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• W2123657381 abstract "Here we provide evidence for an interaction-dependent subnuclear trafficking of the human La (hLa) protein, known as transient interaction partner of a variety of RNAs. Among these, precursor transcripts of certain RNAs are located in the nucleoplasm or nucleolus. Here we examined which functional domains of hLa are involved in its nuclear trafficking. By using green fluorescent-hLa fusion proteins, we discovered a nucleolar localization signal and demonstrated its functionality in a heterologous context. In addition, we revealed that the RRM2 motif of hLa is essential both for its RNA binding competence in vitro and in vivo and its exit from the nucleolus. Our data imply that hLa traffics between different subnuclear compartments, which depend decisively on a functional nucleolar localization signal as well as on RNA binding. Directed trafficking of hLa is fully consistent with its function in the maturation of precursor RNAs located in different subnuclear compartments. Here we provide evidence for an interaction-dependent subnuclear trafficking of the human La (hLa) protein, known as transient interaction partner of a variety of RNAs. Among these, precursor transcripts of certain RNAs are located in the nucleoplasm or nucleolus. Here we examined which functional domains of hLa are involved in its nuclear trafficking. By using green fluorescent-hLa fusion proteins, we discovered a nucleolar localization signal and demonstrated its functionality in a heterologous context. In addition, we revealed that the RRM2 motif of hLa is essential both for its RNA binding competence in vitro and in vivo and its exit from the nucleolus. Our data imply that hLa traffics between different subnuclear compartments, which depend decisively on a functional nucleolar localization signal as well as on RNA binding. Directed trafficking of hLa is fully consistent with its function in the maturation of precursor RNAs located in different subnuclear compartments. The human La protein (hLa) 1The abbreviations used are: hLa, human La autoantigen; FRAP, fluorescence recovery after photobleaching analysis; GFP, green fluorescent protein; EGFP, enhanced GFP; RNP, ribonucleoprotein particles; RRM, RNA recognition motif; WAM, Walker-A-motif; WT, wild type; NoLS, nucleolar localization signal; snoRNA, small nucleolar RNA; snRNA, small nuclear RNA; PBS, phosphate-buffered saline; DAPI, 4,6-diamidino-2-phenylindole. is a 47-kDa phosphoprotein predominantly localized in the nucleus. It was first discovered as an autoantigen recognized by antibodies present in the sera of patients suffering from systemic lupus erythematosus and Sjogren's syndrome (1Mattioli M. Reichlin M. Arthritis Rheum. 1974; 17: 421-429Crossref PubMed Scopus (217) Google Scholar, 2Alspaugh M.A. Talal N. Tan E.M. Arthritis Rheum. 1976; 19: 216-222Crossref PubMed Scopus (238) Google Scholar). The La protein is a member of a large group of RNA-binding proteins containing RNA recognition motifs (RRMs) (3Chan E.K. Tan E.M. Mol. Cell. Biol. 1987; 7: 2588-2591Crossref PubMed Scopus (26) Google Scholar, 4Chambers J.C. Kenan D. Martin B.J. Keene J.D. J. Biol. Chem. 1988; 263: 18043-18051Abstract Full Text PDF PubMed Google Scholar, 5Chan E.K. Sullivan K.F. Tan E.M. Nucleic Acids Res. 1989; 17: 2233-2244Crossref PubMed Scopus (80) Google Scholar, 6Birney E. Kumar S. Krainer A.R. Nucleic Acids Res. 1993; 21: 5803-5816Crossref PubMed Scopus (589) Google Scholar, 7Query C.C. Bentley R.C. Keene J.D. Cell. 1989; 57: 89-101Abstract Full Text PDF PubMed Scopus (442) Google Scholar) and interacts with a variety of small RNAs (for reviews see Refs. 8Maraia R.J. Intine R.V. Mol. Cell. Biol. 2001; 21: 367-379Crossref PubMed Scopus (108) Google Scholar, 9Maraia R.J. Intine R.V. Gene Expr. 2002; 10: 41-57PubMed Google Scholar, 10Wolin S.L. Cedervall T. Annu. Rev. Biochem. 