Subcellular Distribution of ADAR1 Isoforms Is Synergistically Determined by Three Nuclear Discrimination Signals and a Regulatory Motifстатья из журнала
Аннотация: ADAR1 is an RNA-specific adenosine deaminase that edits RNA sequences. We have demonstrated previously that different ADAR1 isoforms are induced during acute inflammation. Here we show that the mouse ADAR1 isoforms are differentially localized in cellular compartments and that their localization is controlled by several independent signals. Nuclear import of the full-length ADAR1 is predominantly regulated by a nuclear localization signal at the C terminus (NLS-c), which consists of a bipartite basic amino acid motif plus the last 39 residues of ADAR1. Deletion of the NLS-c causes the truncated ADAR1 protein to be retained in the cytoplasm. The addition of this sequence to pyruvate kinase causes the cytoplasmic protein to be localized within the nucleus. The localization of nuclear ADAR1 is determined by a dynamic balance between the nucleolar binding activity of the nucleolar localization signal (NoLS) in the middle of the protein and the exporting activity of the nuclear exporter signal (NES) near the N terminus. The NoLS consists of a typical monopartite cluster of basic residues followed by the third double-stranded RNA-binding domain. These signals act independently; however, NES function can be completely silenced by the NLS-c when a regulatory motif within the catalytic domain and the NoLS are deleted. Thus, the intracellular distribution of the various ADAR1 isoforms is determined by NLS-c, NES, NoLS, and a regulatory motif. ADAR1 is an RNA-specific adenosine deaminase that edits RNA sequences. We have demonstrated previously that different ADAR1 isoforms are induced during acute inflammation. Here we show that the mouse ADAR1 isoforms are differentially localized in cellular compartments and that their localization is controlled by several independent signals. Nuclear import of the full-length ADAR1 is predominantly regulated by a nuclear localization signal at the C terminus (NLS-c), which consists of a bipartite basic amino acid motif plus the last 39 residues of ADAR1. Deletion of the NLS-c causes the truncated ADAR1 protein to be retained in the cytoplasm. The addition of this sequence to pyruvate kinase causes the cytoplasmic protein to be localized within the nucleus. The localization of nuclear ADAR1 is determined by a dynamic balance between the nucleolar binding activity of the nucleolar localization signal (NoLS) in the middle of the protein and the exporting activity of the nuclear exporter signal (NES) near the N terminus. The NoLS consists of a typical monopartite cluster of basic residues followed by the third double-stranded RNA-binding domain. These signals act independently; however, NES function can be completely silenced by the NLS-c when a regulatory motif within the catalytic domain and the NoLS are deleted. Thus, the intracellular distribution of the various ADAR1 isoforms is determined by NLS-c, NES, NoLS, and a regulatory motif. ADAR1 deaminates adenosines in mRNA, thereby altering codons and giving rise to different protein isoforms (1Yang J.H. Sklar P. Axel R. Maniatis T. Nature. 1995; 374: 77-81Crossref PubMed Scopus (115) Google Scholar, 2Yang J.H. Sklar P. Axel R. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4354-4359Crossref PubMed Scopus (41) Google Scholar). This deaminase activity was first identified in Xenopus embryos, which unwind dsRNA 1The abbreviations used are: dsRNA, double-stranded RNA; dsRBD, dsRNA-binding domain; ADAR, adenosine deaminases acting on RNA; NLS, nuclear localization signal; NLS-c, NLS at the C terminus; NoLS, nucleolar localization signal; NES, nuclear exporter signal; cNES, leucine-rich nuclear export consensus; LMB, leptomycin B; GFP, green fluorescent protein; EGFP, enhanced GFP; PK, pyruvate kinase; m, mouse. 