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• W2034339628 abstract "MLN51 is a nucleocytoplasmic shuttling protein that is overexpressed in breast cancer. The function of MLN51 in mammals remains elusive. Its fly homolog, named barentsz, as well as the proteins mago nashi and tsunagi have been shown to be required for proper oskar mRNA localization to the posterior pole of the oocyte. Magoh and Y14, the human homologs of mago nashi and tsunagi, are core components of the exon junction complex (EJC). The EJC is assembled on spliced mRNAs and plays important roles in post-splicing events including mRNA export, nonsense-mediated mRNA decay, and translation. In the present study, we show that human MLN51 is an RNA-binding protein present in ribonucleo-protein complexes. By co-immunoprecipitation assays, endogenous MLN51 protein is found to be associated with EJC components, including Magoh, Y14, and NFX1/TAP, and subcellular localization studies indicate that MLN51 transiently co-localizes with Magoh in nuclear speckles. Moreover, we demonstrate that MLN51 specifically associates with spliced mRNAs in co-precipitation experiments, both in the nucleus and in the cytoplasm, at the position where the EJC is deposited. Most interesting, we have identified a region within MLN51 sufficient to bind RNA, to interact with Magoh and spliced mRNA, and to address the protein to nuclear speckles. This conserved region of MLN51 was therefore named SELOR for speckle localizer and RNA binding module. Altogether our data demonstrate that MLN51 associates with EJC in the nucleus and remains stably associated with mRNA in the cytoplasm, suggesting that its overexpression might alter mRNA metabolism in cancer. MLN51 is a nucleocytoplasmic shuttling protein that is overexpressed in breast cancer. The function of MLN51 in mammals remains elusive. Its fly homolog, named barentsz, as well as the proteins mago nashi and tsunagi have been shown to be required for proper oskar mRNA localization to the posterior pole of the oocyte. Magoh and Y14, the human homologs of mago nashi and tsunagi, are core components of the exon junction complex (EJC). The EJC is assembled on spliced mRNAs and plays important roles in post-splicing events including mRNA export, nonsense-mediated mRNA decay, and translation. In the present study, we show that human MLN51 is an RNA-binding protein present in ribonucleo-protein complexes. By co-immunoprecipitation assays, endogenous MLN51 protein is found to be associated with EJC components, including Magoh, Y14, and NFX1/TAP, and subcellular localization studies indicate that MLN51 transiently co-localizes with Magoh in nuclear speckles. Moreover, we demonstrate that MLN51 specifically associates with spliced mRNAs in co-precipitation experiments, both in the nucleus and in the cytoplasm, at the position where the EJC is deposited. Most interesting, we have identified a region within MLN51 sufficient to bind RNA, to interact with Magoh and spliced mRNA, and to address the protein to nuclear speckles. This conserved region of MLN51 was therefore named SELOR for speckle localizer and RNA binding module. Altogether our data demonstrate that MLN51 associates with EJC in the nucleus and remains stably associated with mRNA in the cytoplasm, suggesting that its overexpression might alter mRNA metabolism in cancer. Human metastatic lymph node (MLN) 1The abbreviations used are: MLN, metastatic lymph node; EJC, exon junction complex; PBS, phosphate-buffered saline; mRNP, mRNA protein particle; RT, reverse transcriptase; EYFP, enhanced yellow fluorescent protein; LMB, leptomycin B; NES, nuclear export signal; NLS, nuclear localization signal; GST, glutathione S-transferase; TAP, tandem affinity purification; EST, expressed sequence tag; oligos, oligonucleotides; AdML, adenovirus major late; PABP, poly (A)-binding protein. 