2002; 71: 375-403Crossref PubMed Scopus (339) Google Scholar). The La protein is implicated in several aspects of the RNA metabolism, including processing of precursors of tRNAs, U3 snoRNA, U1 RNA, U6 snRNA (11Fan H. Goodier J.L. Chamberlain J.R. Engelke D.R. Maraia R.J. Mol. Cell. Biol. 1998; 18: 3201-3211Crossref PubMed Scopus (101) Google Scholar, 12Intine R.V. Sakulich A.L. Koduru S.B. Huang Y. Pierstorff E. Goodier J.L. Phan L. Maraia R.J. Mol. Cell. 2000; 6: 339-348Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 13Pannone B.K. Xue D. Wolin S.L. EMBO J. 1998; 17: 7442-7453Crossref PubMed Scopus (170) Google Scholar, 14Lerner M.R. Boyle J.A. Hardin J.A. Steitz J.A. Science. 1981; 211: 400-402Crossref PubMed Scopus (383) Google Scholar, 15Rinke J. Steitz J.A. Cell. 1982; 29: 149-159Abstract Full Text PDF PubMed Scopus (237) Google Scholar, 16Yoo C.J. Wolin S.L. Cell. 1997; 89: 393-402Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 17Kufel J. Allmang C. Chanfreau G. Petfalski E. Lafontaine D.L. Tollervey D. Mol. Cell. Biol. 2000; 20: 5415-5424Crossref PubMed Scopus (117) Google Scholar, 18Rinke J. Steitz J.A. Nucleic Acids Res. 1985; 13: 2617-2629Crossref PubMed Scopus (93) Google Scholar, 19Madore S.J. Wieben E.D. Pederson T. J. Biol. Chem. 1984; 259: 1929-1933Abstract Full Text PDF PubMed Google Scholar, 20Verheggen C. Lafontaine D.L. Samarsky D. Mouaikel J. Blanchard J.M. Bordonne R. Bertrand E. EMBO J. 2002; 21: 2736-2745Crossref PubMed Scopus (151) Google Scholar), and stabilization of viral (21Spangberg K. Wiklund L. Schwartz S. J. Gen. Virol. 2001; 82: 113-120Crossref PubMed Scopus (58) Google Scholar, 22Heise T. Guidotti L.G. Chisari F.V. J. Virol. 1999; 73: 5767-5776Crossref PubMed Google Scholar, 23Heise T. Guidotti L.G. Cavanaugh V.J. Chisari F.V. J. Virol. 1999; 73: 474-481Crossref PubMed Google Scholar) as well as cellular RNAs (24McLaren R.S. Caruccio N. Ross J. Mol. Cell. Biol. 1997; 17: 3028-3036Crossref PubMed Scopus (53) Google Scholar). In the cytoplasm, La has been implicated in the translational control of viral (25Meerovitch K. Svitkin Y.V. Lee H.S. Lejbkowicz F. Kenan D.J. Chan E.K. Agol V.I. Keene J.D. Sonenberg N. J. Virol. 1993; 67: 3798-3807Crossref PubMed Google Scholar, 26Ali N. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2249-2254Crossref PubMed Scopus (248) Google Scholar, 27Pudi R. Abhiman S. Srinivasan N. Das S. J. Biol. Chem. 2003; 278: 12231-12240Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and cellular RNAs (28Trotta R. Vignudelli T. Candini O. Intine R.V. Pecorari L. Guerzoni C. Santilli G. Byrom M.W. Goldoni S. Ford L.P. Caligiuri M.A. Maraia R.J. Perrotti D. Calabretta B. Cancer Cells. 2003; 3: 145-160Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 29Kim Y.K. Back S.H. Rho J. Lee S.H. Jang S.K. Nucleic Acids Res. 2001; 29: 5009-5016Crossref PubMed Scopus (82) Google Scholar, 30Holcik M. Korneluk R.G. Mol. Cell. Biol. 2000; 20: 4648-4657Crossref PubMed Scopus (197) Google Scholar). The human La protein contains a nuclear localization sequence at the very C-terminal end, and the staining for endogenous La as well as the distribution of GFP-tagged La reveal a predominantly diffuse nuclear staining, although this may appear different under certain conditions or treatments (31Deng J.S. Takasaki Y. Tan E.M. J. Cell Biol. 1981; 91: 654-660Crossref PubMed Scopus (59) Google Scholar, 32Ayukawa K. Taniguchi S. Masumoto J. Hashimoto S. Sarvotham H. Hara A. Aoyama T. Sagara J. J. Biol. Chem. 2000; 275: 34465-34470Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 33Rutjes S.A. Utz P.J. van der Heijden A. Broekhuis C. van Venrooij W.J. Pruijn G.J. Cell Death Differ. 