1The abbreviations used are: dsRNA, double-stranded RNA; dsRBD, dsRNA-binding domain; ADAR, adenosine deaminases acting on RNA; NLS, nuclear localization signal; NLS-c, NLS at the C terminus; NoLS, nucleolar localization signal; NES, nuclear exporter signal; cNES, leucine-rich nuclear export consensus; LMB, leptomycin B; GFP, green fluorescent protein; EGFP, enhanced GFP; PK, pyruvate kinase; m, mouse. by converting adenosines to inosines (3Bass B.L. Weintraub H. Cell. 1988; 55: 1089-1098Abstract Full Text PDF PubMed Scopus (520) Google Scholar, 4Rebagliati M.R. Melton D.A. Cell. 1987; 48: 599-605Abstract Full Text PDF PubMed Scopus (223) Google Scholar). A family of A-to-I RNA editing enzymes exists in mammals (5Chen C.X. Cho D.S. Wang Q. Lai F. Carter K.C. Nishikura K. RNA (N. Y.). 2000; 6: 755-767Crossref PubMed Scopus (366) Google Scholar, 6Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (367) Google Scholar, 7Maas S. Gerber A.P. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8895-8900Crossref PubMed Scopus (70) Google Scholar, 8Melcher 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 (239) Google Scholar, 9Melcher T. Maas S. Herb A. Sprengel R. Seeburg P.H. Higuchi M. Nature. 1996; 379: 460-464Crossref PubMed Scopus (429) Google Scholar) with homologues in Drosophila (10Palladino M.J. Keegan L.P. O'Connell M.A. Reenan R.A. RNA (N. Y.). 2000; 6: 1004-1018Crossref PubMed Scopus (139) Google Scholar), zebrafish (11Slavov D. Clark M. Gardiner K. Gene (Amst.). 2000; 250: 41-51Crossref PubMed Scopus (22) Google Scholar), and Xenopus (12Bass B.L. Hurst S.R. Singer J.D. Curr. Biol. 1994; 4: 301-314Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), suggesting that these enzymes are evolutionarily conserved. ADAR1 is ubiquitously expressed in a variety of cells and tissues (6Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (367) Google Scholar, 8Melcher 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 (239) Google Scholar) and may participate in host defense by targeting viral RNA (13Lei M. Liu Y. Samuel C.E. Virology. 1998; 245: 188-196Crossref PubMed Scopus (47) Google Scholar). It was recently shown that ADAR1 participates in the development of acute inflammation (14Yang 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 (104) Google Scholar, 15Rabinovici R. Kabir K. Chen M. Su Y. Zhang D. Luo X. Yang J.H. Circ. Res. 2001; 88: 1066-1071Crossref PubMed Scopus (42) Google Scholar). ADAR1 knock-out mice die as embryos with immature erythrocytes (16Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (339) Google Scholar), suggesting that ADAR1-mediated RNA editing is critical for normal proliferation and/or differentiation of erythrocytes during development. The sequences of ADAR1 from human, rat, and mouse are highly conserved; a Z-DNA-binding motif, three dsRNA-binding domains (dsRBDs), and a conserved deaminase domain have been identified. Full-length human ADAR1 protein is found in the cytoplasm; it is initially transported into the nucleus and then re-exported by a nuclear export signal (NES) near the N-terminal region (17Poulsen H. Nilsson J. Damgaard C.K. Egebjerg J. Kjems J. Mol. Cell. Biol. 2001; 21: 7862-7871Crossref PubMed Scopus (116) Google Scholar). An atypical nuclear localization signal (NLS) with nuclear import activity occurs within dsRBDIII so that human ADAR1 displays the characteristics of a shuttling protein (18Eckmann C.R. Neunteufl A. Pfaffstetter L. Jantsch M.F. Mol. Biol. Cell. 2001; 12: 1911-1924Crossref PubMed Scopus (87) Google Scholar, 19Strehblow A. Hallegger M. Jantsch M.F. Mol. Biol. Cell. 2002; 13: 3822-3835Crossref PubMed Scopus (89) Google Scholar). A different NLS within the N-terminal 269 amino acids is reported to direct active import of human ADAR1 (17Poulsen H. Nilsson J. Damgaard C.K. Egebjerg J. Kjems J. Mol. Cell. Biol. 2001; 21: 7862-7871Crossref PubMed Scopus (116) Google Scholar). In addition, a human ADAR1 fragment lacking the NES has been shown to localize predominantly within the nucleus (19Strehblow A. Hallegger M. Jantsch M.F. Mol. Biol. Cell. 2002; 13: 3822-3835Crossref PubMed Scopus (89) Google Scholar). A similar fragment of human ADAR1 was found exclusively in the nucleus, implicating another NLS at the C-terminal end of human ADAR1 (17Poulsen H. Nilsson J. Damgaard C.K. Egebjerg J. Kjems J. Mol. Cell. Biol. 2001; 21: 7862-7871Crossref PubMed Scopus (116) Google Scholar). The existence of a