51 cDNA was identified from a breast cancer-derived metastatic lymph node cDNA library by differential hybridization of malignant (metastatic lymph node) versus nonmalignant (breast fibroadenoma and normal lymph node) tissues (1Tomasetto C. Regnier C. Moog-Lutz C. Mattei M.G. Chenard M.P. Lidereau R. Basset P. Rio M.C. Genomics. 1995; 28: 367-376Crossref PubMed Scopus (228) Google Scholar). MLN51 presents a correlated pattern of gene amplification and transcript overexpression in breast cancers and cancer-derived cell lines (1Tomasetto C. Regnier C. Moog-Lutz C. Mattei M.G. Chenard M.P. Lidereau R. Basset P. Rio M.C. Genomics. 1995; 28: 367-376Crossref PubMed Scopus (228) Google Scholar, 2Bieche I. Tomasetto C. Regnier C.H. Moog-Lutz C. Rio M.C. Lidereau R. Cancer Res. 1996; 56: 3886-3890PubMed Google Scholar, 3Kauraniemi P. Barlund M. Monni O. Kallioniemi A. Cancer Res. 2001; 61: 8235-8240PubMed Google Scholar). In addition, elevated quantities of MLN51 protein have been found in 30% of primary breast tumor samples tested, although no correlation between MLN51 overexpression and a specific histological tumor type or grade has been found (4Degot S. Regnier C.H. Wendling C. Chenard M.P. Rio M.C. Tomasetto C. Oncogene. 2002; 21 (and references therein): 4422-4434Crossref PubMed Scopus (32) Google Scholar). MLN51 is a nucleocytoplasmic protein containing, within its amino-terminal half, a coiled-coil domain followed by two nuclear localization signals responsible for its nuclear localization. Its carboxyl-terminal half contains putative Src homology domains 2 and 3 binding sites and mediates its cytoplasmic retention (4Degot S. Regnier C.H. Wendling C. Chenard M.P. Rio M.C. Tomasetto C. Oncogene. 2002; 21 (and references therein): 4422-4434Crossref PubMed Scopus (32) Google Scholar). Finally, MLN51 is well conserved during evolution in mammals as well as in more distant species such as worm and fly. From these results, we proposed previously (4Degot S. Regnier C.H. Wendling C. Chenard M.P. Rio M.C. Tomasetto C. Oncogene. 2002; 21 (and references therein): 4422-4434Crossref PubMed Scopus (32) Google Scholar) that MLN51 might have a basal cellular function and that its overexpression in cancer cells may have deleterious effects. The MLN51 counterpart in the fly, called Barentsz, has been isolated from a functional genetic screening, as a gene essential for oskar mRNA localization (5van Eeden F.J. Palacios I.M. Petronczki M. Weston M.J. St. Johnston D. J. Cell Biol. 2001; 154: 511-523Crossref PubMed Scopus (118) Google Scholar). Messenger RNA localization to discrete cellular regions is an important process well described during oogenesis in Drosophila melanogaster. For instance, localization of bicoid and oskar mRNAs to the anterior and posterior poles of the oocyte, respectively, defines the anteriorposterior axis of the embryo (6St. Johnston D. Cell. 1995; 81: 161-170Abstract Full Text PDF PubMed Scopus (510) Google Scholar). Both mRNAs are synthesized in nurse cells and then routed to the oocyte at opposite poles where they remain (7Driever W. Nusslein-Volhard C. Cell. 1988; 54: 95-104Abstract Full Text PDF PubMed Scopus (712) Google Scholar, 8Ephrussi A. Lehmann R. Nature. 