1999; 6: 976-986Crossref PubMed Scopus (86) Google Scholar). Subcellular distribution of human and yeast La was assumed to be independent of phosphorylation (34Broekhuis C.H. Neubauer G. van Der Heijden A. Mann M. Proud C.G. van Venrooij W.J. Pruijn G.J. Biochemistry. 2000; 39: 3023-3033Crossref PubMed Scopus (40) Google Scholar, 35Long K.S. Cedervall T. Walch-Solimena C. Noe D.A. Huddleston M.J. Annan R.S. Wolin S.L. RNA (New York). 2001; 7: 1589-1602PubMed Google Scholar); however, recent studies (36Raats J.M. Roeffen W.F. Litjens S. Bulduk I. Mans G. van Venrooij W.J. Pruijn G.J. Eur. J. Cell Biol. 2003; 82: 131-141Crossref PubMed Scopus (15) Google Scholar, 37Intine R.V. Tenenbaum S.A. Sakulich A.L. Keene J.D. Maraia R.J. Mol. Cell. 2003; 12: 1301-1307Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) revealed that phosphorylation at Ser-366 may well influence subcellular localization of La. A regulated, nonhomogeneous nuclear distribution of La may also be predicted from the distinct nuclear distribution of its various RNA interaction partners. For instance, pre-tRNAs are most likely synthesized in the nucleoplasm (38Pombo A. Jackson D.A. Hollinshead M. Wang Z. Roeder R.G. Cook P.R. EMBO J. 1999; 18: 2241-2253Crossref PubMed Scopus (207) Google Scholar) but are processed, at least in part, in the nucleolus (39Bertrand E. Houser-Scott F. Kendall A. Singer R.H. Engelke D.R. Genes Dev. 1998; 12: 2463-2468Crossref PubMed Scopus (182) Google Scholar, 40Wolin S.L. Matera A.G. Genes Dev. 1999; 13: 1-10Crossref PubMed Scopus (116) Google Scholar), whereas in yeast the La-associated precursor of U3 snoRNA is probably processed in the nucleolar body and nucleolus (20Verheggen C. Lafontaine D.L. Samarsky D. Mouaikel J. Blanchard J.M. Bordonne R. Bertrand E. EMBO J. 2002; 21: 2736-2745Crossref PubMed Scopus (151) Google Scholar). Nevertheless, interactions with precursor RNAs in different nuclear compartments implicate a highly dynamic and regulated nuclear distribution of La. Although a nucleolar localization of La has been reported (for review see Ref. 9Maraia R.J. Intine R.V. Gene Expr. 2002; 10: 41-57PubMed Google Scholar), the hLa protein was not identified among the recently resolved protein composition of the nucleolus (41Andersen J.S. Lyon C.E. Fox A.H. Leung A.K. Lam Y.W. Steen H. Mann M. Lamond A.I. Curr. Biol. 2002; 12: 1-11Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar). Understanding the nuclear distribution of La and its dynamics requires a more detailed knowledge of its functional domains engaged in RNA binding and localization. Thus far it is known that the human La protein contains three RRMs differently involved in the binding of RNAs (for review see Ref. 8Maraia R.J. Intine R.V. Mol. Cell. Biol. 2001; 21: 367-379Crossref PubMed Scopus (108) Google Scholar). Two conserved amino acid signatures within the RRMs, referred to as RNP-1 and RNP-2 (RNP consensus sequence), are essential for RNA binding (42Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1731) Google Scholar). Recently, we have shown that the RNP-2 of RRM2 in hLa is required for binding of hepatitis B virus RNA (43Horke S. Reumann K. Rang A. Heise T. J. Biol. Chem. 2002; 277: 34949-34958Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and it seems reasonable to assume that this motif is also required for the binding of cellular precursor transcripts. La interacts with RNA polymerase III products by binding the poly(U) stretch at the 3′-end common to this class of transcripts (44Stefano J.E. Cell. 1984; 36: 145-154Abstract Full Text PDF PubMed Scopus (285) Google Scholar) or, in the case of the RNA polymerase II product U3 snoRNA, after partial processing exposing a poly(U) stretch (17Kufel J. Allmang C. Chanfreau G. Petfalski E. Lafontaine D.L. Tollervey D. Mol. Cell. Biol. 2000; 20: 5415-5424Crossref PubMed Scopus (117) Google Scholar). Because elements for nucleolar localization have been described for other proteins (45Annilo T. Karis A. Hoth S. Rikk T. Kruppa J. Metspalu A. Biochem. Biophys. Res. Commun. 