nucleolar localization signal (NoLS) further complicates the localization of ADAR1. The NoLS is capable of targeting ADAR1 to the nucleolus where rRNA interacts with imported ribosomal proteins to form preribosomal particles (20Melese T. Xue Z. Curr. Opin. Cell Biol. 1995; 7: 319-324Crossref PubMed Scopus (240) Google Scholar). Nucleolar localization of ADARs may suggest that RNA editing occurs during transcription or RNA processing in the nucleolus. Alternatively, it may regulate protein synthesis (21Herbert A. Wagner S. Nickerson J.A. Mol. Cell. 2002; 10: 1235-1246Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Furthermore, the Xenopus ADAR1 contains a distinctive NLS downstream from the Z-DNA-binding motif, which targets the enzyme to the nascent ribonucleoprotein matrix on lampbrush chromosomes, where it is specifically associated with active transcriptional sites (22Doyle M. Jantsch M.F. J. Cell Biol. 2003; 161: 309-319Crossref PubMed Scopus (20) Google Scholar, 23Eckmann C.R. Jantsch M.F. J. Cell Biol. 1999; 144: 603-615Crossref PubMed Scopus (31) Google Scholar). This observation suggests that RNA editing by Xenopus ADAR1 is coupled with transcriptional events or targets newly synthesized RNAs. Since human ADAR1 contains an NES that has not been identified in Xenopus ADAR1, this protein may function differently in mammalian cells. Nevertheless, it is likely that RNA editing by ADAR1 is dynamically regulated in different cellular compartments by these localization signals. Certain patterns of basic amino acid residues are necessary for proteins to be imported into the nucleus, although no precise consensus amino acid sequence has been identified (24Nigg E.A. Baeuerle P.A. Luhrmann R. Cell. 1991; 66: 15-22Abstract Full Text PDF PubMed Scopus (165) Google Scholar). Many variable NLS sequences have been identified in viral and cellular proteins; these sequences are classified as monopartite or bipartite (25Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1242) Google Scholar, 26Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1708) Google Scholar). The typical monopartite NLS is a cluster of basic residues starting with Pro and followed by 5 residues, of which at least 3 are either Lys or Arg. The bipartite pattern (e.g. KRXKKXXKX) begins with 2 basic residues (Lys or Arg) followed by a 10-residue spacer and finally a cluster in which at least 3 out of 5 residues are Lys or Arg (25Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1242) Google Scholar). Proteins containing these signals are transported into the nucleus by ATP-dependent translocation through the nuclear pore complexes (27Newmeyer D.D. Forbes D.J. Cell. 1988; 52: 641-653Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 28Richardson W.D. Mills A.D. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1988; 52: 655-664Abstract Full Text PDF PubMed Scopus (372) Google Scholar). A number of NLS-binding proteins have been reported to function as cytoplasmic receptors that deliver karyophilic proteins to the transport machinery of nuclear pore complexes. No precise NoLS consensus amino acid sequence has been identified, although NoLS signals usually reside within an NLS sequence. An atypical NoLS in human immunodeficiency virus type 1 contains two stretches of basic amino acids (GRKKRRQRRRAHQN, basic residues are underlined) to target the Tat protein to the nucleolus (29Siomi H. Shida H. Maki M. Hatanaka M. J. Virol. 1990; 64: 1803-1807Crossref PubMed Google Scholar). We have shown previously that multiple short versions of ADAR1 are induced during acute inflammation (30Yang 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 (45) Google Scholar). Deletions often occur in these inducible isoforms within the regions that are important for nuclear export or import, suggesting that the intracellular localization of ADAR1 may be differentially regulated under various pathological conditions. Here we show that localization of the ADAR1 isoforms is regulated by several nuclear discrimination signals. Furthermore, nuclear import of the full-length ADAR1 is predominantly determined by an essential NLS within the last 56 residues of the C