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J. Biol. Chem., Vol. 279, Issue 32, 33702-33715, August 6, 2004
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From the
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Département de Pathologie Moléculaire, UPR 6520 CNRS/U596 INSERM/Université Louis Pasteur, BP 10142, 67404 Illkirch, C.U. de Strasbourg and ¶Centre de Génétique Moléculaire, CNRS UPR2167, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
Received for publication, March 11, 2004 , and in revised form, May 11, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The MLN51 counterpart in the fly, called Barentsz, has been isolated from a functional genetic screening, as a gene essential for oskar mRNA localization (5). 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 (6). Both mRNAs are synthesized in nurse cells and then routed to the oocyte at opposite poles where they remain (7, 8). 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 (9). 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 (10).
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 (11, 12), 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) (13–16). This complex contains at least six proteins as follows: Aly/REF, SRm160, RNPS1, UAP56, Y14, and Magoh (13, 14, 16–19). 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 (13). In addition, Y14 and Magoh remain stably associated with mRNA after export to the cytoplasm (15–17). Thus, the EJC provides a link between pre-mRNA splicing and downstream events, including mRNA nuclear export, nonsense-mediated mRNA decay (reviewed in Refs. 20 and 21), and translation (22, 23).
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 (5), 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 (24). However, MLN51 cannot rescue the barentsz-mutant phenotype, as oskar mRNA remains at the anterior pole of the oocyte in these mutant flies (24). 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 (5), 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.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-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 (GenBankTM accession numbers AI842301 [GenBank] and AI876771 [GenBank] ), 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 (GenBankTM accession number AJ292072 [GenBank] ). The fish MLN51 counterpart (GenBankTM accession number AJ555546 [GenBank] ) 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 (GenBankTM accession numbers BN000153 [GenBank] and BN000152 [GenBank] ).
To identify and define the MLN51 protein family members, we used PipeAlign (25). 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 (26). 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 (27). The Protparam and Profilescan softwares were used to determine the molecular weight and the pHi. Prosite (28), PSORTII (29), and Coils (30) 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 (GenBankTM accession number NM_002370 [GenBank] ) (31), Y14/RNA-binding motif protein 8 (RBM8) (Gen-BankTM accession number NM_005105 [GenBank] ) (14), and nuclear RNA export factor 1 (NXF1/TAP) (32) (GenBankTM accession number NM_006362 [GenBank] ) 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 (4, 33, 34).
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 (35). 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 1.5x 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 (4). 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 x 106) were plated on 10-cm dishes and transfected using JetPEITM 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 1x 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; 1x protease inhibitor mixture). Cellular debris was removed by centrifugation at 10,000 x 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 
-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 1x protease inhibitor mixture). Cell lysates were then homogenized with 15 strokes in a 5-ml Dounce homogenizer and centrifuged at 10,000 x 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 1x protease inhibitor mixture) and centrifuged at 10,000 x 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 x 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 (36), 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 (37). Briefly, recombinant control and MLN51-derived proteins were quantified, separated on SDS-PAGE, and electroblotted onto nitrocellulose membranes. After overnight renaturation, the blots were incubated for 1 h at room temperature with a 32P-labeled riboprobe, washed, and autoradiographed at -80 °C for 4 h.
Immunocytochemistry and LMB Treatment—Five thousand to ten thousand HeLa cells were plated on glass coverslips in 24-well plates and transfected using JetPEITM transfection reagent (Polyplus transfection) with 3 µg of various plasmids. After 24 h of culture, cells were washed with PBS, fixed for 5 min at room temperature in 4% paraformaldehyde in PBS, and permeabilized for 10 min with 0.1% Triton X-100 in PBS. After blocking in 1% bovine serum albumin in PBS, cells were incubated at room temperature with the primary antibodies mouse anti-FLAGM2 and rabbit anti-MLN51 antibodies for 1 h. Cells were washed three times in PBS and incubated for 1 h with Cy3, Cy5, and AlexaFluor 488-conjugated appropriate secondary antibody (1:400). Cells were washed three times in PBS, and nuclei were counterstained with Hoechst 33258 dye. Slides were mounted in Vectashield (Poly-sciences Inc., Warrington, PA). Observations were made with a fluorescence microscope (Leica DMLB 30T, Leica Microsystem, Wetzlar Germany) or confocal microscopes (Leica SP1 and Leica SP2 UV, Leica Microsystem). To study CRM1-dependent nucleocytoplasmic transport of MLN51, transfected HeLa cells as described above were incubated, 24 h after transfection, for 1 h with 10 µg/ml cycloheximide (Sigma), and then 20 ng/ml leptomycin B (Sigma) were added for 5 h (38).
