Mammalian Smaug Is a Translational Repressor That Forms Cytoplasmic Foci Similar to Stress Granules
2005; Elsevier BV; Volume: 280; Issue: 52 Linguagem: Inglês
10.1074/jbc.m508374200
ISSN1083-351X
AutoresMaría Verónica Báez, Graciela L. Boccaccio,
Tópico(s)Genomics and Chromatin Dynamics
ResumoCytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile α motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons. Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile α motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons. Messenger RNA localization, translation activation, silencing, and controlled degradation contribute to the fine-tuning of gene expression in time and space. All these processes depend on several families of RNA-binding proteins that are of comparable importance to transcription factors in regulating gene expression (1Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7018-7024Crossref PubMed Scopus (199) Google Scholar). Sterile α motif (SAM) 3The abbreviations used are: SAMsterile α motifSRESmaug recognition elementECFPenhanced cyan fluorescent proteinPABPpoly(A)-binding proteinSGstress granulesPBprocessing bodiesSMNsurvival motor neuron proteinTIA-1T-cell intracytoplasmic antigenTIARTIA-1-related proteinPBSphosphate-buffered salineRTreverse transcriptionBHKbaby hamster kidneydDrosophilahhumanmmurine.-containing RNA binding domains define a novel family of RNA-binding proteins that function as post-transcriptional regulators (2Aviv T. Lin Z. Lau S. Rendl L.M. Sicheri F. Smibert C.A. Nat. Struct. Biol. 2003; 10: 614-621Crossref PubMed Scopus (158) Google Scholar). They bind to an RNA sequence motif known as SRE (Smaug recognition element), the Drosophila protein Smaug being the first member that was identified (2Aviv T. Lin Z. Lau S. Rendl L.M. Sicheri F. Smibert C.A. Nat. Struct. Biol. 2003; 10: 614-621Crossref PubMed Scopus (158) Google Scholar, 3Smibert C.A. Lie Y.S. Shillinglaw W. Henzel W.J. Macdonald P.M. RNA (N. Y.). 1999; 5: 1535-1547Crossref PubMed Scopus (113) Google Scholar, 4Dahanukar A. Walker J.A. Wharton R.P. Mol. Cell. 1999; 4: 209-218Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 5Green J.B. Gardner C.D. Wharton R.P. Aggarwal A.K. Mol. Cell. 2003; 11: 1537-1548Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Drosophila Smaug is involved in translational repression of the maternal mRNA encoding nanos, a posterior determinant, and thus plays a role in defining embryo polarity. Smaug recruits Cup, an eIF4E-binding protein that prevents the association of eIF4E with eIF4G, thus blocking initiation of the translation of SRE-containing messengers (6Nelson M.R. Leidal A.M. Smibert C.A. EMBO J. 2004; 23: 150-159Crossref PubMed Scopus (197) Google Scholar). In addition, it has been reported recently that Drosophila Smaug mediates degradation of maternal Hsp83 mRNAs by an independent mechanism that involves the CCR4 deadenylase and does not require Cup nor SRE motifs (7Semotok J.L. Cooperstock R.L. Pinder B.D. Vari H.K. Lipshitz H.D. Smibert C.A. Curr. Biol. 2005; 15: 284-294Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The yeast homologue Vts1 stimulates degradation of SRE-containing messengers by a similar mechanism (2Aviv T. Lin Z. Lau S. Rendl L.M. Sicheri F. Smibert C.A. Nat. Struct. Biol. 2003; 10: 614-621Crossref PubMed Scopus (158) Google Scholar). sterile α motif Smaug recognition element enhanced cyan fluorescent protein poly(A)-binding protein stress granules processing bodies survival motor neuron protein T-cell intracytoplasmic antigen TIA-1-related protein phosphate-buffered saline reverse transcription baby hamster kidney Drosophila human murine. Two Smaug homologous genes of unknown function are present in the mammalian genome (2Aviv T. Lin Z. Lau S. Rendl L.M. Sicheri F. Smibert C.A. Nat. Struct. Biol. 2003; 10: 614-621Crossref PubMed Scopus (158) Google Scholar, 3Smibert C.A. Lie Y.S. Shillinglaw W. Henzel W.J. Macdonald P.M. RNA (N. Y.). 