terminus. A regulatory motif may be required for nuclear export of the NES, and this motif can be completely silenced by the NLS. Finally, we show that a signal near the dsRBDIII mediates nucleolar recruitment of the short version of ADAR1. Western Blotting—Samples containing 60 μg of total protein derived from thymocytes of BALB/c mice (Jackson Laboratories) were resolved on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. ADAR1 immunoreactivity was detected using rabbit antiserum raised against the C terminus of recombinant mouse ADAR1 expressed in Escherichia coli (2765-3459, AF291050) or against the N terminus of a synthetic mouse ADAR1 peptide (Santa Cruz Biotechnology). Construction of ADAR1 Chimeras—Different mADAR1 fragments were generated by PCR using the pCRII-ADAR1Lb plasmid (AF291050) as a template. A BamHI sequence was added to each end of the amplified fragments. The 5′-primers included a start codon with a KoZak consensus sequence ((A/G)NNATGG) (31Louis B.G. Ganoza M.C. Mol. Biol. Rep. 1988; 13: 103-115Crossref PubMed Scopus (7) Google Scholar). The stop codon in the 3′-primer was removed to create an in-frame fusion to enhanced green fluorescent protein (EGFP). The primer sequences are shown in Table I. PCR products were then digested with BamHI and directly cloned into pEGFP-N1 or pEGFP-N2 vectors (Clontech) at the N-terminal end of EGFP. For the ΔNLS and NoLS-I constructs, fragments were excised from the mADAR1 cDNA with the following restriction enzymes: HindIII or XbaI/HindIII, respectively. The resulting inserts were then ligated into the pEGFP-N1 or pEGFP-N2 vector. Positive clones were identified by BamHI digestion, and the reading frames were confirmed by sequencing. ADAR1-EGFP DNAs with the correct sequences were transfected into mouse fibroblasts (3T3) or other cell lines, and the expression of protein chimeras was confirmed by the presence of green signals under fluorescence or confocal microscopy.Table IPrimers used for construction of ADAR1-EGFP chimerasName5′ primer3′ primerADAR1atcgggatccactatggctcaagggttcaggggacccgtggatccagtcattgggtactggacagaggΔNES-IgtggatccagtcattgggtactggacagaggaactcgagccaagcaggacgcagcagtgaaagccΔNES-IIaaactcgagctatggctcccaacaagatcagggtggatccagtcattgggtactggacagaggNESgaattctcgagaccatggggctttgctcacacttccgggagcgcggatccaaaggctccacaaaggaggtttcccNoLSaaactcgagctatggctcccaacaagatcaggtggatcccagacttgtcatccgctgaactggcNLS-IaaactcgagctatggctcccaacaagatcagggtggatccagtcattgggtactggacagaggNLS-cactcgagactatggagttgtctcgggtgtccaaggtggatccagtcattgggtactggacagaggNLS-IIIgaattctcgagaccatggcccgcagagatttactgcagctctcttatggtgaacgcggatccagtcacgggcagctttcttggcttcaccataagagagctgEN3229gaattcggatccgagttgtctcgggtgtccaagtggatccagtcattgggtactggacagaggEN2983gaattcggatccaccttttcagccaagggcatctgtggatccagtcattgggtactggacagaggEN2741gaattcggatccaccctgtctttgaaaatcccaagtggatccagtcattgggtactggacagaggEN2413gaattcggatccacatgggtgttgtcgtgagtttgtggatccagtcattgggtactggacagaggEN1975gaattcggatccatggctcccaacaagatcaggagtggatccagtcattgggtactggacagaggEO2414gaattcggatccccaacaagatcaggaggattggtgaattcggatcccctctggatctcttttcataaEO2263gaattcggatccccaacaagatcaggaggattggttggatccaacccaactgctctgccttctcgct Open table in a new tab Two chimeric pyruvate kinase constructs, Myc-PK-NLS and Myc-PK-NoLS-NLS, were generated to test the function of NLS and NoLS. The NLS fragment, which consists of the last 56 residues of ADAR1, was amplified by PCR to add EcoRI and XhoI sites at its 5′- and 3′-ends, respectively. This fragment was then ligated into the EcoRI and XhoI sites of Myc-PK pcDNA3 (a kind gift from Dr. M. Matunis, The Johns Hopkins University) (32Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (377) Google Scholar). The NoLS fragment (including the P-cluster and dsRBDIII) was amplified to add an EcoRI site at both ends; this fragment was ligated into the EcoRI site of Myc-PK-NLS pcDNA3. These constructs were then transfected into 3T3 cells, and localization of the chimeric proteins was evaluated by immunofluorescence with an anti-Myc antibody. Construction of Bifunctional Chimeras—The NES fragment (385-603, GenBank™ accession number AF291050), which contains the leucine-rich nuclear export consensus (cNES) consensus