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Characterization of the SECIS Binding Protein 2 Complex Required for the Co-Translational Insertion of Selenocysteine in Mammals Scott A

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Characterization of the SECIS Binding Protein 2 Complex Required for the Co-Translational Insertion of Selenocysteine in Mammals Scott A 5172–5180 Nucleic Acids Research, 2005, Vol. 33, No. 16 doi:10.1093/nar/gki826 Characterization of the SECIS binding protein 2 complex required for the co-translational insertion of selenocysteine in mammals Scott A. Kinzy, Kelvin Caban and Paul R. Copeland* Department of Molecular Genetics, Microbiology and Immunology, UMDNJ—Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA Received June 16, 2005; Revised August 3, 2005; Accepted August 23, 2005 ABSTRACT insertion sequence (SECIS) element that is found in all euka- ryotic selenoprotein mRNA 30 untranslated regions, and was Selenocysteine is incorporated into at least 25 originally thought to be the sole RNA element required for Sec human proteins by a complex mechanism that is a incorporation. This concept has been recently challenged, unique modification of canonical translation elonga- however, by the discovery of a stem–loop sequence in the tion. Selenocysteine incorporation requires the con- coding region of selenoprotein N that is able to support certed action of a kink-turn structural RNA (SECIS) UGA readthrough in the absence of a SECIS element, albeit element in the 30 untranslated region of each seleno- for both UGA and UAG codons (3). Mammalian Sec incorp- protein mRNA, a selenocysteine-specific translation oration also requires the function of a Sec-specific translation elongation factor (eEFSec/mSelB) that specifically and elongation factor (eEFSec) and a SECIS binding [Ser]Sec protein (SBP2). Here, we analyze the molecular con- exclusively binds to the Sec-tRNA (4,5). In addition, text in which SBP2 functions. Contrary to previous two specific binding partners have been identified for the SECIS element: SECIS binding protein 2 (SBP2) and ribo- findings, a combination of gel filtration chromato- somal protein L30 (rpL30). SBP2 is required for Sec incorp- graphy and co-purification studies demonstrates oration (6), while L30 has thus far been shown to enhance that SBP2 does not self-associate. However, SBP2 Sec incorporation in transfected cells (7). is found to be quantitatively associated with SBP2 from the rat (Rattus norvegicus) is a 94 kDa protein ribosomes. Interestingly, a wild-type but not mutant with orthologues found in species ranging from Plasmodium SECIS element is able to effectively compete with falciparum to mammals. Structure–function analysis of SBP2 the SBP2 ribosome interaction, indicating that SBP2 has shown that it is comprised of three distinct domains: an cannot simultaneously interact with the ribosome N-terminal domain for which a function has not yet been and the SECIS element. This data also supports the identified, a central ‘functional domain’ that is required for hypothesis that SBP2 interacts with one or more Sec incorporation but not SECIS element binding, and a kink turns on 28S rRNA. Based on these results, C-terminal SECIS element binding domain (8). The latter two domains comprising the C-terminal 447 amino acids we propose a revised model for selenocysteine are sufficient for all three known biological activities of incorporation where SBP2 remains ribosome SBP2: Sec incorporation, SECIS element binding and ribo- bound except during selenocysteine delivery to the some binding (8,9). The SBP2 SECIS element binding domain ribosomal A-site. has at its core an L7Ae RNA binding motif that is known to be required for interacting with RNA elements known as kink turns for several ribosomal proteins including rpL30 (10,11), INTRODUCTION although this core motif is not sufficient for SECIS element Selenocysteine (Sec), the twenty-first amino acid, is incorpor- binding (8). The L7Ae RNA binding motif is used by rpL30 ated into at least 25 human proteins at specific UGA codons for both mRNA and 28S rRNA interactions (12), thus provid- that are decoded by the Sec-tRNA[Ser]Sec (1,2). This tRNA is ing the best explanation for the competition between SBP2 not sufficient for nonsense suppression because at least two and L30 for SECIS element binding (7). SBP2 also binds to protein factors and one additional cis-sequence are required ribosomes and 28S rRNA (8), and it has been proposed that for Sec incorporation. The cis-sequence is termed a Sec SBP2 makes ribosome contacts at one or more kink-turn *To whom correspondence should be addressed. Tel: +732 235 