1992; 358: 387-392Crossref PubMed Scopus (498) Google Scholar). Bicoid mRNA translation occurs at the anterior pole only after egg fertilization, whereas oskar mRNA translation is coupled to its localization at the posterior pole and starts at mid-oogenesis. The mechanisms that underlie oskar mRNA localization have been elucidated in part by the identification of mutants bearing oskar mRNA localization defects in D. melanogaster (9van Eeden F. St. Johnston D. Curr. Opin. Genet. Dev. 1999; 9: 396-404Crossref PubMed Scopus (160) Google Scholar). To date, many mutants, including those with defects in the kinesin heavy chain, staufen, mago nashi, tsunagi, cytoplasmic tropomyosin II and barentsz genes, have been identified (10Martin S.G. Leclerc V. Smith-Litiere K. St. Johnston D. Development (Camb.). 2003; 130: 4201-4215Crossref PubMed Scopus (58) Google Scholar). The trans-acting factors involved in oskar mRNA localization complex have diverse functions and are conserved in mammals. Kinesin heavy chain and tropomyosin II are related to cytoskeletal filaments, whereas staufen, mago nashi, and tsunagi are RNA-binding proteins. Although the kinesin heavy chain and staufen mammalian orthologs have also been shown to be implicated in mRNA localization (11Severt W.L. Biber T.U. Wu X. Hecht N.B. DeLorenzo R.J. Jakoi E.R. J. Cell Sci. 1999; 112: 3691-3702Crossref PubMed Google Scholar, 12Roegiers F. Jan Y.N. Trends Cell Biol. 2000; 10: 220-224Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), a similar role for the mago nashi and tsunagi human orthologs (Magoh and Y14) is not known. In contrast, the function of Magoh and Y14 in mRNA metabolism is well described. The stable Magoh/Y14 heterodimer is a core component of the exon junction complex (EJC) (13Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (708) Google Scholar, 14Kataoka N. Yong J. Kim V.N. Velazquez F. Perkinson R.A. Wang F. Dreyfuss G. Mol. Cell. 2000; 6: 673-682Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 15Le Hir H. Gatfield D. Izaurralde E. Moore M.J. EMBO J. 2001; 20: 4987-4997Crossref PubMed Scopus (605) Google Scholar, 16Kataoka N. Diem M.D. Kim V.N. Yong J. Dreyfuss G. EMBO J. 2001; 20: 6424-6433Crossref PubMed Scopus (171) Google Scholar). This complex contains at least six proteins as follows: Aly/REF, SRm160, RNPS1, UAP56, Y14, and Magoh (13Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (708) Google Scholar, 14Kataoka N. Yong J. Kim V.N. Velazquez F. Perkinson R.A. Wang F. Dreyfuss G. Mol. Cell. 2000; 6: 673-682Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 16Kataoka N. Diem M.D. Kim V.N. Yong J. Dreyfuss G. EMBO J. 2001; 20: 6424-6433Crossref PubMed Scopus (171) Google Scholar, 17Le Hir H. Gatfield D. Braun I.C. Forler D. Izaurralde E. EMBO Rep. 2001; 2: 1119-1124Crossref PubMed Scopus (144) Google Scholar, 18Gatfield D. Le Hir H. Schmitt C. Braun I.C. Kocher T. Wilm M. Izaurralde E. Curr. Biol. 2001; 11: 1716-1721Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 19Luo M.L. Zhou Z. Magni K. Christoforides C. Rappsilber J. Mann M. Reed R. Nature. 2001; 413: 644-647Crossref PubMed Scopus (307) Google Scholar). As a consequence of splicing, EJC is assembled onto nascent mRNA in a sequence-independent manner at a defined position located 20–24 nucleotides upstream of the exon-exon junction (13Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (708) Google Scholar). In addition, Y14 and Magoh remain stably associated with mRNA after export to the cytoplasm (15Le Hir H. Gatfield D. Izaurralde E. Moore M.J. EMBO J. 2001; 20: 4987-4997Crossref PubMed Scopus (605) Google Scholar, 16Kataoka N. Diem M.D. Kim V.N. Yong J. Dreyfuss G. EMBO J. 2001; 20: 6424-6433Crossref PubMed Scopus (171) Google Scholar, 17Le Hir H. Gatfield D. Braun I.C. Forler D. Izaurralde E. EMBO Rep. 2001; 2: 1119-1124Crossref PubMed Scopus (144) Google Scholar). Thus, the EJC provides a link between pre-mRNA splicing and downstream events, including mRNA nuclear export, nonsense-mediated mRNA decay (reviewed in Refs. 20Dreyfuss G. Kim V.N. Kataoka N. Nat. Rev. Mol. Cell. Biol. 2002; 3: 195-205Crossref PubMed Scopus (1122) Google Scholar and 21Le Hir H. Nott A. Moore M.J. Trends Biochem. Sci. 2003; 28: 215-220Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar), and translation (22Wiegand H.L. Lu S. Cullen B.