as well as the N-terminal bipartite signal (Bipartite N) and the Z-DNA-binding domain (Zα), was amplified by PCR using the pCRII-ADAR1Lb plasmid as a template. XhoI and BamHI sites were added to the primers to generate restriction enzyme sites at the 5′- or 3′-ends, respectively. The amplified fragment was then subcloned into the BamHI site of the pEGFP-N1 vector to generate the pEGFP-NES construct. Various NLS-c fragments, extending from 3229, 2983, 2741, 2414, or 1975 to the stop codon of mouse ADAR1, were amplified by PCR using the same template. The stop codon was replaced with TGG to generate chimeras in-frame with EGFP. A BamHI was added to each end of the amplified fragment, and the insert was then ligated into the unique BamHI site of the pEFGP-NES construct to generate pEGFP-NES-NLS bifunctional chimeras. Similarly, the NoLS fragments were amplified from 1983 to 2414 or 1983 to 2263 of mouse ADAR1 and inserted into the pEFGP-NES construct to generate pEGFP-NES-NoLS bifunctional chimeras. After transformation and purification, clones with inserts were selected by restriction enzyme digestion, and the reading frames were confirmed by DNA sequencing. Cell Culture and Transfection—Mouse fibroblasts (3T3), mouse neuroblastoma cells (N18), human HeLa, or embryonic kidney 293 cells were cultured to the logarithmic stage in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Approximately 2 × 105 cells were placed in each well of 24-well plates and cultured overnight in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. In a typical transfection, 2 μg of the pEGFP-ADAR1 chimeric construct was mixed with 3 μl of LipofectAMINE 2000 (Invitrogen) in 0.5 ml of serum-free Opti-MEM, according to the manufacturer's recommended protocol. After incubation at 37 °C for 5 h, the cells were washed and recultured with fresh Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for 12-24 h. Green fluorescence usually appeared 7-9 h after transfection; photographs were taken 12-24 h after transfection using a fluorescence or confocal microscope (Zeiss). Blocking CRM1-mediated Nuclear Export by Leptomycin B—To block nuclear export with leptomycin B (LMB, a kind gift of Minoru Yoshida, University of Tokyo), 3T3, N18, or 293 cells (2 × 105 cells/well) were incubated for 5-6 h with 2 μg of pEGFP-ADAR1 DNA and 6 μl of LipofectAMINE 2000 (Invitrogen), as described above. After washing, the transfected cells were cultured in appropriate medium in either the presence or the absence of LMB (1 ng/ml). Green fluorescence was examined 12-24 h after transfection. ADAR1 Variants Truncated at the N and C Termini Are Differentially Localized—We have shown that two major short forms of mouse ADAR1 truncated from the N-terminal (mADAR1 p100 and p80) are expressed in spleen and thymus during endotoxin-induced acute inflammation (30Yang 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 (45) Google Scholar). Using an antibody directed against the N-terminal region of ADAR1, we also detected short isoforms truncated from the C-terminal (Fig. 1). The anti-C-terminal antibody detected full-length ADAR1 and isoforms truncated from the N terminus; the anti-N-terminal antibody detected full-length ADAR1 and two isoforms truncated from the C terminus. We reasoned that the isoforms derived from truncation of the N and C termini might be localized in different cellular compartments. mADAR1 chimeras containing fragments truncated from the N or C terminus of mADAR1 and fused to EGFP (Fig. 2, ΔNES and ΔNLS) were transiently expressed in various cell lines, and their localization was determined by fluorescence microscopy. Consistent with previous studies, full-length mADAR1 was found in the cytoplasm (Fig. 2A) (18Eckmann C.R. Neunteufl A. Pfaffstetter L. Jantsch M.F. Mol. Biol. Cell. 2001; 12: 1911-1924Crossref PubMed Scopus (87) Google Scholar, 30Yang 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 (45) Google Scholar), whereas the ADAR1 fragment from which the N-terminal region was deleted was localized in the cytoplasm and nucleolus (Fig. 2B, ΔNES) (17Poulsen H. Nilsson J. Damgaard C.K. Egebjerg J. Kjems J. Mol. Cell. Biol. 2001; 21: 7862-7871Crossref PubMed Scopus (116) Google Scholar, 30Yang 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 (45) Google Scholar, 33Desterro J.M. Keegan L.P. Lafarga M. Berciano M.T. O'Connell M. Carmo-Fonseca M. J. Cell Sci. 2003; 116: 1805-1818Crossref PubMed Scopus (194) Google Scholar). LMB, an antagonist of the nuclear receptor exportin CRM1 (34Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Crossref PubMed Scopus (620) Google Scholar, 35Fukuda M. Asano S. Nakamura T. Adachi M. Yoshida M. Yanagida M. Nishida E. Nature. 