1998; 249: 759-766Crossref PubMed Scopus (32) Google Scholar, 46Ueki N. Kondo M. Seki N. Yano K. Oda T. Masuho Y. Muramatsu M. Biochem. Biophys. Res. Commun. 1998; 252: 97-102Crossref PubMed Scopus (24) Google Scholar, 47Scott M. Boisvert F.M. Vieyra D. Johnston R.N. Bazett-Jones D.P. Riabowol K. Nucleic Acids Res. 2001; 29: 2052-2058Crossref PubMed Scopus (102) Google Scholar, 48Rizos H. Darmanian A.P. Mann G.J. Kefford R.F. Oncogene. 2000; 19: 2978-2985Crossref PubMed Scopus (91) Google Scholar, 49Von Kobbe C. Bohr V.A. J. Cell Sci. 2002; 115: 3901-3907Crossref PubMed Scopus (68) Google Scholar, 50Etheridge K.T. Banik S.S. Armbruster B.N. Zhu Y. Terns R.M. Terns M.P. Counter C.M. J. Biol. Chem. 2002; 277: 24764-24770Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 51Lohrum M.A. Ashcroft M. Kubbutat M.H. Vousden K.H. Nat. Cell Biol. 2000; 2: 179-181Crossref PubMed Scopus (171) Google Scholar, 52Schmidt-Zachmann M.S. Nigg E.A. J. Cell Sci. 1993; 105: 799-806Crossref PubMed Google Scholar), it is conceivable to assume that hLa bears such a signal to ensure its interaction with pre-tRNAs, for example, in the nucleolus besides the binding of pre-U3 snoRNA or U6 snRNA in the nucleoplasm. In this report we identified a yet unknown nucleolar localization signal (NoLS) in the C-terminal region of hLa, and we show that it functions in a heterologous context. In addition, we demonstrate the accumulation of hLa mutants deficient in interaction with pre-tRNA in vivo in the nucleolus. Based on these and additional data, we propose a model for the dynamic intranuclear distribution of hLa which is consistent with the complex network of functional interactions between La and precursor transcripts occurring in various subnuclear compartments. Plasmid Constructs and Mutagenesis—Expression plasmid pET28a(+)-hLa-Δ2 was described previously (43Horke S. Reumann K. Rang A. Heise T. J. Biol. Chem. 2002; 277: 34949-34958Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Plasmids described herein were produced with the same strategy. Oligonucleotides used are as follows: for hLa-Δ6.3, 5′-TTG GTC TTC TAT TAT TTT CTT CAG TGC-3′ and 5′-GTA CAG TTT CAG GGC AAG-3′; for hLa-F118A, 5′-GTA AAA AAC AGA TCT GTT TAT ATT AAA GGC GCC CCA ACT GAT GCA ACT C-3′ and 5′-GAG TTG CAT CAG TTG GGG CGC CTT TAA TAT AAA CAG ATC TGT TTT TTA C-3′. For expression of hLa mutants as GFP fusion proteins by a eukaryotic expression vector, the respective hLa DNAs were amplified from the pET28a(+)-hLa plasmids mentioned above via PCR by using the primers 5′-AGA TCT CGAGCTCAA ATG GCT GAA AAT GGT GAT-3′ and 5′-GCT CCC AAGCTT GCC CCG CAA ACA AAA GTC G-3′, containing SacI and HindIII restriction sites (underlined), respectively. Purified PCR products were digested with the appropriate restriction enzymes and cloned into the HindIII and SacI linearized vector pEGFP-C1 (Clontech) by using the rapid ligation kit (Roche Applied Science). Finally, in-frame cloning was controlled by sequencing. For fusion of hLa amino acids 323-354 to the N terminus (hLa-NoLSPTB-GFP) or C terminus (PTB-hLa-NoLS-GFP) of PTB cloned into the eukaryotic expression vector EGFP-N1 ((Clontech), PTB-GFP), the respective hLa sequence was amplified by PCR from the pET-28a(+)-hLa plasmid using the following primers: 5′-CCT ACA CTCGAGGCCACCATG CAA GAA TCC CTA AAC AAA TGG-3′ (containing an XhoI restriction site (underlined), a Kozak sequence (underlined), and a start codon (boldface), respectively) and 5′-CCT ACA AAGCTT TTT TCC TTT ACC AGA CCC AGG-3′ (containing a HindIII