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11327-11332Crossref PubMed Scopus (178) Google Scholar, 23Nott A. Le Hir H. Moore M.J. Genes Dev. 2004; 18: 210-222Crossref PubMed Scopus (316) Google Scholar). The functional characterization of barentsz, the fly ortholog of MLN51, was especially supportive because it gave clues regarding MLN51 function. Barentsz was found to be in the oskar mRNA localization complex (5van Eeden F.J. Palacios I.M. Petronczki M. Weston M.J. St. Johnston D. J. Cell Biol. 2001; 154: 511-523Crossref PubMed Scopus (118) Google Scholar), indicating that this protein acts directly on the fate of some mRNAs. Introduction of the mouse Mln51 gene into barentsz-deficient flies showed that MLN51 recapitulates some barentsz-specific features such as its subcellular localization, its interaction with staufen, and its incorporation into the oskar mRNA localization complex (24Macchi P. Kroening S. Palacios I.M. Baldassa S. Grunewald B. Ambrosino C. Goetze B. Lupas A. St. Johnston D. Kiebler M. J. Neurosci. 2003; 23: 5778-5788Crossref PubMed Google Scholar). However, MLN51 cannot rescue the barentsz-mutant phenotype, as oskar mRNA remains at the anterior pole of the oocyte in these mutant flies (24Macchi P. Kroening S. Palacios I.M. Baldassa S. Grunewald B. Ambrosino C. Goetze B. Lupas A. St. Johnston D. Kiebler M. J. Neurosci. 2003; 23: 5778-5788Crossref PubMed Google Scholar). Although MLN51 and barentsz have common structural elements, the low overall homology score between both proteins might underline species-specific functions. In fly oocytes, barentsz subcellular localization is strongly altered in mago nashi mutants (5van Eeden F.J. Palacios I.M. Petronczki M. Weston M.J. St. Johnston D. J. Cell Biol. 2001; 154: 511-523Crossref PubMed Scopus (118) Google Scholar), suggesting a potential relationship between these proteins. The role of human MLN51 remains elusive. However, as Magoh is a core EJC component, we have investigated whether human MLN51 is functionally linked to Magoh in mammalian cells by interacting with EJC components and/or as part of the EJC. Probes, cDNA Library Screening, cDNA Cloning, and Sequence Analysis—For cDNA library screening, 100,000 plaque-forming units were plated on LB agar, and the nylon filter replicas (Hybond N, Amersham Biosciences) were hybridized at 42 °C in 50% formamide, 5× SSC, 0.4% Ficoll, 0.4% polyvinylpyrrolidone, 20 mmol/liter sodium phosphate, pH 6.5, 0.5% SDS, 10% dextran sulfate, and 100 μg/ml denatured salmon sperm DNA for 36–48 h with 32P-labeled MLN51-specific mouse and fish probes diluted to 1 × 106 cpm/ml. Stringent washes were performed at 60 °C in 0.1× SSC and 0.1% SDS. Filters were autoradiographed at -80 °C for 24–72 h. Plaques that gave a signal were subjected to a secondary screening using the same conditions. Pure plaques were directly recovered as bacterial colonies using the pBluescript/λ-ZAPII in vivo excision system (Stratagene, La Jolla, CA). The mouse MLN51 cDNA was isolated in two steps. First, by using two overlapping mouse ESTs (GenBank™ accession numbers AI842301 and AI876771), we designed two oligonucleotides primers and amplified an 800-bp internal cDNA fragment from mouse stomach mRNA by RT-PCR. Second, this fragment was used as a probe to screen a mouse stomach cDNA