Mammalian Smaug Is a Translational Repressor That Forms Cytoplasmic Foci Similar to Stress Granules

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. 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. 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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 agai

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