Rna-protein Interaction in the Selenoprotein Synthesis Machinery -2

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3’UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding. INTRODUCTION Selenoproteins are a diverse family of proteins characterized by the presence of the 21st amino acid selenocysteine (Sec). This amino acid is co-translationally incorporated into the growing peptide chain in response to a UGA Sec codon, otherwise read as a signal for termination of translation. In eukaryotes, the correct recoding event of UGA stop to UGA Sec relies on specific, conserved RNA structures and proteins. The tRNA and the SECIS element, an RNA hairpin in the 30UTR of selenoprotein mRNAs, and two trans-acting proteins, the specialized translation elongation factor eEFSec and the SECIS Binding Protein 2 (SBP2), are the key players of the recoding machinery (1). Specialized protein complexes that involve SECp43, the Phosphoserine tRNA Kinase (PSTK) and the Sec synthase are recruited to the tRNA to ensure proper selenocysteine synthesis (2–4). Ribosomal protein L30 has also been implicated in this mechanism and shown to compete with SBP2 for SECIS binding (5). There are two types of functional SECIS RNAs, forms 1 and 2, classified according to their different apex: form 2 has an additional helix, and its apical loop is shorter than in form 1 (6,7). Structure-based alignments in the currently available eukaryotic selenoproteome identified form 2 SECIS as the most widespread element (8). Except for the apex, SECIS RNA hairpins share common structural features, in particular four consecutive non-Watson–Crick base pairs (the quartet) composed of a central tandem of sheared G.A/A.G base pairs (7–10). *To whom correspondence should be addressed. Tel: +33 3 88 41 70 50; Fax: +33 3 88 60 22 18; Email: [email protected] Present address: David Schmitt, Novartis, Basel, Switzerland. 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research Advance Access published February 17, 2009 -59te l-0 04 55 00 2, v er si on 1 9 Fe b 20 10 Such non-canonical base pairs are characteristic of K (kink)-turn motifs which are recurrent in a variety of RNAs (11–13). The SECIS RNA has been proposed to contain a K-turn like motif (14) that is essential for SBP2 interaction and selenoprotein incorporation in vivo (10). A number of proteins fulfilling different functions such as snRNPs, snoRNPs or ribosomal proteins bind K-turn RNA motifs (15,16). They all carry the L7Ae RNA-binding domain (or module) (17) that contains a restricted set of amino acids that establish base-specific contacts with the sheared G.A/A.G base pairs (11,18,19). SBP2 also has the L7Ae module in its RNA-binding domain (20–22). In an earlier work, we predicted the human SBP2 (hSBP2) amino acids that contact the SECIS RNA at the K-turn like motif (20). However, while sharing some RNA-binding properties with other proteins of the L7Ae family, SBP2 possesses its own specificities (23). The known functions of SBP2, comprising SECIS and ribosome binding, and Sec incorporation, reside in the C-terminal two-thirds of the protein (21,22,24). However, no function has been attributed to the remaining N-terminal section which has been shown to be dispensable for Sec incorporation in rabbit reticulocyte lysate (25). Selenoproteins exist in the three domains of life. Vertebrate genomes encode up to 25–26 selenoproteins but surprisingly larger selenoproteomes can be found in aquatic unicellular organisms (26). Only three selenoprotein genes have been discovered in Drosophila melanogaster, SPS2, SelH and SelK (27,28). SPS2 is the selenophosphate synthetase involved in selenocysteine biosynthesis. SelH and SelK are poorly characterized functionally but they seem nevertheless to play an antioxidant role (29,30). In each case however, only form 2 SECIS RNAs were found in the 30UTR of the selenoprotein mRNAs. Some of us have recently published the annotation and multiple sequence alignments of insect selenoprotein synthesis factors, especially in 12 Drosophila genomes (31). Among these factors, our attention was attracted by the putative Drosophila SBP2 because they lack the sequence homologous to the N-terminus of hSBP2. Although a number of SBP2 sequences from mammals, non-mammalian vertebrates or even unicellular organisms are