In Vitro Splicing, Affinity Co-precipitation, and X. laevis Microinjections—DNA templates for radiolabeled RNA synthesis, in vitro splicing, activation of RNase H activity and RNA analysis on denaturing PAGE were previously described (13, 15, 17). Briefly, 10-µl splicing reactions were supplemented with 10 ng/µl of purified recombinant TAP-MLN51 proteins. After a 2-h incubation at 30 °C, reactions were either left on ice or supplemented with 1 µM cDNA oligos and incubated for an additional 10 min at 30 °C for activation of RNase H activity. For affinity co-precipitations, samples were then diluted in 300 µl of IPP150 (10 mM Tris·Cl, pH 8.0, 150 mM NaCl, 0.1% Nonidet P-40) combined with 10 µl of 50% slurry IgG-Sepharose (Amersham Biosciences). After gentle mixing for 1 h at 4 °C, and three washes with 500 µl of IPP150, co-precipitated RNAs were phenol/chloroform-extracted and precipitated before analysis on denaturing PAGE and visualized with a PhosphorImager (Amersham Biosciences). Oocyte microinjections and analysis of microinjected RNAs were performed as described previously (15). For protein injections, 25 nl of recombinant TAP-MLN51 at 0.5 µg/µl in 1.5x PBS, 10% glycerol were injected into oocyte cytoplasm 2 h before RNA mixture injection into oocyte nuclei.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Taken together, these data show that MLN51 is mainly found in human cells as part of large RNA-protein particles (RNPs). These particles are most probably enriched in mRNAs, because the sedimentation profile of MLN51 is very similar to the profile of the mRNA-binding protein PABP.
MLN51 Is Associated with mRNPs in Vivo—In order to confirm that MLN51 is associated with mRNPs (mRNA-protein particles), we performed oligo(dT)-cellulose chromatography on HeLa cell fractions enriched in ribosomes. After sedimentation on linear sucrose gradient, fractions containing the ribosomal L7a protein were pooled and incubated in the presence of EDTA with oligo(dT)-cellulose. The unadsorbed material was washed off, and the adsorbed material was recovered by elution with a buffer containing 25% formamide. Detection of MLN51, PABP, L7a, and FMRP (as positive control) were performed on each sample by Western blotting. The ribosomal L7a protein, used here as a negative control, does not bind to the oligo(dT)-cellulose (41), indicating that ribosomes are no longer associated with mRNAs (Fig. 2A). Conversely, both PABP (39) and FMRP (41), bound to oligo(dT)-cellulose, were released by formamide containing elution buffer (Fig. 2A, lane 4). Similarly, MLN51 was bound to oligo(dT)-cellulose and released by formamide containing elution buffer (Fig. 2A, lane 4). This result confirms that MLN51 is present in mRNP complexes.
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We next performed Northwestern analysis to confirm that MLN51 directly binds RNA. For this purpose, we expressed and purified bacterial recombinant TAP-MLN51 using a modified TAP method (35) (see "Experimental Procedures"). Approximately 1 µg of recombinant MLN51 was separated on a 10% SDS-PAGE and electroblotted onto nitrocellulose membranes. After renaturation of the membrane, Northwestern analysis was performed using an in vitro transcribed, 240-nucleotide 32P-labeled riboprobe from pBluescript (Fig. 2C). We observed that the riboprobe was efficiently bound to the full-length protein, TAP-MLN51/1–703 (Fig. 2, C lanes 2 and 7), indicating that recombinant MLN51 possesses an RNA-binding motif. In an attempt to define more precisely the region responsible for RNA binding, we expressed several truncated versions of TAP-MLN51 that we subjected to Northwestern analysis. The amino-terminal half of MLN51 (TAP-MLN51/1–351) showed a clear interaction with RNA (Fig. 2, lane 8) whereas the carboxyl-terminal half (TAP-MLN51/352–703) did not (lane 10), indicating that the RNA binding domain is contained within the amino-terminal half of the protein. We then narrowed our construct to obtain a recombinant protein encompassing amino acids 137–283, containing the most conserved region between MLN51 and barentsz (5, 42). We observed that this region was still able to interact with RNA (Fig. 2, lane 9), whereas no binding was detected with the negative control bovine serum albumin (lane 6) nor with the recombinant TAP tag protein, purified independently (not shown). Although no typical RNA-binding motifs were found in the MLN51 primary sequence, these results show that MLN51 can directly bind RNA via a region spanning residues 137–283. Nevertheless, direct comparison of riboprobe binding (Fig. 2, lanes 7–10) with the different recombinant proteins (lanes 2–5) revealed that the strongest binding was found with the complete protein, suggesting that additional parts of the protein cooperate and/or bind to RNA. Taken together, these results indicate that MLN51 is an RNA-binding protein.