restriction site (underlined)), or 5′-CCT ACA GAATTC TAC AAG AAT CCC TAA ACA AAT GG-3′ (containing an EcoRI restriction site (underlined)) and 5′-CCT ACA GTCGAC GCT TTT CCT TTA CCA GAC CCA GG-3′ (containing a SalI restriction site (underlined)). PCR was performed, and purified products were digested with XhoI and HindIII for N-terminal fusion to PTB-GFP cDNA or with EcoRI and SalI for C-terminal fusion and cloned into the linearized, respective PTB-GFP vector. In the case of PTB-hLa-NoLS-GFP an extra alanine or leucine was added for in-frame cloning at both fusion sites. Expression and Purification of Recombinant Proteins—His-tagged recombinant WT and hLa mutant proteins were purified and analyzed as described previously (43Horke S. Reumann K. Rang A. Heise T. J. Biol. Chem. 2002; 277: 34949-34958Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Immunoblot signals were quantified with the Fluor-S MultiImager system (Bio-Rad) to ensure that equal amounts were used in subsequent experiments. For all experiments at least 2 to 3 different protein preparations were used. In Vitro Transcription and UV Cross-linking Assays—DNA templates used for in vitro transcription of pre-tRNAVal were produced by PCR as described previously (22Heise T. Guidotti L.G. Chisari F.V. J. Virol. 1999; 73: 5767-5776Crossref PubMed Google Scholar). Plasmid HtV1 (a kind gift of H. Beier, Institute for Biochemistry, University of Würzburg, Germany (53Arnold G.J. Schmutzler C. Thomann U. van Tol H. Gross H.J. Gene (Amst.). 1986; 44: 287-297Crossref PubMed Scopus (39) Google Scholar)) containing the complete tRNAVal sequence served as template, and the primers used for generation of pre-tRNAVal DNA template were 5′-CCATCGATT AAT ACG ACT CAC TAT AGT TGG TTT CCG TAG TGT AGT GG-3′ containing a ClaI restriction site (underlined) and the T7-RNA polymerase promotor (boldface), and 5′-AAA GCG ACT CTC TTT GTT TCC-3′. In vitro transcription was performed as mentioned elsewhere (22Heise T. Guidotti L.G. Chisari F.V. J. Virol. 1999; 73: 5767-5776Crossref PubMed Google Scholar). For UV cross-links, recombinant hLa proteins (amounts as indicated in Fig. 2A) were incubated for 10 min at room temperature with excess amounts of 32P-labeled (300,000 cpm) and in vitro transcribed pre-tRNAVal in 20-μl reactions containing 10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 0.5% Nonidet P-40, 3 mm MgCl2, 0.5 mm ETDA, and 2 μg of poly(rC) (Sigma). Afterward, cross-linking occurred with 0.6 J in a Stratalinker-1800 (Stratagene), followed by RNase A (15 μg) treatment for 20 min at 40 °C, addition of loading buffer, incubation at 60 °C for 5 min, and electrophoretic separation in 12.5% SDS-PAGE. After drying of gels, signals were evaluated by PhosphorImager analysis using a FujiX-BAS 2000 system (Fuji, Germany), provided with the Tina2.0d software (Raytest, Germany). Cell Culture, Transfection Procedures, Immunofluorescence, and Co-immunoprecipitation—4 × 104 Huh7, U2OS, or HeLa cells per well of a 24-well plate were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transfection of GFP plasmids (1 μg each) was performed using FuGENE 6 Transfection Reagent (Roche Applied Science), and subsequent experiments were performed 1 day after transfection. For fixation, cells grown on coverslips were immediately placed on ice, washed twice with chilled PBS buffer (137 mm NaCl, 3 mm KCl, 10 mm Na2HPO4, 2 mm KH2PO4, pH 7.4), incubated 5 min with chilled methanol and 30 s with chilled acetone, and dried at room temperature. After rehydration with PBS buffer, cells were incubated for 1 h at room temperature with primary monoclonal mouse-α-NOH61 antibody (kindly provided by M. S. Schmidt-Zachmann, Deutsches Krebsforschungzentrum, Heidelberg, Germany (54Zirwes R.F. Eilbracht J. Kneissel S. Schmidt-Zachmann