1999; 5: 1535-1547Crossref PubMed Scopus (113) Google Scholar). Here we show that Smaug 1, encoded in human chromosome 14, represses translation of SRE-containing messengers in fibroblast cell lines. Both hSmaug 1 and Drosophila Smaug form cytoplasmic granules when expressed in fibroblasts and colocalize when cotransfected. Furthermore, hSmaug 1 foci contain polyadenylated mRNAs, and their size and number depend on polysome integrity, as described in the cases of stress granules (SG) and processing bodies (PB) (8Anderson P. Kedersha N. J. Cell Sci. 2002; 115: 3227-3234Crossref PubMed Google Scholar, 9Kedersha N.L. Gupta M. Li W. Miller I. Anderson P. J. Cell Biol. 1999; 147: 1431-1442Crossref PubMed Scopus (902) Google Scholar, 10Kimball S.R. Horetsky R.L. Ron D. Jefferson L.S. Harding H.P. Am. J. Physiol. 2003; 284: C273-C284Crossref PubMed Google Scholar). We found that murine Smaug 1 is expressed in the brain and is abundant in synaptoneurosomes, a subcellular region where translation is tightly regulated by synaptic stimulation (reviewed in Refs. 11Steward O. Schuman E.M. Annu. Rev. Neurosci. 2001; 24: 299-325Crossref PubMed Scopus (597) Google Scholar, 12Bailey C.H. Kandel E.R. Si K. Neuron. 2004; 44: 49-57Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 13Ostroff L.E. Fiala J.C. Allwardt B. Harris K.M. Neuron. 2002; 35: 535-545Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 14Takei N. Inamura N. Kawamura M. Namba H. Hara K. Yonezawa K. Nawa H. J. Neurosci. 2004; 24: 9760-9769Crossref PubMed Scopus (357) Google Scholar, 15Si K. Giustetto M. Etkin A. Hsu R. Janisiewicz A.M. Miniaci M.C. Kim J.H. Zhu H. Kandel E.R. Cell. 2003; 115: 893-904Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 16Krichevsky A.M. Kosik K.S. Neuron. 2001; 32: 683-696Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 17Menon K.P. Sanyal S. Habara Y. Sanchez R. Wharton R.P. Ramaswami M. Zinn K. Neuron. 2004; 44: 663-676Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 18Gebauer F. Hentze M.W. Nat. Rev. Mol. Cell Biol. 2004; 5: 827-835Crossref PubMed Scopus (716) Google Scholar). Our results suggest a role for Smaug 1 in RNA granule formation and translation regulation of SRE-containing transcripts at post-synaptic sites. Plasmids and Library Screening—A pCDNA3.0 vector (Invitrogen) encoding Drosophila Smaug was generated by subcloning the coding region from a dSmaug cDNA kindly provided by Dr. C. Smibert (University of Toronto, Canada) using the primers 5′-TAAGAACTATCCCGGTACCACAA-3′ and 5′-GATCAAATTTGCTCGAGTTCTCC-3′. Firefly luciferase reporters carrying three copies of either wild type or mutated SRE were constructed by subcloning of the BamHI/HindIII fragment of C145 and C146 plasmids, a generous gift of C. Smibert (19Smibert C.A. Wilson J.E. Kerr K. Macdonald P.M. Genes Dev. 1996; 10: 2600-2609Crossref PubMed Scopus (185) Google Scholar), into a pcDNA3.0 vector. A pCDNA6.0 encoding murine Staufen 1 (GenBank™ accession number AF395842) (20Thomas M.G. Martinez Tosar L.J. Loschi M. Pasquini J.M. Correale J. Kindler S. Boccaccio G.L. Mol. Biol. Cell. 2005; 16: 405-420Crossref PubMed Scopus (115) Google Scholar) was used. The predicted coding region of hSmaug 1 from the AK034323 EST was subcloned between HindIII and SacII sites in the pECFP-N1 vector (Clontech) and KpnI and XhoI sites in the pcDNA6.0 vector (Invitrogen). Screening of the mouse brain, heart, kidney, testis, and embryo cDNA libraries was performed at OriGene Technologies, Inc. (Rockville, MD) using three pairs of primers: 5′-GTGGAGTAGTGATTGCCGCTTG-3′ and 5′-CACTCGTTCCAGCCCTTAAACC-3′; 5′-CAGTCCAACTCCCTCCCAACAG-3′ and 5′-AGTCTCTGCAACCCTGAAGATGG-3′; and 5′-AGACTGTTGCACTGCTGTCG-3′ and 5′-TCCAATCGTGTTGATTGTGG-3′. Primary Antibody against Mammalian Smaug 1—Rabbit polyclonal antisera were raised against the hSmaug 1 SAM domain, which was prepared as similarly described for the Drosophila SAM domain (21Green J.B. Edwards T.A. Trincao J. Escalante C.R. Wharton R.P. Aggarwal A.K. Biochem. Biophys. Res. Commun. 