120-1127Crossref PubMed Scopus (226) Google Scholar). A cloning protocol that depended upon an inosine-specific cleavage of RNA detected a large number of editing sites in non-coding regions of RNAs from Caenorhabditis elegans and humans that included sites in 5′- and 3′-untranslated regions and introns (9Morse D.P. Bass B.L. Biochemistry. 1997; 36: 8429-8434Crossref PubMed Scopus (87) Google Scholar). Recent bioinformatic studies have suggested the presence of more than 12,000 editing sites corresponding to non-coding regions of the human genome (10Athanasiadis A. Rich A. Maas S. PLoS. Biol. 2004; 2: 1-15Crossref Scopus (540) Google Scholar, 11Blow M. Futreal P.A. Wooster R. Stratton M.R. (2004) Genome Res. 2004; 14: 2379-2387Crossref PubMed Scopus (239) Google Scholar, 12Kim D.D. Kim T.T. Walsh T. Kobayashi Y. Matise T.C. Buyske S. Gabriel A. Genome Res. 2004; 14: 1719-1725Crossref PubMed Scopus (400) Google Scholar, 13Levanon E.Y. Eisenberg E. Yelin R. Nemzer S. Hallegger M. Shemesh R. Fligelman Z.Y. Shoshan A. Pollock S.R. Sztybel D. Olshansky M. Rechavi G. Jantsch M.F. Nat. Biotechnol. 2004; 22: 1001-1005Crossref PubMed Scopus (611) Google Scholar). The importance of editing in development has been demonstrated by studies showing that deletion of ADAR1 is an embryonic lethal event in mice; fibroblasts derived from ADAR1–/– embryos are subject to stress-induced apoptosis (14Wang Q. Miyakoda M. Yang W. Khillan J. Stachura D.L. Weiss M.J. Nishikura K. J. Biol. Chem. 2004; 279: 4952-4961Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Finally, it has been suggested that A to I modification antagonizes RNA interference: editing could regulate endogenous micro RNA mediated gene silencing or limit RNA interference resulting from nonspecific antisense transcription (15Tonkin L.A. Bass B.L. Science. 2003; 302: 1725Crossref PubMed Scopus (128) Google Scholar, 16Kent O.A. MacMillan A.M. Org. Biomol. Chem. 2004; 2: 1957-1961Crossref PubMed Scopus (13) Google Scholar, 17DeCerbo J. Carmichael G.G. Curr. Opin. Cell Biol. 2005; 17: 302-308Crossref PubMed Scopus (45) Google Scholar).One difficulty in identifying the biological targets and, hence, roles of A to I editing is that little is understood about the sequence determinants for editing of known substrates beyond the fact that editing sites are found within dsRNAs. The first RNA deaminase to be cloned, ADAR1 (18Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (365) Google Scholar, 19O'Connell M.A. Krause S. Higuchi M. Hsuan J.J. Totty N.F. Jenny A. Keller W. Mol. Cell. Biol. 1995; 15: 1389-1397Crossref PubMed Google Scholar, 20Hough R.F. Bass B.L. RNA. 1997; 3: 356-370PubMed Google Scholar, 21Liu Y. George C.X. Patterson J.B. Samuel C.E. J. Biol. Chem. 1997; 272: 4419-4428Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), was originally identified as an activity responsible for unwinding double-stranded RNA (22Bass B.L. Weintraub H. Cell. 1987; 48: 607-613Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 23Rebagliati M.R. Melton D.A. Cell. 1987; 48: 599-605Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 24Bass B.L. Weintraub H. Cell. 1988; 55: 1089-1098Abstract Full Text PDF PubMed Scopus (517) Google Scholar); subsequently, this unwinding activity was correlated with deamination of A to I within these RNAs (25Polson A.G. Crain P.F. Pomerantz S.C. McCloskey J.A. Bass B.L. Biochemistry. 1991; 30: 11507-11514Crossref PubMed Scopus (107) Google Scholar). Constitutively expressed ADAR1 is a 110-kDa nuclear protein that contains three N-terminal double-stranded RNA binding domains, a C-terminal deaminase domain, and one N-terminal Z-DNA binding domain. ADAR1 exhibited low deaminase activity with a number of specific substrates, including the Q/R site of the GluR-B pre-mRNA, but has been shown to efficiently edit the R/G site of GluR-B as well as the anti-genome of Hepatitis δ virus (3Polson A.G. Bass B.L. Casey J.L. Nature. 