M.S. Mol. Biol. Cell. 2000; 11: 1153-1167Crossref PubMed Scopus (55) Google Scholar)), washed three times for 10 min with PBS buffer, incubated with secondary goat α-mouse rhodamine Alexa-Fluor 594 antibody (Molecular Probes) and Hoechst-staining solution (4 ng/ml, Sigma) for 1 h, washed three times with PBS buffer, and mounted with Mowiol-mounting solution (Calbiochem). Fluorescence images were taken with a Zeiss Axiophot microscope (Zeiss, Germany) equipped with the AxioVision 3.0.6.38 software. For co-immunoprecipitation, 1.7 × 106 293T cells were plated and transfected with GFP-hLa expression plasmids according to Ca2+-phosphate standard procedures. The next day, cells were washed twice with PBS buffer and resolved in 200 μl of lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 20% protease/inhibitor mix Complete™ (Roche Applied Science), 100 units/ml RNasin (Promega, Germany), 0.4% vanadyl ribonucleoside complex (Sigma), 1 mm dithiothreitol). Lysate was cleared, and 2 mg of cell extract was incubated with 5 μg of the respective antibody for 1 h at 4 °C. Anti-GFP mouse monoclonal antibodies (Roche Applied Science) and mouse monoclonal isotype control IgG2a (Dianova, Germany) were used. After addition of 50 μl of protein A-G-agarose+ (Santa Cruz Biotechnology), mixtures were incubated by end-over-end rotation at 4 °C for 12 h. Agarose beads were pelleted and washed twice with lysis buffer and three times with wash buffer (50 mm Tris-HCl, pH 7.4, 200 mm NaCl, 0.1% Nonidet P-40, 0.05% deoxycholate, 10% protease/inhibitor mix, 100 units/ml RNasin, 0.4% vanadyl ribonucleoside complex, 1 mm dithiothreitol) each with 1 ml and 10 min end-over-end rotation. Pellets were dissolved in SDS loading buffer, electrophoresed on 12.5% SDS-PAGE, and electrotransferred, and hLa was visualized with mouse monoclonal α-hLa 3B9. RNA was extracted using TriPure reagent (Roche Applied Science) according to the manufacturer's instructions except for the following: (i) pellets were dissolved in 100 μl of lysis buffer and digested with 30 μg of proteinase K (Roche Applied Science) for 30 min at 50 °C with addition of 0.1% SDS; (ii) 20 μg of each glycogen (Roche Applied Science) was added for precipitation. RNAs were separated on 12% polyacrylamide, 7 m urea gels, blotted to Hybond-N+ membranes (Amersham Bioscience), and detected with radioactively end-labeled oligonucleotides (pre-tRNATyr,5′-GGA TGT CTC CTG CTG AGG AAG TAG CTA C-3′; mature tRNATyr, 5′-GGA TGT CTC CTG CTG AGG AAG TAG CTA C-3′). Signals were quantified using a Fuji-X BAS 2000 PhosphorImaging system (Fuji). Note, the estimated transfection efficiency of 293T cells was ∼80% in contrast to ∼15% for HeLa/Huh7 cells because of different cell lines and transfection procedures. Fluorescence Recovery after Photobleaching (FRAP) Analysis—HeLa cells were plated at a density of 4 × 104 cells in Lab-Tek double chamber systems (Nunc, Germany) and transfected as mentioned above, and FRAP analysis was performed 1 day after transfection on a Leica TCS-SP2 confocal microscope (Leica, Germany) using the 488 nm laser line of the built-in argon laser (nominal output 65 milliwatts). A time resolution of 657 ms per interval was chosen, and five images were taken before bleaching, followed by a triple bleach pulse at 488 nm in the indicated area and subsequent image collection for at least 30 s. Quantification of the fluorescence intensity detected in a region of interest was normalized to the loss in total fluorescence because of bleaching and imaging as described by others (55Phair R.D. Misteli T. Nature. 