2002; 297: 1085-1088Crossref PubMed Scopus (7) Google Scholar). Briefly, XL1-Blue cells were transformed with a pET22b expression vector (Novagen, San Diego) encoding the hSmaug 1-(306-371) fragment. Bacteria were induced with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside for 12-16 h at 18 °C. After cell harvesting by centrifugation, recombinant protein was purified using a nickel-nitrilotriacetic acid column (Qiagen, Germantown, MD), recovered by elution in 300 mm imidazole, and then dialyzed against PBS. Animals were bled after two boosters. Translation and Real Time PCR Assays—The following three plasmids were cotransfected into BHK-21 cells plated in 60-mm dishes (106 cells per dish) using Lipofectamine 2000 (Invitrogen) in Dulbecco's modified Eagle's medium (Sigma) without serum and antibiotics: 1) 20-500 ng of pCDNA3.0 vector carrying the firefly-luciferase coding region fused to a SRE tandem repeat, either wild type or mutated (2Aviv T. Lin Z. Lau S. Rendl L.M. Sicheri F. Smibert C.A. Nat. Struct. Biol. 2003; 10: 614-621Crossref PubMed Scopus (158) Google Scholar); 2) 10 μg of pECFP-hSmaug 1 or 6.7 μg of pECFP vector (Clontech); and 3) 50 ng of pRL-CMV (Promega, Madison WI) encoding Renilla luciferase. Twenty four hours after transfection, firefly and Renilla luciferase activities were quantified using the dual-luciferase reporter assay system (Promega). Cell lysis was performed as indicated by the manufacturers, and measurements were both taken immediately and after freeze-thawing, with no significant variations. Transfections were performed in triplicate, and results were expressed as the average ratio of firefly to Renilla luciferase activity. Real time RT-PCR using SYBR Green (Molecular Probes, Eugene, OR) was performed in triplicates of 1:10 serial dilutions of RNA using the following primers: 5′-tcgcggttgttacttgactg-3′ and 5′-cgatcttgttacaacacccc-3′ for firefly and 5′-ggaaacggatgataactggtcc-3′ and 5′-aggccgcgttaccatgta-3′ for Renilla luciferase. Following Equation 2 in Ref. 22Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar, cycle threshold (CT) versus log [RNA dilution] was plotted. Only dilutions where plot slopes (= 1 + E (where E is the efficiency of target amplification), according to Ref. 22Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar, were close to 2 and similar for the two templates (difference lower than 5%) were considered, indicating similar amplification efficiencies (22Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar). A linear regression was applied to calculate the ΔCT (firefly minus Renilla) and the corresponding S.D. The ratio of firefly luciferase mRNA to Renilla luciferase mRNA was calculated as (1 + E)ΔCT, where (1 + E) was calculated from the slope, and the standard deviation was calculated by error propagation. Cell Transfection, Drug Treatment, and Immunofluorescence—HeLa, BHK-21, and COS-7 cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Natocor, Córdoba, Argentina), penicillin, and streptomycin (Sigma). Plasmid transfection was performed in subconfluent cells plated onto poly-l-lysine-coated coverslips using Lipofectamine 2000 (Invitrogen). Cells were harvested or processed for immunofluorescence 8-16 h after transfection. Fixation in 4% paraformaldehyde at 37 °C, 4% sucrose in PBS was performed with or without prior extraction of living cells in CSK buffer (CSKB: 25 mm KCl, 1 mm HEPES, pH 6.8, 1 mm EGTA, 5 mm MgCl2) containing 0.25 m sucrose and Triton X-100 0.1% for 1 min. After fixation, cells were permeabilized in 0.1% Triton X-100 in PBS and blocked in 2% bovine serum albumin. Primary antibodies were diluted as follows: anti-mammalian Smaug 1, 1:500 to 1:1000, and