library, and from the four independent clones that were isolated, the complete mouse MLN51 cDNA sequence was established (GenBank™ accession number AJ292072). The fish MLN51 counterpart (GenBank™ accession number AJ555546) was isolated by using a similar strategy. A zebrafish MLN51-specific probe was obtained similarly by RT-PCR using 14-h post-fertilization fish mRNA as template and the two following synthetic oligonucleotides: 5′ primer ACT132 (5′-GAGCATGACGTGAGAGGCCAGG-3′) and reverse primer ACT133 (5′-GGCTCTCGACTGGGTGAACCGG-3′). This probe was used to screen an 18–40-h post-fertilization zebrafish cDNA library. Inserts contained in the mouse- and zebrafish-positive clones were sequenced on both strands, and a consensus cDNA nucleotide sequence was established from the different clones using the Autoassembler software (Applied Biosystem, Foster City, CA). To construct the frog MLN51 cDNA sequences, we aligned overlapping ESTs from either Silurana tropicalis or Xenopus laevis, and we established the complete MLN51 sequences from both frog species (GenBank™ accession numbers BN000153 and BN000152). To identify and define the MLN51 protein family members, we used PipeAlign (25Plewniak F. Bianchetti L. Brelivet Y. Carles A. Chalmel F. Lecompte O. Mochel T. Moulinier L. Muller A. Muller J. Prigent V. Ripp R. Thierry J.C. Thompson J.D. Wicker N. Poch O. Nucleic Acids Res. 2003; 31: 3829-3832Crossref PubMed Scopus (104) Google Scholar). PipeAlign is a protein family analysis tool that integrates a five-step process ranging from the search for sequence homologs in protein sequence and three-dimensional structure data bases to the definition of the hierarchical relationship between and within subfamilies. Research for other MLN51 homologous sequences in all available data bases, at both the nucleotide and protein levels, were performed using BLAST software (26Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59926) Google Scholar). Multiple alignments and phylogenic trees were built using ClustalW and Phylowin (Genetics Computer Group, Wisconsin package version 10), respectively. The ESPript program was used for multiple alignment representation (27Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2530) Google Scholar). The Protparam and Profilescan softwares were used to determine the molecular weight and the pHi. Prosite (28Bairoch A. Bucher P. Hofmann K. Nucleic Acids Res. 1996; 24: 189-196Crossref PubMed Scopus (271) Google Scholar), PSORTII (29Nakai K. Horton P. Trends Biochem. Sci. 1999; 24: 34-36Abstract Full Text Full Text PDF PubMed Scopus (1831) Google Scholar), and Coils (30Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3470) Google Scholar) software allowed the finding of putative motifs for post-translational modifications, the prediction of the subcellular localization, and the identification of coiled-coil domains in all MLN51 counterparts, respectively. These software packages are available from the Expasy Molecular Biology Server. Cloning and Constructs—Poly(A)+ mRNAs from HeLa cells were subjected to first strand cDNA synthesis using oligo(dT) or random primers and Expand reverse transcriptase (Roche Diagnostics). The complete open reading frames of Magoh (GenBank™ accession number NM_002370) (31Zhao X.F. Colaizzo-Anas T. Nowak N.J. Shows T.B. Elliott R.W. Aplan P.D. Genomics. 1998; 47: 319-322Crossref PubMed Scopus (34) Google Scholar), Y14/RNA-binding motif protein 8 (RBM8) (Gen-Bank™ accession number NM_005105) (14Kataoka N. Yong J. Kim V.N. Velazquez F. Perkinson R.A. Wang F. Dreyfuss G. Mol. Cell. 2000; 6: 673-682Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), and nuclear RNA export factor 1 (NXF1/TAP) (32Yoon D.W. Lee H. Seol W. DeMaria M. Rosenzweig M. Jung J.U. Immunity. 