MLN51 Is a Nucleocytoplasmic Protein That Co-localizes with Magoh in Nuclear Speckles—In human cells, MLN51 is mainly cytoplasmic (4), whereas Magoh is mainly nuclear (17). To clarify the relationship between both proteins, we have studied the nucleocytoplasmic shuttling property of MLN51 by immunofluorescence. A potential nuclear export signal (NES) consensus sequence is present within the carboxyl-terminal half of MLN51 (24) at positions 462–472 (Fig. 3A). Accordingly, when the carboxyl-terminal half of the protein is deleted, MLN51 becomes mainly nuclear (4, 24). Several full-length and truncated MLN51 constructs were transiently expressed in HeLa cells in the presence or in absence of leptomycin B (LMB), a chromosome region maintenance 1 (CRM1)-specific inhibitor. The protein CRM1 is implicated in the nuclear export of NES-containing proteins (43). Indeed, a fusion protein between the enhanced green fluorescent protein and the NES of the Rev protein efficiently accumulated in the nucleus upon LMB treatment (33) (Fig. 3B, s–x). Similarly, the full-length MLN51protein (MLN51/1–703) accumulated in the nucleus upon LMB treatment (Fig. 3B, a–f), as does its carboxyl-terminal half that contains the NES (MLN51/352–703, Fig. 3B, m–r). Although the carboxyl-terminal half of MLN51 is devoid of NLS, we noticed that under LMB conditions the protein entered in the nucleus, potentially through passive diffusion. Most interesting, we noted that after LMB treatment MLN51 appeared as labeled dots in the nucleus (Fig. 3B, d). This distribution was also found for the amino-terminal half (MLN51/1–351) independently of LMB treatment (Fig. 3B, g–l), whereas the signal obtained with the carboxyl-terminal half (MLN51/352–703) shows an even nuclear distribution after LMB treatment (Fig. 3B, p). To determine which region of the protein is responsible for the punctate subnuclear localization, we generated additional truncated MLN51 proteins fused to the EYFP. Cells transfected with constructs bearing the NES were treated with LMB to prevent their nuclear egress, whereas the constructs devoid of NES were not treated. A minimal region of 147 amino acids spanning residues 137–283 was sufficient to address the fusion protein to these subnuclear structures (Fig. 4A, m–o). In agreement with this, only constructs bearing this region showed a punctate nuclear staining (Fig. 4A, a, d, g, j, and m).
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As Magoh is mostly addressed to nuclear speckles (17), we next examined MLN51 and Magoh co-localization. Full-length MLN51 and FLAG-tagged Magoh were transiently expressed in HeLa cells in the absence or in presence of LMB (Fig. 4C). In the absence of LMB, MLN51 was detected in the cytoplasm, and Magoh was detected in the nucleus (Fig. 4C, a–d). In contrast, in the presence of LMB both proteins co-localized and appeared as ring-shaped structures in the nucleus (Fig. 4C, e–h). We then confirmed that MLN51 and Magoh were present in nuclear speckles in cells overexpressing both EYFP-MLN51/137–283 and FLAG-tagged Magoh. The localization of Magoh and nuclear speckles was detected using anti-FLAG and anti-9G8 antibodies, respectively. Then the signals corresponding to the three proteins, MLN51, Magoh, and 9G8, were examined in 0.2-µm sections obtained by confocal microscopy. When using regular microscopy the MLN51/137–283 or FLAG-Magoh appeared as dots (not shown), using fine sections (Fig. 4D); MLN51 and Magoh co-localized in a ring at the periphery of 9G8.
These results confirm that MLN51 is shuttling between the cytoplasm and the nucleus (4) and indicate that in the nucleus MLN51 co-localizes with Magoh in speckles where SR proteins such as 9G8 are present. Moreover, we have defined a region of 147 amino acids within the MLN51 protein that is sufficient to address the protein to these subnuclear structures.