RLS1, 1:500; anti-PABP rabbit polyclonal antibody (kindly provided by Dr. Evita Mohr, University of Hamburg, Germany), 1:500; anti-TIA-1 goat polyclonal antibody 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA). Monoclonal antibodies and suppliers are as follows: anti-TIAR, SMN (BD Biosciences); anti-HuR and anti-GW182 (Cytostore, Calgary, Alberta, Canada), anti-hPABP (ImmuQuest, Cleveland; UK), 1:100, and anti-V5 (Invitrogen), 1:500. Secondary antibodies were from Molecular Probes or Jackson ImmunoResearch (West Grove, PA). Cells were mounted in Mowiol 4-88 (Calbiochem, EMD Biosciences, Inc). Images were acquired using an LSM-5 PASCAL confocal microscope (Carl Zeiss, Oberkochen, Germany). Proper equipment adjustment was ensured using 1-μm FocalCheck fluorescent microspheres (Molecular Probes). Cycloheximide and puromycin (Sigma) were added to conditioned media from stock aqueous solutions. Treatment with 50 ng/ml leptomycin B (Sigma) was performed in conditioned media for 2 h. Biochemical Fractionation, Sedimentation Velocity Centrifugation, and Western Blotting—Brains from 6-week-old mice were homogenized in 0.8 m sucrose, in CSKB containing the following protease inhibitors: 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin (all from Sigma). Synaptoneurosomes, post-synaptic densities, and a membrane fraction were isolated as described previously (23Ma L. Huang Y.Z. Pitcher G.M. Valtschanoff J.G. Ma Y.H. Feng L.Y. Lu B. Xiong W.C. Salter M.W. Weinberg R.J. Mei L. J. Neurosci. 2003; 23: 3164-3175Crossref PubMed Google Scholar). For sedimentation velocity centrifugation, brain post-synaptic densities or post-nuclear extracts from cultured cells were prepared in CSKB containing 1% Triton X-100, 0.25 m sucrose. Samples of 0.5-1 mg of protein (determined by the bicinchoninic acid protein kit assay; Sigma) were loaded onto continuous 13-ml sucrose gradients (20-60, 15-45, or 10-30% w/v in CSKB) and centrifuged at 220,000 × g for either 2 or 4 h. When required, 1 unit of β-galactosidase was added as a 20 S marker. The polysomal profile was monitored by absorbance at 260 nm and β-galactosidase activity by colorimetric o-nitrophenyl β-d-galactopyranoside reaction. Protein from 1-ml fractions was precipitated in chloroform/methanol (1:2) using 20 μg of lysozyme as carrier and analyzed by Western blot. Briefly, protein was resuspended in Laemmli sample buffer, separated by SDS-PAGE, and electrotransferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA). The following primary antibodies were used: anti-mammalian Smaug 1, diluted 1:10,000 to 1:20,000; anti-Staufen 1 RLS1, 1:5000 (20Thomas M.G. Martinez Tosar L.J. Loschi M. Pasquini J.M. Correale J. Kindler S. Boccaccio G.L. Mol. Biol. Cell. 2005; 16: 405-420Crossref PubMed Scopus (115) Google Scholar); rabbit anti-S6 (Cell Signaling, Beverly, MA), 1:1000; and anti-Drosophila Smaug (generous gift from C. Smibert,), 1:10,000. Detection of peroxidase-conjugated anti-V5 antibody (Invitrogen) and peroxidase-coupled secondary antibodies (Sigma) was performed by chemiluminescence using the LumiGlo system (Cell Signaling). Mammalian Smaug 1 Represses Translation of SRE-containing mRNAs—The two genes homologous to Drosophila Smaug present in mammalian genomes were named as Smaug 1, located in human and mouse chromosome 14, and Smaug 2, located in human chromosome 19 and mouse chromosome 7 (Fig. 1A). We performed a screening of mouse brain, liver, testis, and whole embryo libraries and found two mSmaug 1 transcripts. The most frequent of them corresponds to a previously reported EST (GenBank™ accession number AK034323), and the second most frequent corresponds to a novel late embryonic 5′-untranslated region splicing variant (Fig. 1B). No splicing variants involving the coding region were found in our screening nor in the available data bases, including human and mouse sequences. We confirmed the