1997; 6: 571-582Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (GenBank™ accession number NM_006362) were obtained by RT-PCR using HeLa cells single strand cDNA as template and the following synthetic oligonucleotides: 5′ primer AEI-184 (5′-CCTCTCGAGATGGACTACAAGGACGACGATGACAAGCTTATGGAGAGTGACTTTTATCTG-3′) and reverse primer AEI183 (5′-CACCAATATTCAGTCTAGATTGG-3′); 5′ primer AEI181 (5′-CCTCTCGAGATGGACTACAAGGACGACGATGACAAGCTTATGGCGGACGTGCTAGATCTT-3′) and reverse primer AEI180 (5′-GAGGACCTGTCAGCGACGTCTC-3′); and 5′ primer AEI187 (5′-CCTCTCGAGATGGACTACAAGGACGACGATGACAAGCTTATGGCGGACGAGGGGAAGTCG-3′) and reverse primer AEI186 (5′-GACTACGATCACTTCATGAATGC-3′), respectively. To generate FLAG-tagged fusion proteins, synthetic oligonucleotides containing the nucleotide sequence encoding the FLAG peptide in-frame with 5′ end of each cDNA of interest were used as 5′ PCR primers. After amplification, the purified products (Nucleospin, Macherey Nagel, Duren, Germany) were directly cloned into the pCR3.1 expression vector (BD Biosciences, Palo Alto, CA), generating pCR3.1-FLAG-magoh, pCR3.1-FLAG-Y14, and pCR3.1-FLAG-TAP. The vectors encoding MLN51 and truncated version of the protein fused to the enhanced yellow fluorescent protein (EYFP) were constructed by PCR (using pCR3.1-MLN51 as template). PCR fragments corresponding to MLN51/1–703 and 1–383 were obtained using the following oligonucleotides: AEG200 (5′-GAGACAATTGCGTTCTCCGTAAGATGGCGGAC-3′) as forward primer, AGF274 (5′-CAATTGTTAACTGGAACCCCTGCTTACAAC-3′) and AGF273 (5′-CAATTGTTATGGCTCTGAGGCTGCCTCTTC-3′) as reverse primers, respectively. PCR fragments corresponding to MLN51/137–703, 137–383, and 137–283 were obtained by using AGF268 (5′-AACAATTGGGACACCAAAAGCACTGTGACT-3′) as forward primer and AGF274, AGF273, and AGF-272 (5′-CAATTGTTAGCGATGAGACTTGTTTAGCCG-3′) as reverse primers, respectively. PCR fragments corresponding to MLN-51/277–703 and 277–481 were obtained using AGF269 (5′-AACAATTGGCGGCTAAACAAGTCTCATCGC-3′) as forward primer and AGF274 (5′-AACAATTGTTATGGCTCTGAGGCTGCC-3′) as reverse primer, respectively. Finally, MLN51/377–703 was obtained using AGF270 (5′-AACAATTGGGAAGAGGCAGCCTCAGAGCCA-3′) and AGF274 as forward and reverse primers, respectively. Each fragment was directly cloned into pST1-Blue (Novagen, Darmstadt, Germany). The inserts were released by MunI digestion and inserted in-frame into the EcoRI site of the pEYFP-C1 expression vector (Clontech). Expression vectors allowing the production and purification of recombinant full-length (1–703) or truncated (1–351, 352–703, and137–183) proteins from Escherichia coli were generated as described above. The coding region was inserted in fusion with the tandem affinity purification (TAP) protein (Euroscarf, Frankfurt, Germany) and the polyhistidine tag at the amino- and carboxyl-terminal ends, respectively. All vectors were verified by sequencing from both strands. The pCR3.1-MLN51, pEGFP-Rev-NES, and pSG5-Lasp-1 expression vectors have been described before (4Degot S. Regnier C.H. Wendling C. Chenard M.P. Rio M.C. Tomasetto C. Oncogene. 