annotated in databases, only the rat and hSBP2 have been so far isolated and functionally characterized (20–25,32,33). This prompted us to study Drosophila SBP2s and in particular Drosophila melanogaster. In this work, we report that despite its shorter length, D. melanogaster SBP2 (dSBP2) retains functional properties similar to its mammalian counterpart. However, dSBP2 exhibits selective affinities toward SECIS RNAs, being almost unable to bind form 1 SECIS. We determined that the discriminating amino acids reside in a K (lysine)-rich region that we also identified in hSBP2 as essential for SECIS RNA binding. In addition we showed that, in hSBP2, mutating the K-rich region affected form 1 and form 2 SECIS interaction differently, and that this region also plays a crucial role in 60S ribosomal subunit binding. MATERIALS AND METHODS Strains and growth conditions The Escherichia coli TG2 strain was used as the host strain for plasmid construction. Growth was performed at 378C in LB medium, complemented with 100 mg/ml ampicillin. The E. coli strain BL21(DE3)-star was used for production of Drosophila SBP2 proteins at 258C in ZYM-5052 auto-induction medium as described by Studier et al. (34). The E. coli strain BL21(DE3)RIL (Novagen) was used for production of hSBP2 proteins at 188C in LB medium. Bioinformatic analyses Alignment of human/pig/rat/insect SBP2s. Annotated SBP2 sequences from human (gb|AAK57518.1|AF38 0995), rat (sp|Q9QX72.1|SEBP2_RAT), pig (ef|XP_0019 28402.1) and chicken (ref|XP_424425.2|) were aligned against the putative SBP2 sequences found in three Drosophila species, D. melanogaster, D. pseudoobscura and D. sechelia (31) by Kalign (35). Alignment of the K-rich domain. All annotated members of the SBP2 family in Ensembl (ENSF00000007674) were extracted and all those with no ‘X’ in the relevant region were kept. The search was extended by blasting the D. melanogaster SBP2 against the nr database of NCBI. Of the resulting hits, only those containing the IHSRRF motif (positions 624–629 in hSBP2) characteristic of SBP2 proteins (C.A and A.K., unpublished data) were kept. We subsequently used the L7Ae RNA-binding module of SBP2 (33) to query the nr database using Hmmer (36). Finally, we also added the insect SBP2 sequences (31). The resulting 40 sequences were aligned using mafft (37). The alignment images shown in Figures 1 and 7 were produced by Jalview (38). cDNA cloning and recombinant DNA Drosophila melanogaster SBP2 ORF (Genebank accession # AI062219) was amplified from pOT2 cDNA clone GH01354 (Research Genetics) in a two-step PCR reaction and introduced into the pHMGWA vector (39) using the Gateway Technology (Invitrogen). The resulting pHMdSBP2 vector contains a 6 His-tag and MaltoseBinding Protein coding-frame upstream of the dSBP2 ORF and allows E. coli expression of the protein. Human SBP2 ORF was amplified by PCR from plasmid pA11 (33) and subsequently cloned into pET32b (Novagen), generating phSBP2-FL (full-length), as well as plasmids encoding the N-terminal truncated proteins phSBP2 344–854, phSBP2 399–854, phSBP2 515–854, phSBP2 525–854, phSBP2 545–854, phSBP2 625–854 and phSBP2 674–854, and C-terminal truncated proteins phSBP2 344–674, phSBP2 344–790 and phSBP2 344–820. Alanine scanning mutants in hSBP2 were generated in phSBP2 344–854 using the Kunkel mutagenesis method (40). Amino acids swapping mutants exchanging hSBP2 aa535–539 for dSBP2 aa95–99 (phSBP2-SVRVY) and dSBP2 aa95–99 for hSBP2 aa535–539 (pdSBP2IILKE), were generated by site-directed mutagenesis of phSBP2 344–854 and pHMdSBP2, respectively, 2 Nucleic Acids Research, 2009 -60te l-0 04 55 00 2, v er si on 1 9 Fe b 20 10 using the QuickChange XL Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Plasmids pT7SelN, pT7GPx1 and pT7PHGPx were used for T7 transcription of human SelN, rat GPx1 and PHGPx SECIS RNAs, respectively (7,9). To allow in vitro transcription of Drosophila SECIS RNAs, D. melanogaster SelK and SelH SECIS elements were PCR amplified from pOT2 cDNA clones GH03581 and SD09114, respectively (generously provided by M. Corominas, University of Barcelona) and introduced into the BclI-EcoRI sites of pT7-Bck vector (9) to create pT7dSelK and pT7dSelH. A point mutant in dSelH SECIS (dSelHmut), and the SECIS RNA apex-swapped mutants PHGPx-ApSelN and SelN-ApPHGPx SECIS