presence of mSmaug 1 transcripts in brain, kidney, heart, and liver by RT-PCR analysis (Fig. 1C). We sought to investigate the effect of mammalian Smaug 1 on translation and stability of SRE-containing messengers. Although the direct interaction of hSmaug 1 with SRE-containing messengers was not addressed in this study nor in previous reports, it has been shown previously that SAM domains of vertebrate Smaug homologues specifically recognize SREs (2Aviv T. Lin Z. Lau S. Rendl L.M. Sicheri F. Smibert C.A. Nat. Struct. Biol. 2003; 10: 614-621Crossref PubMed Scopus (158) Google Scholar, 5Green J.B. Gardner C.D. Wharton R.P. Aggarwal A.K. Mol. Cell. 2003; 11: 1537-1548Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). BHK cells were cotransfected with plasmids encoding an enhanced cyan fluorescence protein (ECFP) or a hSmaug 1-ECFP chimera together with firefly luciferase-translational reporters bearing either wild type or mutated SREs in a trimeric tandem array (3xSRE+ and 3xSRE-, respectively) (19Smibert C.A. Wilson J.E. Kerr K. Macdonald P.M. Genes Dev. 1996; 10: 2600-2609Crossref PubMed Scopus (185) Google Scholar). A plasmid encoding Renilla luciferase to normalize for transfection efficiency was cotransfected. We found that in the presence of hSmaug 1-ECFP the firefly luciferase activity yielded from 3xSRE+-containing transcripts was reduced to 43% relative to the ECFP control. In contrast, firefly luciferase activity yielded from 3xSRE-reporters was the same in the presence of hSmaug 1-ECFP or ECFP (Fig. 2A). When extracts of cells expressing firefly luciferase were preincubated with extracts of cells expressing hSmaug 1-ECFP, no effects on luciferase activity levels were observed (not shown), further indicating that luciferase reporters are not modulated at the protein level by hSmaug 1. Simultaneously, firefly and Renilla luciferase mRNA levels were determined by real time PCR. The abundance of the firefly reporters relative to the coexpressed control Renilla mRNA was not affected by the expression of hSmaug 1 (Fig. 2B). As expected, the translational repression was proportional to the relative amounts of hSmaug 1 (Fig. 2C). These results indicate that human Smaug 1 represses translation of SRE-containing messengers without affecting their stability. The possibility of an effect on mRNA decay or deadenylation by recruitment of the CCR4 deadenylase, as is the case of the Drosophila molecule (7Semotok J.L. Cooperstock R.L. Pinder B.D. Vari H.K. Lipshitz H.D. Smibert C.A. Curr. Biol. 2005; 15: 284-294Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), in a different molecular or cell context was not analyzed, and thus this remains open. Murine Smaug 1 Is Present in Brain Synaptoneurosomes and Forms Small Particles—An antibody raised in our laboratory against the native SAM domain of hSmaug 1 specifically recognized the human protein in Western blot assays of BHK cells expressing hSmaug 1-V5. Expression of endogenous Smaug 1 was not detected in BHK or other fibroblasts cell lines (Fig. 3A and data not shown). Next, we evaluated the expression of Smaug 1 in the brain, as translational regulation is frequent in neurons (11Steward O. Schuman E.M. Annu. Rev. Neurosci. 2001; 24: 299-325Crossref PubMed Scopus (597) Google Scholar, 13Ostroff L.E. Fiala J.C. Allwardt B. Harris K.M. Neuron. 2002; 35: 535-545Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 14Takei N. Inamura N. Kawamura M. Namba H. Hara K. Yonezawa K. Nawa H. J. Neurosci. 2004; 24: 9760-9769Crossref PubMed Scopus (357) Google Scholar, 15Si K. Giustetto M. Etkin A. Hsu R. Janisiewicz A.M. Miniaci M.C. Kim J.H. Zhu H. Kandel E.R. Cell. 2003; 115: 893-904Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 17Menon K.P. Sanyal S. Habara Y. Sanchez R. Wharton R.P. Ramaswami M. Zinn K. Neuron. 