2002; 21 (and references therein): 4422-4434Crossref PubMed Scopus (32) Google Scholar, 33Ducret C. Maira S.M. Dierich A. Wasylyk B. Mol. Cell. Biol. 1999; 19: 7076-7087Crossref PubMed Scopus (60) Google Scholar, 34Schreiber V. Moog-Lutz C. Regnier C.H. Chenard M.P. Boeuf H. Vonesch J.L. Tomasetto C. Rio M.C. Mol. Med. 1998; 4: 675-687Crossref PubMed Google Scholar). Purification of Recombinant Proteins in E. coli—Plasmids allowing the synthesis of full-length (1–703) or truncated (1–351, 352–703, and 137–283) MLN51 recombinant proteins together with a control plasmid encoding the TAP protein fused to a polyhistidine tag were all derivatives of pET28a (Novagen). Proteins were purified first on nickel-nitrilotriacetic acid affinity resin (Qiagen, Valencia, CA) or on HIS select HC nickel affinity gel (Sigma) as described by the manufacturers, and then eluted proteins were purified on calmodulin affinity resin (Stratagene) as described previously (35Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1423) Google Scholar). Recombinant proteins were analyzed by surface-enhanced laser desorption ionization-mass spectrometry on normal phase NP20 ProteinChip® arrays (Ciphergen Biosystems, Fremont, CA) and by Coomassie Blue staining after SDS-PAGE. Finally, recombinant proteins were dialyzed against 1.5× PBS containing 10% glycerol. Antibodies—The polyclonal anti-green fluorescent protein and the monoclonal anti-FLAG M2 or polyclonal anti-FLAG antibodies were from Cliniscience (Montrouge, France) and Sigma, respectively. Detection of MLN51 was done using anti-MLN51Ct or anti-MLN51Nt (4Degot S. Regnier C.H. Wendling C. Chenard M.P. Rio M.C. Tomasetto C. Oncogene. 2002; 21 (and references therein): 4422-4434Crossref PubMed Scopus (32) Google Scholar). The rabbit anti-PABP, anti-FMRP, anti-9G8, and anti-L7a antibodies were a kind gift of N. Sonenberg, B. Bardoni, R. Gattoni, and A. Ziemiecki, respectively. Cy3- or Cy5-conjugated affinity-purified goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch (West Grove, PA), Amersham Biosciences, and Molecular Probes (Eugene, OR), respectively. Immunoprecipation and Immunoblotting—For the co-immunoprecipitation of transfected proteins, HeLa cells (2 × 106) were plated on 10-cm dishes and transfected using JetPEI™ transfection reagent (Polyplus transfection, Illkirch, France) with a total of 10 μg of DNA containing various expression plasmids. After 24 h, cells were washed twice in serum-free medium and incubated for 24 h in complete medium. Cells were collected and washed in 1× PBS. Lysis was performed by incubating the cells 15 min at 4 °C in 150 μl of lysis buffer (50 mm Tris·HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100; 1× protease inhibitor mixture). Cellular debris was removed by centrifugation at 10,000 × g for 10 min. One mg of total protein extract was incubated for 10–12 h at 4 °C with 40 μl of anti-FLAG M2 monoclonal antibody affinity resin (Sigma). The beads were washed extensively with lysis buffer, and bound proteins were eluted by SDS sample buffer (50 mm Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 1.4 m β-mercaptoethanol, bromphenol blue). Eluted proteins were recovered from the beads by centrifugation. Total proteins, input proteins, or eluted proteins were resolved by 12 to 8% SDS-PAGE and electrotransferred to nitrocellulose sheets (Schleicher & Schuell). The membrane was blocked in PBS containing 3% nonfat dry milk and 0.1% Tween 20. Rabbit anti-MLN51Ct, anti-Lasp-1, anti-PABP, anti-L7a, anti-FLAG polyclonal sera, mouse anti-FMRP, and anti-FLAG monoclonal sera were used as primary antibodies at dilutions of 1/2000, 1/5000, 1/1000, 1/2000, 1/2000, 1/5000 and 1/10000, respectively. After washing, the blots were incubated with appropriate secondary antibodies. Horseradish peroxidase-conjugated AffiniPure donkey anti-rabbit or goat anti-mouse at 1/10000 (Jackson ImmunoResearch) and horseradish peroxidase-conjugated donkey antigoat at 1/1000 (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Finally, protein-antibody complexes were visualized by an enhanced chemiluminescence detection system (SuperSignal West Pico, Pierce). Cell Fractionation Using Sucrose Gradient Sedimentation—Confluent 