2000; 404: 604-609Crossref PubMed Scopus (964) Google Scholar) using the Scion Image Beta 4.0.2 software. Consistent results were obtained in three independent experiments with at least 10 cells measured for each of the described hLa proteins. Results shown indicate representative experiments. As a control, the temperature was decreased to 24 °C before and during analysis (not shown). RRM2 of Human La Is Essential for RNA Binding—To identify hLa domains required for subnuclear localization, we tested whether its broad RNA binding activity might play a role for its localization. First, we studied by UV cross-linking assays the binding of recombinant wild-type (WT) and mutant hLa (outlined in Fig. 1) to 32P-labeled and in vitro transcribed pre-tRNAVal. The specificity of interaction is demonstrated by much weaker binding of mature tRNA (data not shown); this as well as the analysis of additional hLa mutants will be substantiated by electrophoretic mobility shift assays and BIAcore surface plasmon resonance experiments. 2S. Horke, K. Reumann, C. Schulze, F. Grosse, H. Will, and T. Heise, manuscript in preparation. Recently, we have reported (43Horke S. Reumann K. Rang A. Heise T. J. Biol. Chem. 2002; 277: 34949-34958Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) that the RNP-2 consensus sequence of RRM2 is essential for hepatitis B virus RNA binding. Here we tested the ability of this RNP-2 deletion mutant (referred to as hLa-Δ2, Fig. 1A) and of a mutant protein with a single amino acid substitution (referred to as hLa-F118A; Fig. 1A) to bind pre-tRNAValin vitro (Fig. 2A). As expected, incubation of recombinant hLa-WT with labeled pre-tRNAVal followed by UV cross-linking and electrophoretic separation led to a dose-dependent formation of hLa-WT·pre-tRNAVal complexes as shown in Fig. 2A (upper left panel). However, deletion of the RNP-2 motif of RRM2, referred to as hLa-Δ2, revealed that amino acids 113VYIKGF118 are essential for binding to pre-tRNAVal (Fig. 2A, upper right panel). With this mutant, no RNA binding was detectable independent of the amount of protein or the technique used (data not shown). Because aromatic residues are likely to be important for RRM-mediated RNA binding (42Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1731) Google Scholar), we substituted phenylalanine 118 by alanine and studied the ability of this mutant (hLa-F118A) to bind pre-tRNAValin vitro. As shown in Fig. 2A (lower left panel), this mutation caused a strong decrease in RNA binding activity (∼50% when compared with hLa-WT), indicating that phenylalanine 118 contributes to efficient pre-tRNA recognition. Taken together, we found that RNP-2 of RRM2 is essential for the interaction with RNA polymerase III transcripts, such as pre-tRNAVal. Recently, we have reported that this is also the case for hepatitis B virus RNA (43Horke S. Reumann K. Rang A. Heise T. J. Biol. Chem. 2002; 277: 34949-34958Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), a RNA polymerase II transcript, indicative of the general importance of RRM2 for RNA binding of human La. Human La Defective in RNA Binding Accumulates in Nucleoli—We have identified hLa mutants with complete or partially reduced RNA binding activity in vitro. To study the cellular localization of those hLa mutants, we cloned EGFP fusion proteins, referred to as GFP-hLa-WT, GFP-hLa-Δ2, and GFP-hLa-F118A. To show no interference of the GFP tag in RNA binding and to confirm the in vitro binding results, we first performed co-immunoprecipitations, thereby analyzing the RNA binding capability of the respective GFP-hLa proteins in vivo. Hence, the eukaryotic expression plasmids were transiently transfected, and anti-GFP antibodies were used for co-immunoprecipitations. Specific GFP-hLa precipitation was confirmed by immunoblotting using La-specific antibodies (Fig. 2B, upper panel), and co-precipitated pre-tRNAs were detected by Northern blot analysis (Fig. 2B, lower panel). Our results indicate an efficient and specific GFP-hLa