1996; 380: 454-456Crossref PubMed Scopus (265) Google Scholar, 26Hurst S.R. Hough R.F. Aruscavage P.J. Bass B.L. RNA. 1995; 1: 1051-1060PubMed Google Scholar, 27Maas 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 (115) Google Scholar, 28Herb A. Higuchi M. Sprengel R. Seeburg P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1875-1880Crossref PubMed Scopus (135) Google Scholar). Screening of a rat hippocampal cDNA library with probes complementary to the deaminase domain of ADAR1 resulted in the cloning of ADAR2 deaminase (29Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (428) Google Scholar). The shorter of two isoforms, ADAR2a, is an ∼80-kDa protein containing two N-terminal double-stranded RNA binding domains as well as a C-terminal deaminase domain (30Gerber A. O'Connell M.A. Keller W. RNA. 1997; 3: 453-463PubMed Google Scholar). ADAR2a (henceforth ADAR2) has been shown to efficiently edit both the Q/R and R/G sites of the GluR-B transcript and, in contrast to ADAR1, does not exhibit activity at a position within intron 11 of GluR-B (29Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (428) Google Scholar). ADAR1 and ADAR2 are both expressed ubiquitously, although ADAR2 is enriched in the brain.During a study of the in vitro editing of the GluR-B R/G site by ADAR2, we observed that efficient editing required dimerization of the enzyme on the RNA substrate (31Jaikaran D.C. Collins C.H. MacMillan A.M. J. Biol. Chem. 2002; 277: 37624-37629Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This conclusion was based on a combination of kinetic and gel mobility shift analyses as well as RNA-dependent ADAR2·ADAR2 cross-linking. Subsequently, it was reported that recombinant, tagged human ADAR1 and ADAR2 could be purified as RNA-independent homodimers from Sf9 cells (32Cho D.S. Yang W. Lee J.T. Shiekhattar R. Murray J.M. Nishikura K. J. Biol. Chem. 2003; 278: 17093-17102Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). At the same time, O'Connell and co-workers (33Gallo A. Keegan L.P. Ring G.M. O'Connell M.A. EMBO J. 2003; 22: 3421-3430Crossref PubMed Scopus (96) Google Scholar) found that the Drosophila ADAR dimerized in an RNA-dependent fashion and that this self-association was required for editing.The fact that specific non-neural substrates have not been identified for either ADAR1 or ADAR2, despite the ubiquitous expression profile of these enzymes in all tissue types, coupled with a possible RNA dependence of their dimerization complicates study of ADAR self-association. We therefore decided to create fusion proteins of both ADAR family members with cyan and yellow fluorescent protein (CFP and YFP) and probe their association by fluorescence resonance energy transfer (FRET) in human cells (34Bastiaens P.I. Majoul I.V. Verveer P.J. Soling H.D. Jovin T.M. EMBO J. 1996; 15: 4246-4253Crossref PubMed Scopus (239) Google Scholar). Using this approach, we have been able to observe both homo- and heterodimerization of ADAR1 and ADAR2 in HeLa cells. The results of RNase treatment before FRET measurement suggest that homodimerization of ADAR1 and ADAR2 as well as heterodimerization are independent of RNA binding. Homo- and heterodimerization of ADAR family members, most likely in association with other cellular proteins, probably represent critical regulatory mechanisms governing A to I editing in mammalian cells.EXPERIMENTAL PROCEDURESMammalian Expression Constructs—Human ADAR1 p110 was amplified by PCR from pJEL/hADAR1/H6 using primers containing HindIII and BamHI restriction sites. Human ADAR2 was PCR amplified from a previously described template (31Jaikaran D.C. Collins C.H. MacMillan A.M. J. Biol. Chem. 2002; 277: 37624-37629Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) using primers containing EcoRI and SalI restriction sites. ADAR1 and ADAR2 were then inserted into pEYFP-C1, pEYFP-N1, pECFP-C1, and pECFP-N1 (Clontech) using the appropriate restriction enzymes. Plasmids were transformed into chemically competent DH5α Escherichia coli, amplified, and purified using a plasmid mini-prep kit (Sigma).Cell Culture and Transfection—HeLa cells were cultured as monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml of penicillin, and 50 μg/ml of streptomycin at 37 °C and 5% CO2. Cells (2 × 105) were plated on 25-mm coverslips (Fisher Scientific) in six-well tissue culture dishes and were allowed to adhere for 24 h. The cells were transiently transfected with 1–5 μg of plasmid DNA using Perfectin (Gene Therapy Systems) according to the manufacturer's protocol and analyzed 20–24 h post-transfection. To examine the RNA dependence of localization and ADAR association, HeLa cells transfected with 1–5 μg of plasmid DNA were permeabilized in Gal-Screen Buffer B (Applied Biosystems) for 10 min. They were then washed twice with phosphate-buffered saline before being treated with an RNase mixture (RNase A, 200 μg/μl; RNase T1, 100 units/μl; RNase V1, 0.5 units/μl) for 30 min.Fluorescence Microscopy—Transiently transfected cells on coverslips were rinsed three times in phosphate-buffered saline and fixed for 15 min in freshly prepared 4% formaldehyde (Sigma) at room temperature. Coverslips were washed a further three times in phosphate-buffered saline before being mounted onto slides using Vectashield with DAPI (Vector Laboratories). Images were collected with a Zeiss laser scanning confocal microscope (LSM 510 NLO Meat) mounted on a Zeiss Axiovert 200 M inverted microscope with a ×40 F-fluar lens (N.A. 1.3) equipped with four visible lasers with five laser lines and a spectral Meta detector. The 458- and 514-nm laser lines (emitted from a 25-milliwatt argon laser) were used to image CFP and YFP. Band pass filters of 462–484 and 580–612 nm were used in collecting emission from CFP and YFP, respectively.Fluorescence Resonance Energy Transfer—FRET experiments were performed on fixed cells using the donor recovery after acceptor photobleach method (34Bastiaens P.I. Majoul I.V. Verveer P.J. Soling H.D. Jovin T.M. EMBO J. 1996; 15: 4246-4253Crossref PubMed Scopus (239) Google Scholar). First, images were obtained in the CFP and YFP channels and the intensities of the signals were calculated. YFP was then selectively photobleached at 514 nm in a defined region of the cell. A second set of images was then obtained using the same conditions as prior to photobleaching. FRET efficiency was calculated as shown in Equation 1FRET efficiency=(Dpost−Bpost)−(Dpre−Bpre)(Dpost−Bpost)(Eq. 1) where D is the donor channel intensity, B is the background intensity, and “pre” and “post” indicate measurements before and after photobleaching. A non-bleached area of the same cell served as an internal control.RESULTSCellular Localization of ADAR1 and ADAR2 Fluorescent Fusion Proteins—To examine both cellular localization and associations of ADAR1 and ADAR2 by fluorescence, we cloned the cDNA for human ADAR1 and ADAR2 into the mammalian expression vectors ECFP and EYFP to produce ADAR fusion proteins with N-terminal CFP and YFP under the control of a cytomegalovirus promoter. We also prepared expression constructs with CFP C-terminal to the deaminase sequence in order to be able to probe for directionality in any observed association.Previous studies have revealed a dynamic association of ADAR1 and ADAR2 with the nucleolus; ADAR2 shuttles between the nucleolus and the nucleoplasm, whereas ADAR1 shuttles between the nucleolus, nucleoplasm, and cytoplasm (an interferon-inducible form of ADAR1 is cytoplasmic) (35Sansam C.L. Wells K.S. Emeson R.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14018-14023Crossref PubMed Scopus (142) Google Scholar, 36George C.X. Samuel C.