RNAs, were generated by site-directed mutagenesis of pT7dSelH, pT7PHGPx and pT7SelN, respectively, using the QuickChange XL SiteDirected Mutagenesis kit. To generate selenoprotein mRNA reporter constructs for in vitro translation assays, D. melanogaster SelH ORF and 30UTR (Genebank accession #AI542675) were amplified from pOT2 cDNA clone SD09114 and cloned into the HindIII-KpnI sites of the pXJ(HA)3 eukaryotic expression vector (41) to create pHAdSelH. Rat selenoprotein reporter constructs pGPx1-GPx1SECIS and pGPx1SECIS were as described in (10). To create pGPx1-PHGPxSECIS and pGPx1-SelNSECIS, the GPx1 SECIS element of the pGPx1-GPx1SECIS reporter construct was exchanged for PHGPx and SelN SECIS elements from pT7PHGPx and pT7SelN, respectively, using BclI-EcoRI sites. Oligonucleotides used for PCR and mutagenesis are listed in Supplementary Data. Recombinant protein preparation Drosophila SBP2 recombinant proteins expressed in E. coli were purified using Amylose Resin column (NEB). 6 His and MBP tags were cleaved from the dSBP2 protein by thrombin (Sigma) when used for electrophoretic mobility shift assays. hSBP2 recombinant proteins expressed in E. coli were purified using Ni-NTA agarose (Qiagen). Elution buffer was exchanged to dialysis buffer containing 20mM Tris–HCl pH 7.5, 100mM NaCl, 10mM MgCl2, 20% glycerol, 1mM DTT and Cocktail inhibitor (Sigma). Electrophoretic mobility shift assay Plasmids yielding PHGPx, SelN, GPx1, dSelK, dSelH, dSelHmut, PHGPx-ApSelN and SelN-ApPHGPx SECIS RNAs were linearized by EcoRI. Internally labeled SECIS RNAs were obtained by T7 transcription using 80 mCi of [a-P]-ATP (3000Ci/mmol). SECIS RNA-SBP2 complexes were formed as described in (20). Routinely, 30 000 cpm of P-labeled SECIS RNA were incubated for 30min at 308C with various concentrations of purified SBP2 protein (from 0 to 2000 nM), in 7.5ml of phosphate buffer saline pH 7.4, 2mMDTT. RNA–protein complexes were separated on 6% non-denaturing polyacrylamide gel electrophoresis in 0.5 TBE, 5% glycerol buffer. The intensities of free and bound RNAs were quantitated with the Fujifilm FLA-5100 Imaging system. Kds were determined from three independent experiments. In vitro selenoprotein synthesis assays In vitro translation of Drosophila (dSelH) or rat GPx1 from selenoprotein encoding plasmids carrying (or lacking) wild-type SECIS elements were performed using TNT Coupled Reticulocyte Lysate Systems (Promega). One microgram of each of the selenoprotein plasmid DNA was used as the template in 50 ml in vitro transcription/ translation reactions in the presence of 25 ml rabbit reticulocyte lysate, 20 mCi of S-methionine and 120–240 nM of purified SBP2 protein. In vitro translated HA-tagged dSelH and hGPx1 proteins were purified using microMACS Epitope Tag Protein Isolation Kits (Miltenyi Biotec), resolved by 10% SDS–PAGE and detected with the Fujifilm FLA-5100 Imaging system. Quantification of selenoprotein synthesis was performed from two to three independent experiments. Ribosome-binding assays 60S and 40S ribosomal subunits were isolated from full term human placenta according to ref. (42). Human recombinant ribosomal protein p40 was a kind gift of Dr Alexey Malygin (ICBFM, Novosibirsk, Russia). Monoclonal antibodies against human p40 were provided by Dr Valery Loktev. Binding mixtures (50ml) containing 30 pmol of 60S or 40S subunits were reactivated at 378C for 10min in PBSD buffer (150mM NaCl, 27mM KCl, 8mM Na2HPO4, 1.7mM KH2PO4 and 2mM DTT) containing 0.5mM MgCl2. Then 3.5mg SBP2 (or SBP2 mutants) or 2 mg ribosomal protein p40 were added and incubated at 228C for 20min. The mixtures were loaded onto a 15–30% linear sucrose gradient in PBSD with 0.5mM Mg and centrifuged in a SW41 rotor at 23 000 rpm for 15 h. Fractions corresponding to 60S and 40S subunits were precipitated by 10% trichloroacetic acid, and the pellet content loaded onto 10% SDS– PAGE which was blotted onto nitrocellulose membranes. SBP2 was detected with an anti-SBP2 polyclonal antibody (1/5000–1/10 000 dilution) in PBST (1 PBS containing 0.1% Tween 20, 3% dry milk), p40 with a monoclonal anti-p40 (1/3000 dilution). Membranes were treated with anti-rabbit HRP-conjugated secondary antibody (1/10 000 dilution), revealed with the ECL plus kit (GE HealthCare) and exposed to either X-ray film or ChemDoc XRS.