15-cm dishes of HeLa or MCF7 cells were washed twice in cold PBS, and cells were collected by gentle scraping in PBS. After centrifugation at 1500 rpm for 5 min, cells were resuspended in 1 ml of lysis buffer (25 mm Hepes, pH 6.8, 50 mm KCl, 1 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 250 mm sucrose, and 1× protease inhibitor mixture). Cell lysates were then homogenized with 15 strokes in a 5-ml Dounce homogenizer and centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant was collected and centrifuged again. The supernatant was aliquoted, frozen in liquid N2, and stored at -80 °C. For cell fractionation, 150 μl of 10 mg/ml cell extract was mixed with an equal volume of gradient buffer (25 mm Hepes, pH 6.8, 50 mm KCl, 1 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 1× protease inhibitor mixture) and centrifuged at 10,000 × g for 5 min at 4 °C. The supernatant was loaded on 4 ml of 10–40% linear sucrose gradient. One-tenth of the collected fractions was analyzed by Western blot. For cell fractionation after RNase treatment, cell extracts were incubated with 10 μg of RNase A and 50 units of RNase T1 for 15 min at 37 °C before loading on the gradient as described above. For cell fractionation under EDTA treatment, cell extracts were mixed with an equal volume of gradient buffer containing 30 mm EDTA and devoid of MgCl2, incubated on ice 15 min, and loaded on a 4-ml 10–40% linear sucrose gradient containing 30 mm EDTA. After centrifugation at 200,000 × g for 2 h at 4 °C, 300-μl aliquots were collected from the top to the bottom of the gradient and labeled as fraction 1–14. To visualize ribosomal RNA, RNA extraction was performed using the RNASolv® reagent (Omega Bio-tek, Lilburn, GA) from one-tenth of each collected fraction. RNAs were analyzed by denaturing formaldehyde-agarose gel electrophoresis in the presence of ethidium bromide. Oligo(dT) Chromatography, RNA Homopolymers Binding Assay, and Northwestern Analysis—Purification of poly(A)-containing mRNPs present in polyribosomes was done as described by Lindberg and Sundquist (36Lindberg U. Sundquist B. J. Mol. Biol. 1974; 86: 451-468Crossref PubMed Scopus (113) Google Scholar), with minor modifications. Briefly, fractions from sucrose gradient containing L7a were pooled, dialyzed, concentrated, and incubated in 1 ml of binding buffer containing 100 mm KCl, 25 mm Tris·HCl, pH 7.4, and 25 mm EDTA in the presence of 20 mg of oligo(dT)-cellulose (type 7, Amersham Biosciences) for 30 min at room temperature. After 2 washes with 1 ml of binding buffer, elution of the adsorbed mRNP complexes was done in elution buffer containing 25 mm Tris·HCl, pH 7.4, and 25% formamide. Fractions corresponding to input, unbound, wash, and eluted proteins were analyzed by Western blot for the presence of MLN51, PABP, L7a, and FMRP. MLN51 and Lasp-1 were transcribed-translated using the TnT rabbit reticulocyte lysate or wheat germ systems (Promega, Madison, WI) from pCR3.1-MLN51 and pSG5-Lasp-1 vectors according to the manufacturer's conditions. For RNA homopolymers binding assays, RNA homopolymer-conjugated agarose beads poly(A), poly(G), poly(C)m and poly(U) (Sigma) were mixed to the in vitro transcribed-translated MLN51 and Lasp-1 proteins in RNA binding buffer (10 mm Tris·HCl, pH 8, 2.5 mm MgCl2, 0.5% Triton X-100, 150 mm NaCl) for 30 min at 20 °C. After three washes in RNA binding buffer, bound proteins were eluted with SDS sample buffer and analyzed by Western blot. Northwestern analysis was performed as described (37Bertrandy S. Burlet P. Clermont O. Huber C. Fondrat C. Thierry-Mieg D. Munnich A. Lef" @default.
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