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4621-4626Crossref PubMed Scopus (221) Google Scholar, 37Yang J.H. Nie Y. Zhao Q. Su Y. Pypaert M. Su H. Rabinovici R. J. Biol. Chem. 2003; 278: 45833-45842Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We determined the localization of the N-terminal CFP ADAR fusion proteins in transfected HeLa cells by confocal fluorescence microscopy and observed, as expected, a predominantly nucleolar localization for these proteins (Fig. 1). Identical results were achieved using either C-terminal CFP or N-terminal YFP expression systems (data not shown).Homo- and Heterodimerization of ADAR1 and ADAR2—Having established the nucleolar localization of the fluorescently tagged ADAR1 and ADAR2, we performed co-transfections of HeLa cells using expression vectors for N-terminal CFP/YFP ADAR1 together and CFP/YFP ADAR2 together as well as control transfections with plasmids expressing CFP and YFP alone. A FRET experiment in this system involves the direct or indirect measurement, using fluorescence, of energy transfer between CFP and YFP following specific excitation of the CFP fluorophore. To simplify analysis, FRET signals were quantified by measurement of donor recovery following photobleaching. Briefly, CFP fluorescence was measured at 475 nm, following excitation at 458 nm with a laser, both before and after specific photobleaching of YFP. The increase in CFP fluorescence after YFP photobleaching corresponds to the original amount of energy transferred from CFP to YFP; thus, the difference in CFP fluorescence prior to and after photobleaching corresponds to the level of FRET. Using this method, we were not able to measure any FRET between CFP and YFP alone. However, when N-terminal CFP ADAR1 was excited in the presence of N-terminal YFP ADAR1, we measured a FRET efficiency, between the fusion proteins localized to the nucleolus, of 18% (Fig. 2, A and B; Table 1). For comparison, in a system expressing tandem CFP·YFP joined by a short 10-amino acid linker, FRET efficiencies of 35% were measured (data not shown). When we analyzed cells transfected with N-terminal CFP ADAR2 and N-terminal YFP ADAR2, we were able to measure FRET efficiencies of 19% within the nucleolus (Fig. 2, C and D; Table 1). Thus, as assayed by FRET, both ADAR1 and ADAR2 form homodimers in the nucleolus.FIGURE 2Homodimerization of ADAR1 and ADAR2. A, confocal fluorescence imaging of HeLa cells co-transfected with CFP·ADAR1 and YFP·ADAR1. Shown are emissions filtered at 462–484 nm (top, CFP), emissions filtered at 580–612 nm (middle, YFP), and merge (bottom). Images were taken before (left) and after (right) specifically photobleaching YFP within a nucleolus of the cell. Bar, 5 μm. B, quantification of images before and after photobleaching (indicated by arrow). CFP fluorescence is shown for the photobleached nucleolus (open circles) and a non-bleached nucleolus (closed circles). The fluorescence of YFP is shown for the same photobleached nucleolus (open squares) and non-bleached nucleolus (closed squares). Quantifications corresponding to images at left are indicated (solid diamond, broken diamond). C, experiment as in panel A but for cells co-transfected with CFP·ADAR2 and YFP·ADAR2. D, quantification as in panel B for images shown in panel C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1% CFP·ADAR-YFP·ADAR FRET in HeLa cells-RNase+RNaseCFP·ADAR118 ± 418 ± 3YFP·ADAR1CFP·ADAR219 ± 318 ± 3YFP·ADAR2CFP·ADAR212 ± 315 ± 2YFP·ADAR1 Open table in a new tab We repeated co-transfection experiments with vectors expressing N-terminal CFP ADAR1 or ADAR2 as well as C-terminal YFP ADAR1 or ADAR2. Because the FRET signal varies with r6 (38Förster T. Ann. Physik. 1948; 2: 55-75Crossref Scopus (4843) Google Scholar), we reasoned that measurements made with the fluorophores in different orientations might yield information on the disposition of the ADAR monomers with respect to one another. However, FRET levels measured using these expression constructs were essentially the same as observed in our initial experiment (data not shown). Thus, the FRET experiment does not reveal the orientation of individual ADAR proteins with respect to one another in the dimer.After determining that ADAR1 and ADAR2 form homodimers within the cell, we wanted to examine the possibility of heterodimerization between isoforms. We performed co-transfections in HeLa cells using N-terminal CFP ADAR2 and N-terminal YFP ADAR1. Both isoforms localized to identical nucleoli within the cells, and we were again able to measure a FRET signal between the two