MLN51 Contains a Novel Conserved Protein Module Called the SELOR Module—MLN51 was originally identified as a gene overexpressed in breast cancer. At that time, no functional indication could be deduced from the MLN51 primary sequence because no homology to known proteins was found (1). To identify functional motifs within this protein, we have cloned MLN51 counterpart sequences from the following species: mouse (Mm MLN51), frog (Xl MLN51, St. MLN51), and fish (Dr MLN51). A multialignment of MLN51 protein sequences from man to fish showed a high homology score ranging from 55 to 91% identity (Fig. 5, A and B). A region spanning amino acids 168–246 of the human sequence was found to be more conserved than the rest of the protein in all the MLN51 ortholog sequences available (24, 42). To better define this region, we performed multialignment by using relevant sequences coming from every available species, allowing the definition of the signature of a novel 80-amino acid-long conserved protein region (Fig. 5, C and D). Because this region is only present among MLN51 orthologs, it cannot be considered as a novel protein domain, and for that reason we refer to it as a protein module. This module shares 100% identity among human, mouse, and rat orthologs and exhibits an average score of 95% similarity among fish and frogs, 65% similarity among insects, worms, and sea squirt, and 39% similarity in plant (Fig. 5, C and D). In insects, an insertion of 15 and 33 amino acids splits this region into two halves in mosquito (Ag MLN51) and fly (D. melanogaster MLN51/barentsz and Dp MLN51) orthologs, respectively (Fig. 5C). Because this module contained the RNA-binding and speckles-addressing region of MLN51, as shown above, we named it SELOR as an acronym for speckle localizer and RNA binding module.
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To study whether MLN51 interacts directly with Magoh, we produced Magoh fused to glutathione S-transferase (GST) and GST-Magoh/His-tagged Y14 heterodimers in E. coli. We then performed GST-pulldown analysis with recombinant MLN51 protein. GST-Magoh pulled down His-tagged Y14 (31) but failed to pull down recombinant MLN51 (not shown), thus suggesting that the interaction between MLN51 and Magoh is indirect. Taken together these results indicate that MLN51 is able to interact indirectly with Magoh. Given that Magoh is a core component of the EJC, it is tempting to speculate that MLN51 associates with mRNAs via EJCs.
MLN51 Specifically Associates with EJC Assembled in Vitro—To test this hypothesis, we next examined whether MLN51 interacts with the EJC assembled in vitro. To do so, the radiolabeled pre-mRNA AdML (derived from the adenovirus major late transcription unit) was incubated under splicing conditions in the presence of HeLa cell nuclear extracts to generate spliced mRNAs on which the EJC is assembled (13). The splicing reactions were supplemented with recombinant TAP-tagged MLN51 proteins. After splicing, these reactions were subjected to affinity precipitation with IgG-Sepharose beads, and co-precipitated RNAs were analyzed by denaturing PAGE (Fig. 7A). In splicing reactions supplemented with TAP-MLN51/1–703, AdML spliced mRNAs were specifically and efficiently precipitated (Fig. 7A, lanes 2 and 3), in contrast to RNA molecules not associated to the EJC such as the pre-mRNA or the splicing intermediate (free exon 1 and lariat intermediate). Identical results were obtained when splicing reactions were supplemented with the truncated proteins TAP-MLN51/1–351 (Fig. 7A, lanes 4 and 5) and TAP-MLN51/137–283 (lanes 8 and 9), but no specific precipitations were observed with the truncated protein TAP-MLN51/352–703 (lanes 6 and 7) or the TAP tag alone (lanes 10 and 11). In parallel, we performed the similar experiments with the AdML control mRNA that was transcribed from cDNA and on which the EJC is not assembled under splicing conditions (Fig. 7A, lanes 12–21). We observed that the control mRNA was not co-precipitated when the reaction was supplemented with the full-length recombinant protein TAP-MLN51/1–703 or one of its truncated versions. Thus, these results indicate that recombinant MLN51 proteins containing the SELOR module are specifically incorporated in AdML mRNP complex generated by splicing.
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-globin derived mRNAs, /38 and /17 (Fig. 7C). Because the EJC is assembled more than 20 nucleotides upstream of the exon-exon junction, spliced /17 mRNA, which contained a 17-nucleotide-long 5' exon, did not carry an EJC (15). In contrast, the EJC was efficiently assembled on the slightly longer mRNA /38 containing a 38-nucleotide-long 5' exon (15). A mixture of /38 and /17 pre-mRNAs was incubated under splicing conditions in the presence of TAP-MLN51/1–703, TAP-MLN51/1–351, or TAP-MLN51/352–703, and reactions were then subjected to affinity precipitation. As before, we observed that MLN51/1–703 and TAP-MLN51/1–351 associate specifically with spliced /38 mRNAs carrying the EJC but not TAP-MLN51/352–703. Taken together, these results showed that recombinant MLN51, via its conserved SELOR module, specifically associates with mRNAs as a consequence of splicing and that this association is spatially restricted to the mRNA fragment on which the EJC is assembled.