2004; 44: 663-676Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 24Zalfa F. Giorgi M. Primerano B. Moro A. Di Penta A. Reis S. Oostra B. Bagni C. Cell. 2003; 112: 317-327Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). A protein band of ∼70 kDa was observed in blots of mouse and rat brain extracts, in accordance with the predicted molecular weight of mSmaug 1 (Fig. 3B). We then examined the distribution of mSmaug 1 in brain subcellular fractions. Murine Smaug 1 was enriched in synaptoneurosomes (Fig. 3C), and a further purification indicated that mSmaug 1 concentrates in post-synaptic densities, a fraction from synatoneurosomes almost membrane-free and enriched in cortical cytoskeleton and synapse-associated elements, such as localized mRNAs and polyribosomes and the marker protein PSD-95 (11Steward O. Schuman E.M. Annu. Rev. Neurosci. 2001; 24: 299-325Crossref PubMed Scopus (597) Google Scholar, 13Ostroff L.E. Fiala J.C. Allwardt B. Harris K.M. Neuron. 2002; 35: 535-545Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 16Krichevsky A.M. Kosik K.S. Neuron. 2001; 32: 683-696Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). In contrast, mSmaug 1 was not detected in the synatoneurosomes membrane fraction (Fig. 3C). It also seems to be absent from myelin, as an oligodendrocyte extract gave no signal (Fig. 3C). Taken together, these results suggest that mSmaug 1 is localized in neuronal structures associated with dendritic synapses. Next, we investigated the interaction of mSmaug 1 with the synaptoneurosomal translational apparatus by sedimentation velocity analysis. After centrifugation through a sucrose gradient, mSmaug 1 migrated as a 20 S particle and appeared not to be associated with polyribosomes or with ribosomal subunits (Fig. 3D). As expected, the double-stranded RNA-binding protein Staufen 1 was detected in polysomes and in a faster sedimenting fraction (Fig. 3D), as described previously (16Krichevsky A.M. Kosik K.S. Neuron. 2001; 32: 683-696Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 20Thomas M.G. Martinez Tosar L.J. Loschi M. Pasquini J.M. Correale J. Kindler S. Boccaccio G.L. Mol. Biol. Cell. 2005; 16: 405-420Crossref PubMed Scopus (115) Google Scholar, 25Luo M. Duchaine T.F. DesGroseillers L. Biochem. J. 2002; 365: 817-824Crossref PubMed Scopus (50) Google Scholar, 26Kiebler M.A. Hemraj I. Verkade P. Kohrmann M. Fortes P. Marion R.M. Ortin J. Dotti C.G. J. Neurosci. 1999; 19: 288-297Crossref PubMed Google Scholar, 27Marion R.M. Fortes P. Beloso A. Dotti C. Ortin J. Mol. Cell. Biol. 1999; 19: 2212-2219Crossref PubMed Scopus (154) Google Scholar, 28Wickham L. Duchaine T. Luo M. Nabi I.R. DesGroseillers L. Mol. Cell. Biol. 1999; 19: 2220-2230Crossref PubMed Scopus (209) Google Scholar). These results are compatible with a role of mSmaug 1 in inhibition of translation initiation in neurons. Smaug Forms Cytosolic Granules That Contain Polyadenylated RNA—To analyze further translational repression by mammalian Smaug 1, tagged hSmaug 1 molecules were transiently transfected in fibroblast cell lines, and their subcellular distribution was then analyzed by immunofluorescence and sedimentation velocity gradients. We found that a large proportion of Smaug 1 concentrated in cytoplasmic granules no larger than 0.5-2 μm (Fig. 4A). These granules were observed in COS-7, BHK, and HeLa cells transfected with two different human Smaug 1 chimeras, fused to ECFP or to the small tag His6-V5 (Fig. 4 and data not shown). Most cells (66-85%) showed the representative phenotype depicted in Fig. 4A, granule size, and number as being roughly proportional to expression levels (TABLE ONE). As expected, transfected ECFP presented no granules (data not shown). Human Smaug 1 granules were distributed throughout the cytoplasm and were also observed after Triton X-100 extraction of living cells (Fig. 4B), whereas the nonpunctate hSmaug 1 signal was no longer present (see also TABLE ONE). Treatment with the