1997; 390: 308-311Crossref PubMed Scopus (1022) Google Scholar, 36Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1734) Google Scholar), at a concentration of 1 ng/ml blocked export and resulted in accumulation of full-length ADAR1 within the nucleus (Fig. 2A, +LMB). The fragment lacking the C-terminal region was found within the cytoplasm (Fig. 2C, ΔNLS); however, LMB did not cause ΔNLS to accumulate in the nucleus, even at concentrations up to 10 ng/ml (Fig. 2C, +LMB). The ADAR1 variant ΔNLS, in which the C terminus was truncated, was retained in the cytoplasm after translation without nuclear importation; deletion of the last 56 amino acid residues prevented mADAR1 from being transported into the nucleus. Nuclear Discrimination Signals Are Conserved in Mammalian ADAR1—Since NLS activity was also observed near the third dsRBD of human ADAR1 (18Eckmann C.R. Neunteufl A. Pfaffstetter L. Jantsch M.F. Mol. Biol. Cell. 2001; 12: 1911-1924Crossref PubMed Scopus (87) Google Scholar, 19Strehblow A. Hallegger M. Jantsch M.F. Mol. Biol. Cell. 2002; 13: 3822-3835Crossref PubMed Scopus (89) Google Scholar), we reasoned that multiple signals might exist within mADAR1 that function synergistically in regulating nuclear importation. Using Reinhardt's method (PSORT, bioweb.pasteur.fr/seqanal/interfaces/psort2), we identified three putative nuclear discrimination signals that are conserved in the human, rat, and mouse ADAR1 sequences (Fig. 3). Two of these are located in the N- and C-terminal regions, with the sequence KRDINRILYSLEKKGKL located at position 171 and the sequence RRDLLQLSYGEAKKAAR at position 1097, respectively. Both follow the bipartite pattern (25Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1242) Google Scholar). The N-terminal bipartite signal (Bipartite N) is located within the Z-DNA-binding domain (Zα) and is adjacent to a cNES; this region was shown to mediate nuclear export of ADAR1 (17Poulsen H. Nilsson J. Damgaard C.K. Egebjerg J. Kjems J. Mol. Cell. Biol. 2001; 21: 7862-7871Crossref PubMed Scopus (116) Google Scholar, 18Eckmann C.R. Neunteufl A. Pfaffstetter L. Jantsch M.F. Mol. Biol. Cell. 2001; 12: 1911-1924Crossref PubMed Scopus (87) Google Scholar, 19Strehblow A. Hallegger M. Jantsch M.F. Mol. Biol. Cell. 2002; 13: 3822-3835Crossref PubMed Scopus (89) Google Scholar). The C-terminal bipartite signal (Bipartite C) is found in the adenosine deaminase domain, within the last 56 residues of ADAR1. A similar region was shown to be important for nuclear translation (21Herbert A. Wagner S. Nickerson J.A. Mol. Cell. 2002; 10: 1235-1246Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), which we show to be involved in nuclear import. The third signal (P-cluster) is a typical monopartite cluster of basic residues, with a conserved pattern starting with Pro, followed by Lys or Arg in 3 out of 5 residues (PNKIRRI) (37Hicks G.R. Raikhel N.V. Annu. Rev. Cell Dev. Biol. 1995; 11: 155-188Crossref PubMed Scopus (259) Google Scholar). The P-cluster is adjacent to the third dsRNA-binding domain (dsRBDIII), which has been shown previously to be necessary for nuclear import of human ADAR1 (18Eckmann C.R. Neunteufl A. Pfaffstetter L. Jantsch M.F. Mol. Biol. Cell. 2001; 12: 1911-1924Crossref PubMed Scopus (87) Google Scholar, 19Strehblow A. Hallegger M. Jantsch M.F. Mol. Biol. Cell. 2002; 13: 3822-3835Crossref PubMed Scopus (89) Google Scholar, 33Desterro J.M. Keegan L.P. Lafarga M. Berciano M.T. O'Connell M. Carmo-Fonseca M. J. Cell Sci. 2003; 116: 1805-1818Crossref PubMed Scopus (194) Google Scholar), and which we show in this study is also important for nucleolar localization. These