nucleolar-localized fusion proteins (Fig. 3; Table 1). The amount of FRET observed between ADAR1 and ADAR2 was 12%, which is consistent with the formation of ADAR heterodimers in the nucleolus. Again, no difference in FRET signal was measured when co-transfecting N-terminal CFP ADAR2 and C-terminal YFP ADAR1; therefore, an orientation of dimerization was impossible to determine.FIGURE 3Heterodimerization of ADAR1 and ADAR2. A, confocal images obtained from HeLa cells co-transfected with CFP·ADAR2 (top) and YFP·ADAR1 (middle). A merged image of the two top images is shown in the lower panels. Images were taken before (left) and after (right) specifically photobleaching YFP within a nucleolus of the cell. Bar, 5 μm. B, quantification of images before and after photobleaching (indicated by arrow). CFP fluorescence is shown for the photobleached nucleolus (open circles) and a nonbleached nucleolus (closed circles). The fluorescence of YFP is shown for the same photobleached nucleolus (open squares) and non-bleached nucleolus (closed squares). Quantifications corresponding to images at left are indicated (solid diamond, broken diamond).View Large Image Figure ViewerDownload Hi-res image Download (PPT)RNA Binding and ADAR Dimerization—One of the reasons for examining ADAR dimerization in living cells is the possibility of a dependence on RNA or other proteins for dimerization; in vitro studies of ADAR association are in disagreement in this regard (31Jaikaran D.C. Collins C.H. MacMillan A.M. J. Biol. Chem. 2002; 277: 37624-37629Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 32Cho D.S. Yang W. Lee J.T. Shiekhattar R. Murray J.M. Nishikura K. J. Biol. Chem. 2003; 278: 17093-17102Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 33Gallo A. Keegan L.P. Ring G.M. O'Connell M.A. EMBO J. 2003; 22: 3421-3430Crossref PubMed Scopus (96) Google Scholar). Indeed, studies of ADAR dimerization are complicated by the fact that the endogenous RNA substrates for ADAR1 and for ADAR2 in most cells are unknown. The results of our FRET experiments suggest that ADAR1 and ADAR2 form both homo- and heterodimers in the nucleolus; we also wished to examine whether disruption of RNA binding by ADAR1 or ADAR2 affected cellular localization and formation of homo- or heterodimers. Emeson and co-workers (35Sansam C.L. Wells K.S. Emeson R.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14018-14023Crossref PubMed Scopus (142) Google Scholar) previously noted that ADAR2 localization to the nucleolus was dependent on rRNA synthesis and was also abolished upon RNase treatment of cells. We therefore decided to examine the RNA dependence of ADAR self-association by RNase treatment of cells expressing the fluorescent fusion proteins.We repeated transient transfections, as described above, to yield cells expressing combinations of CFP/YFP ADAR1 and CFP/YFP ADAR2. Transfected cells were permeabilized and treated with a mixture of RNases before being examined to determine the cellular localization of the fusion proteins. Consistent with earlier studies (35Sansam C.L. Wells K.S. Emeson R.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14018-14023Crossref PubMed Scopus (142) Google Scholar), permeabilization of the cells did not affect ADAR localization, but subsequent treatment with RNase abolished the exclusive nucleolar localization of both fusion proteins that was now observed throughout the nucleus and to a small extent within the cytoplasm (Fig. 4, A and B; data not shown). In the case of RNase-treated cells co-expressing CFP ADAR2 and YFP ADAR1, although exclusive nucleolar localization was abolished the proteins remained co-localized (Fig. 4C).FIGURE 4RNA-independent homo- and heterodimerization of ADAR family members. A, confocal fluorescence imaging of HeLa cells co-transfected with CFP·ADAR1 and YFP·ADAR1 and then treated with RNase. Shown are emissions filtered at 462–484 nm (top, CFP), emissions filtered at 580–612 nm (middle, YFP), and merge (bottom). Images were taken before (left) and after (right) specifically