CRM1-inhibitory drug leptomycin B strikingly increased the otherwise minor nuclear hSmaug 1 staining (Fig. 4, B and C), suggesting that this protein is shuttling between the nucleus and the cytoplasm. Smaug 1 foci were extremely infrequent in the nuclear compartment; the protein showed a homogeneous distribution throughout the nucleoplasm (Fig. 4C).TABLE ONEPresence of TIA-1, TIAR, and PABP in hSmaug 1 foci The frequency of hSmaug 1 foci and the presence of marker proteins in those foci were analyzed in independent transient-transfection experiments by immunostaining and confocal microscopy. Determinations were performed after 16 h of expression, unless indicated otherwise. N indicates number of transfected cells analyzed. Cells with more than four hSmaug1 granules larger than 0.5 μm were considered as containing foci. The percentage relative to total transfected cells is indicated. The percentage of cells showing colocalization with the different markers was relative to the number of granule-containing cells. Cells were scored as positive for colocalization when more than 60% of their hSmaug1 foci contained the analyzed marker. Cells positive for PABP colocalization always showed more than 95% of double-stained foci. No differences in colocalization frequency of the distinct markers were observed between high expressing and low expressing cells (not shown). ND indicates not determined.ConstructionCell lineNFociPABPTIARTIA-1%%%%hSmg1-ECFPBHK-21aDeterminations were performed after 8 h of expression40066NDNDND5885100NDND7376ND636916783NDND79BHK-2114183ND65ND114709852NDCOS-7112839464NDhSmg1-V5BHK-211388093ND6612381ND66NDCOS-79983ND68NDhSmg1-ECFP (after Triton X-100)BHK-2190100bNongranular hSmaug1 signal was washed out after Triton extraction of living cells, and therefore the remaining signal was granular in 100% of the cases8477NDCOS-797100bNongranular hSmaug1 signal was washed out after Triton extraction of living cells, and therefore the remaining signal was granular in 100% of the cases9273NDa Determinations were performed after 8 h of expressionb Nongranular hSmaug1 signal was washed out after Triton extraction of living cells, and therefore the remaining signal was granular in 100% of the cases Open table in a new tab Remarkably, Drosophila Smaug also formed cytoplasmic granules when expressed in fibroblast cell lines (Fig. 4D). Moreover, the Drosophila protein colocalized with mammalian Smaug 1 when coexpressed (Fig. 4E), further suggesting a functional similarity between the fly and the mammalian homologues. We then performed a subcellular fractionation of BHK cells expressing hSmaug 1-V5. Transfected hSmaug 1 was quantitatively recovered in the cytosolic fraction. When post-nuclear extracts were analyzed by sedimentation velocity centrifugation, hSmaug 1 migrated close to the 20 S marker (Fig. 4F), similar to the synaptoneurosome-associated mSmaug 1 (Fig. 3D). We simultaneously analyzed the distribution of the Drosophila protein coexpressed in the same conditions. Most interestingly, fly Smaug also migrates as a small ribonucleoparticle or free protein (Fig. 4F). Finally, for comparison, a construct encoding Staufen 1-V5 was cotransfected with hSmaug 1-V5 and analyzed simultaneously. Similarly to the endogenous Staufen 1 (Fig. 3D), Staufen 1-V5 comigrated with polysomes (Fig. 4F), indicating that overexpression is not perturbing the subcellular distribution of the transfected proteins. Furthermore, the absence of the transfected dSmaug and hSmaug 1 in fast sedimentation fractions, together with the plasticity of hSmaug 1 foci (see below), indicate that Smaug foci were not aggregates of mis-folded proteins. Given that hSmaug 1 repressed the translation of SRE-containing transcripts (Fig. 2), we investigated the presence of messenger RNA in the hSmaug 1 foci. Detection of the general PAB
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