consensus sequences are not found in ADAR sequences from Xenopus, Drosophila, Caenorhabditis elegans, or fishes, indicating that the intracellular localization of ADAR1 may be uniquely regulated in mammals, as compared with other taxa. The Last 56 Residues Containing the Bipartite C Domain Predominantly Control Nuclear Importation of Mouse ADAR1—We examined the function of Bipartite C by constructing N-terminal deletions of mADAR1. In agreement with our previous findings, fragments without Bipartite N were consistently transported into the nucleus and bound to the nucleolus (Fig. 4A); fragments lacking Bipartite N and all dsRNA-binding domains were still found in the nucleus at large (Fig. 4B). This suggests that Bipartite C is functionally sufficient for nuclear accumulation of ADAR1. To identify the essential residues for nuclear localization, a fragment containing Bipartite C followed by 39 residues at the C-terminal end of ADAR1 was linked to EGFP. In the same cells, this chimera was found in the nucleus (Fig. 4C). However, the nucleolar localization was not observed when Bipartite C and its N-terminal sequences were completely removed (Fig. 4D). Thus, the sequence containing Bipartite C followed by 39 residues at the C-terminal end of ADAR1, termed NLS-c, is an independent and fully functional signal for nuclear localization. To ascertain whether the nuclear localization activity of NLS-c was mediated by nuclear import rather than nuclear retention, we fused the NLS-c fragment to pyruvate kinase (PK), a well characterized cytoplasmic protein (19Strehblow A. Hallegger M. Jantsch M.F. Mol. Biol. Cell. 2002; 13: 3822-3835Crossref PubMed Scopus (89) Google Scholar, 32Matunis M.J. Wu J. Blobel G. J. Cell Biol. 1998; 140: 499-509Crossref PubMed Scopus (377) Google Scholar). As shown in Fig. 5, PK-NLS-c-Myc was localized within the nucleus, whereas the PK-Myc control was detected in the cytoplasm. The NLS-c Interacts with NES—Since the final localization of ADAR1 may be dynamically balanced by nuclear exportation and importation, we constructed a series of bifunctional ADAR chimeras containing both the NLS-c and NES sequences. An NES fragment containing cNES and Bipartite N (385-603 bp) was linked to a C-terminal fragment containing the minimum NLS-c and followed by GFP. The NES fragment consistently formed a fully functional CRM1-mediated exporter that was exclusively localized in the cytoplasm and accumulated in the nucleus and nucleolus when nuclear exportation was blocked by LMB (Fig. 6, NES). However, the chimeras containing both NES and NLS-c were found within the nucleus (Fig. 6, EN3229 and EN2983). The addition of LMB did not affect the nuclear localization of the EN3229 and EN2983 constructs. Thus, the NLS-c completely masked the function of CRM1-mediated nuclear exportation through an as yet unknown mechanism. Nevertheless, we demonstrated that nuclear exportation of the NES fragment was restored when the NLS-c was further extended to the catalytic domain (Fig. 6, EN2741 and EN2413). These data suggest that a regulatory motif within the catalytic domain (804-994) is involved in the interaction between the NES and the NLS-c. Since the fragment EN2741 lacking the leucine-zipper-like dimerization domain (19Strehblow A. Hallegger M. Jantsch M.F. Mol. Biol. Cell. 2002; 13: 3822-3835Crossref PubMed Scopus (89) Google Scholar) was still predominantly exported to the cytoplasm or accumulated in the nucleus when LMB was added, the regulatory motif is more likely localized between the residues from 914 to 994. The P-cluster and dsRBDIII Provide a Fully Functional Nucleolar Localization Signal—The NLS-c was further extended to include the P-cluster and the dsRBDIII in NES-NLS-c constructs (Fig. 6, EN1975). The resu
Год издания: 2004
Авторы: Yongzhan Nie, Qingchuan Zhao, Yingjun Su, Jing-Hua Yang
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: RNA regulation and disease, RNA Research and Splicing, Viral Infections and Immunology Research
Другие ссылки: Journal of Biological Chemistry (PDF)
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Открытый доступ: hybrid
Том: 279
Выпуск: 13
Страницы: 13249–13255