immunoprecipitation without detectable contaminations by endogenous hLa and, most important, that GFP-hLa is capable of pre-tRNA binding, because pre-tRNATyr (as well as processing intermediates thereof) but not mature tRNATyr can be co-precipitated as would be expected for endogenous hLa. Concerning the hLa mutants, this experiment revealed that GFP-hLa-WT but not GFP-hLa-Δ2.0 was associated with pre-tRNATyrin vivo, thereby reflecting the results obtained in UV cross-links. These data signify that deletion of the RNP-2 motif abolishes the interaction of hLa with pre-tRNATyr and point out the importance of RRM2 in hLa for pre-tRNA binding in vivo. In the case of co-immunoprecipitations with hLa-Δ2 or IgG control, where virtually no RNA was precipitated signal intensities of pre-tRNAs in supernatants were unexpectedly weaker than those in starting material and probably result from minor degradation during the experiment. Next, the subcellular localization of hLa mutants GFP-hLa-WT, GFP-hLa-Δ2, and GFP-hLa-F118A were analyzed in human cell lines Huh7, HeLa, or U2OS by transient transfection and fluorescence microscopy. The GFP-hLa-WT signal was compared with immunostained endogenous hLa in nontransfected cells. In both cases a diffuse and a minor granular nuclear distribution pattern was obtained for all three cell lines (representatively shown for HuH7 cells, Fig. 3A). Of note, to reduce the risk of artifacts caused by overexpression, we chose cells with low expression levels of GFP fusion proteins; however, we observed comparable phenotypes (see below) in all cases. We conclude that the nuclear distribution of endogenous hLa is indistinguishable from exogenously overexpressed GFP-hLa fusion protein. In order to analyze the effect of RNA binding activity of La on its cellular localization, distribution of GFP-hLa-Δ2 and GFP-hLa-F118A was monitored. Merging the GFP-hLa-Δ2 and GFP-hLa-F118A signals with that of nucleolus specific NOH61 revealed that both mutants accumulate in nucleoli (Fig. 3B). To show that ongoing transcription has no detectable effect on the subnuclear distribution of hLa, we treated GFP-hLa-WT transfected cells with actinomycin D (2.5 and 5 μg per ml) for up to 8 h. No change in the distribution of the GFP-hLa proteins within the nucleus compared with nontreated cells was observed (data not shown). In addition to the prominent nucleolar localization, GFP-hLa-Δ2 and GFP-hLa-F118A were to a minor extent visible in the cytoplasm (Fig. 3B), which might indicate that RNA binding contributes to an efficient nuclear import (or retention) of hLa. We conclude from these data that hLa mutants with a reduced or lacking pre-tRNA binding activity in vitro and in vivo (Fig. 2, A and B) accumulate or are retained in the nucleolus, which argues for a role of the RNA binding competence of hLa for maintenance of the predominant nuclear diffuse distribution pattern. Identification of the NoLS of hLa—The C-terminal part of La contains the following functional motifs: a proposed dimerization domain (43Horke S. Reumann K. Rang A. Heise T. J. Biol. Chem." @default.
• W2123657381 created "2016-06-24" @default.
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• W2123657381 creator A5072767663 @default.
• W2123657381 date "2004-06-01" @default.
• W2123657381 modified "2023-10-16" @default.
• W2123657381 title "Nuclear Trafficking of La Protein Depends on a Newly Identified Nucleolar Localization Signal and the Ability to Bind RNA" @default.
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• W2123657381 doi "https://doi.org/10.1074/jbc.m401017200" @default.
• W2123657381 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15060081" @default.
• W2123657381 hasPublicationYear "2004" @default.
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