4670; Fax: +732 235 5223; Email: [email protected] Ó The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2005, Vol. 33, No. 16 5173 motifs, but the specific ribosome binding site has not been For the co-expression of GST-CTSBP2 and XH-CTSBP2, identified. Several reports have also suggested that SBP2 may we created XH-CTSBP2 in pCRII-TOPO (Invitrogen) by exist as a homomultimer based on glycerol gradient sedi- amplifying XH-CTSBP2 out of pTrcHis including the Trc mentation and the appearance of higher molecular weight promoter region. This fragment was TOPO-TA cloned into complexes during electrophoretic mobility shift assays pCRII-TOPO, which lacks a bacterial promoter and contains (7,8,13,14). These results led to a model where SBP2 could both ampicillin (Amp) and kanamycin (Kan) resistance genes. simultaneously interact with the ribosome and a SECIS The ampicillin resistance gene was deleted by digesting the element by having two RNA binding motifs available, one plasmid with Xmn I and Ahd I, polishing overhangs with for SECIS element binding and the other for 28S kink-turn T4 DNA polymerase (Invitrogen) and re-ligating the plasmid binding (15). with blunt-end ligation using T4 DNA ligase as per the In this report, we characterized the aggregate molecular manufacturer’s protocol (Invitrogen). Ligated plasmids were weight of both full-length (FLSBP2) and C-terminal SBP2 transformed into DH5a by electroporation. Kan-resistant (CTSBP2) with a variety of N-terminal tags. These studies colonies were screened for the loss of Amp resistance by were carried out in the context of pull-down and ribosome streaking individual colonies on Amp-containing plates. Plas- binding experiments, which compel us to significantly alter mid DNA was isolated from Kan-resistant/Amp-sensitive our model for Sec incorporation such that SBP2 cannot colonies, and transformed into BL21. The cells were grown simultaneously interact with the ribosome and the SECIS in LB medium containing 100 mg/ml Amp and 50 mg/ml Kan element. to a density of 1.0 OD600 and then induced with L-(+)- arabinose (0.2%; Sigma) and isopropyl-b-D-thiogalactoside (0.24 mg/ml; Fisher Biotech) for 1.5 h at 30C. The cells MATERIALS AND METHODS were pelleted, resuspended in PBS with protease inhibitors and lysed by freeze–thaw followed by sonication for Expression and purification of recombinant proteins 10 s/ml. The sonicate was then centrifuged at 15 000 g for The nucleotide sequence corresponding to full-length rat SBP2 15 min at 4C. The supernatant was applied directly to a 1 ml as well as the fully functional C-terminal half of SBP2 (amino GSTrap FF affinity column (GE Healthcare) equilibrated in acids 399–846) were amplified by PCR and subcloned into PBS, pH 7.3 and tagged protein was eluted in 50 mM Tris– pTrcHis by TOPO-TA cloning according to the manufac- HCl, pH 8.0 containing 10 mM reduced glutathione (Sigma): turer’s protocol (Invitrogen) to generate Xpress/His (XH) 20 ml of each fraction was resolved by 12% SDS–PAGE, tagged constructs (XH-FLSBP2 and XH-CTSBP2), which transferred to nitrocellulose and probed with a 1:10 000 were subsequently transformed into E.coli strain BL21 dilution of anti Xpress-tag antibody (Invitrogen). (Promega). The transformed bacteria were grown in LB medium to a density of 1.0 OD , and then induced with 600 Gel filtration chromatography isopropyl-b-D-thiogalactoside (0.24 mg/ml; Fisher Biotech) for 1.5 h at 30C. The cells were pelleted, resuspended in Two gel filtration chromatography columns were run using a Buffer XH (50 mM Sodium Phosphate, pH 8.0, 1 M NaCl, Beckman System Gold HPLC system (126 pumps and 168 1% Tween-20, 50 mM imidazole) with protease inhibitors diode array detector). A 10 · 300 mm Superdex 200 HR 10/30 (Complete, Roche) and lysed by freeze–thaw followed by (GE Healthcare) column was run at 0.4 ml/min to analyze sonication for 10 s/ml. The sonicate was then centrifuged at proteins in the 10–600 kDa range to yield 0.4 ml fractions. 15 000 g for 15 min at 4C. The supernatant was applied A10· 500 mm column containing Sephacryl S-500 High directly to a 1 ml HiTrap Chelating HP affinity column (GE Resolution resin (GE Healthcare) was run at 0.5 ml/min to Healthcare) charged with 10 mg/ml nickel sulfate hexahydrate analyze proteins in the 40–2000 kDa range to yield 0.5 ml (Sigma), and tagged protein was eluted in Buffer XH with a fractions. Prior to the injection of sample, the columns were linear 50–500 mM imidazole gradient. Strep-tagged CTSBP2 equilibrated with a minimum of 2 column volumes of PBS plus (ST-CTSBP2) was purified as previously described (9). 2 mM DTT (PBSD).
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