photobleaching YFP within a region of the cell nucleus. Bar, 5 μm. B, quantification of images before and after photobleaching (indicated by arrow). CFP fluorescence is shown for the photobleached area (open circles) and a nonbleached area (closed circles). The fluorescence of YFP is shown for the same photobleached area (open squares) and non-bleached area (closed squares). Quantifications corresponding to images at left are indicated (filled diamond, broken diamond). C, experiment as in panel A but for cells co-transfected with CFP·ADAR2 and YFP·ADAR2. D, quantification as in panel B for images shown in panel C. E, experiment as in panel A but for cells co-transfected with CFP·ADAR2 and YFP·ADAR1. F, quantification as in panel B for images shown in panel E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next performed a series of FRET experiments on RNase-treated cells in which we measured donor recovery following photobleaching of YFP in extra-nucleolar regions of the cell. FRET efficiencies similar to those observed in untreated cells were measured for ADAR1-, ADAR2-, and ADAR1/ADAR2-expressing cells (Fig. 4; Table 1). These results suggest that neither homonor heterodimerization of ADAR1/ADAR2 is dependent on RNA binding.A recent paper by Bass and co-workers (39Macbeth M.R. Schubert H.L. Vandemark A.P. Lingam A.T. Hill C.P. Bass B.L. Science. 2005; 309: 1534-1539Crossref PubMed Scopus (312) Google Scholar) reports the crystal structure of the deaminase domain of ADAR2 and suggests that dimerization is not mediated through this domain. We expressed CFP and YFP fusion proteins of the same deaminase construct (ADAR2-(299–701)) used in the structural studies in HeLa cells, determined their localization, and looked for evidence of FRET between the fluorophores. The deaminase fusion proteins did not localize to the nucleolus (instead showing a diffuse expression throughout the cell) and did not undergo FRET (data not shown). This supports the conclusion that dimerization is not mediated by the deaminase domain and previous suggestions from work with Drosophila ADAR that ADAR self-association is mediated by N-terminal regions of the protein (33Gallo A. Keegan L.P. Ring G.M. O'Connell M.A. EMBO J. 2003; 22: 3421-3430Crossref PubMed Scopus (96) Google Scholar).DISCUSSIONWe have examined the localization and association of human ADAR1 and ADAR2 in HeLa cell culture by transient expression of fluorescent fusion proteins followed by confocal fluorescence microscopy. Our observations are in agreement with the previously reported primary localization of both ADAR1 and ADAR2 to the nucleolus. The FRET studies reported here also demonstrate that nucleolar ADAR1 and ADAR2 form both homo- and heterodimers. The observation of ADAR homodimerization is consistent with previous in vitro studies (31Jaikaran D.C. Collins C.H. MacMillan A.M. J. Biol. Chem. 2002; 277: 37624-37629Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 32Cho D.S. Yang W. Lee J.T. Shiekhattar R. Murray J.M. Nishikura K. J. Biol. Chem. 2003; 278: 17093-17102Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 33Gallo A. Keegan L.P. Ring G.M. O'Connell M.A. EMBO J. 2003; 22: 3421-3430Crossref PubMed Scopus (96) Google Scholar); heterodimerization has not been previously observed. To examine the RNA dependence of ADAR localization and dimerization, we treated cells expressing the fluorescent fusions with RNase; this had the effect of abolishing the exclusive nucleolar localization of both proteins but did not affect FRET between them. Thus, as observed by Emeson and co-workers (35Sansam C.L. Wells" @default.
• W1989656471 created "2016-06-24" @default.
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• W1989656471 creator A5012876075 @default.
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• W1989656471 date "2006-06-01" @default.
• W1989656471 modified "2023-09-26" @default.
• W1989656471 title "FRET Analysis of in Vivo Dimerization by RNA-editing Enzymes" @default.
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