MLN51 Associates with Spliced mRNAs Both in the Nucleus and the Cytoplasm—As shown previously, whereas MLN51 is mainly localized in the cytoplasm, it has the ability to shuttle between the nucleus and the cytoplasm. Given that MLN51 binds spliced mRNA in vitro, we next examined in X. laevis oocytes whether MLN51 is specifically found in association with spliced mRNAs both in the nucleus and in the cytoplasm in vivo (Fig. 8). To do so, TAP-MLN51/1–703 and TAP-MLN51/1–351 were separately injected into oocyte cytoplasm. Oocytes were then incubated for 2 h to allow the recombinant proteins to be imported in the nucleus. Subsequently, body-labeled -globin pre-mRNA was injected into oocyte nuclei along with two RNAs serving as controls for nuclear export. U6
ss was not exported to the cytoplasm, whereas the human initiator methionyl-tRNA was rapidly exported. All RNAs were nuclear immediately after injection (Fig. 8, lanes 1 and 2). After a 2-h incubation, all U6ss RNA was still nuclear, and the control tRNA was exclusively cytoplasmic. During the same period, the pre-mRNA was efficiently spliced, and more than half of the mRNA was exported to the cytoplasm (Fig. 8, lanes 3 and 4). Nuclear and cytoplasmic fractions of injected oocytes were then subjected to affinity precipitation with IgG-Sepharose beads, and co-precipitated RNAs were analyzed in parallel. When oocytes were pre-injected with either TAP-MLN51/1–703 or TAP-MLN51/1–351, we observed that spliced -globin mRNAs were the only molecules efficiently co-precipitated both in the nucleus and the cytoplasm. Indeed, the RNA molecules that did not carry the EJC, such as the pre-mRNA, the intron, and the control RNAs U6 and tRNA were not significantly precipitated. This observation was not restricted to -globin mRNA because identical results were obtained when similar experiments were performed with the Fushi Tarazu (Ftz) pre-mRNA (Fig. 8, lanes 11–20). We confirmed that TAP-MLN51 binding to -globin and Ftz mRNAs in oocytes was restricted to mRNAs generated by splicing by analyzing in parallel the -globin and Ftz control mRNAs that were transcribed from the corresponding cDNAs (Fig. 8, lanes 21–30). Indeed, in comparison to in vivo spliced -globin and Ftz mRNAs, the corresponding control mRNAs were not efficiently co-precipitated.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Although no evidence for a direct interaction with RNA had been reported for barentsz and mouse MLN51 (5, 24), we show that MLN51 is an RNA-binding protein. By using different deleted MLN51 constructs, we isolated the protein portion, from amino acids 137 to 283, able to bind RNA. When compared with the full-length protein, this region binds RNA less efficiently, suggesting that different regions of MLN51 might cooperate for binding RNA, as is the case for other RNA-binding proteins such as DAX-1 (49). This region shares no homology with previously known RNA binding domains, and computer analysis failed to predict secondary structural elements within this region. For example, structural analysis of the most common RNA recognition motif from several different proteins showed that these domains are characterized by a four-stranded anti-parallel -sheet packed against two 
-helices arranged in a /
of the initial translation reactions and 
of the bound proteins were analyzed by immunoblotting using anti-MLN51Ct (lanes 1–10) and anti-Lasp-1 (lanes 11–15) antibodies. The protein molecular size markers are indicated on the left, and the positions of MLN51 and Lasp-1 are indicated by an arrowhead and an arrow, respectively. C, Northwestern analysis of MLN51 interaction to RNA. One microgram of purified recombinant proteins, MLN51/1–703 (lanes 2 and 7), MLN51/1–351 (lanes 3 and 8), MLN51/137–283 (lanes 4 and 9), and MLN51/352–703 (lanes 5 and 10), and 1 µg of bovine serum albumin (BSA, lanes 1 and 6) were separated on SDS-PAGE, electroblotted onto nitrocellulose membrane, and renatured. The Ponceau S-stained membrane is shown on the left. Binding assay was performed using a 32P-labeled 240-nucleotide riboprobe transcribed from PvuII-linearized pBluescript. After washing, the membrane has been subjected to autoradiography (right panel). The protein molecular size markers are indicated on the left.
) mixed with either