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Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation

2000; Springer Nature; Volume: 19; Issue: 17 Linguagem: Inglês

10.1093/emboj/19.17.4796

1460-2075

Delphine Fagegaltier, Nadia Hubert, Kenichiro Yamada, Takaharu Mizutani, Philippe Carbon, Alain Krol,

RNA Research and Splicing

Article1 September 2000free access Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation Delphine Fagegaltier Delphine Fagegaltier UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Search for more papers by this author Nadia Hubert Nadia Hubert UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Present address: European Molecular Biology Laboratory, D-69117 Heidelberg, Germany Search for more papers by this author Kenichiro Yamada Kenichiro Yamada UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Present address: Institute for Developmental Research, Aichi, 480-03 Japan Search for more papers by this author Takaharu Mizutani Takaharu Mizutani Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603 Japan Search for more papers by this author Philippe Carbon Philippe Carbon UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Search for more papers by this author Alain Krol Corresponding Author Alain Krol UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Search for more papers by this author Delphine Fagegaltier Delphine Fagegaltier UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Search for more papers by this author Nadia Hubert Nadia Hubert UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Present address: European Molecular Biology Laboratory, D-69117 Heidelberg, Germany Search for more papers by this author Kenichiro Yamada Kenichiro Yamada UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Present address: Institute for Developmental Research, Aichi, 480-03 Japan Search for more papers by this author Takaharu Mizutani Takaharu Mizutani Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603 Japan Search for more papers by this author Philippe Carbon Philippe Carbon UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Search for more papers by this author Alain Krol Corresponding Author Alain Krol UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France Search for more papers by this author Author Information Delphine Fagegaltier1, Nadia Hubert1,2, Kenichiro Yamada1,3, Takaharu Mizutani4, Philippe Carbon1 and Alain Krol 1 1UPR du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, Cedex, France 2Present address: European Molecular Biology Laboratory, D-69117 Heidelberg, Germany 3Present address: Institute for Developmental Research, Aichi, 480-03 Japan 4Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603 Japan ‡N.Hubert and K.Yamada contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4796-4805https://doi.org/10.1093/emboj/19.17.4796 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Decoding of UGA selenocysteine codons in eubacteria is mediated by the specialized elongation factor SelB, which conveys the charged tRNASec to the A site of the ribosome, through binding to the SECIS mRNA hairpin. In an attempt to isolate the eukaryotic homolog of SelB, a database search in this work identified a mouse expressed sequence tag containing the complete cDNA encoding a novel protein of 583 amino acids, which we called mSelB. Several lines of evidence enabled us to establish that mSelB is the bona fide mammalian elongation factor for selenoprotein translation: it binds GTP, recognizes the Sec-tRNASec in vitro and in vivo, and is required for efficient selenoprotein translation in vivo. In contrast to the eubacterial SelB, the recombinant mSelB alone is unable to bind specifically the eukaryotic SECIS RNA hairpin. However, complementation with HeLa cell extracts led to the formation of a SECIS-dependent complex containing mSelB and at least another factor. Therefore, the role carried out by a single elongation factor in eubacterial selenoprotein translation is devoted to two or more specialized proteins in eukaryotes. Introduction Selenocysteine is the major biological form of selenium. Only eubacteria, archaea and animals can incorporate it co-translationally into selenoproteins, a class of enzymes instrumental in oxidation–reduction reactions. Selenocysteine is part of the active site in these proteins, which comprise various prokaryotic enzymes but also the animal glutathione peroxidases and thioredoxin reductases that are of prime importance for maintaining the redox status of the cell (reviewed in Burk and Hill, 1999). The conservation of a complex molecular machinery for designating an internal UGA triplet as the selenocysteine and not the stop codon attests to the crucial role borne by this amino acid (reviewed in Atkins et al., 1999). The mechanism leading to selenoprotein translation has been unraveled in eubacteria (reviewed in Atkins et al., 1999; Commans and Böck, 1999). The tRNASec is charged with serine by the conventional seryl-tRNA synthetase, and the seryl residue is converted to selenocysteine on the tRNASec by selenocysteine synthase. The general translation factor EF-Tu does not participate in this process where it is replaced by the specialized elongation factor SelB (Forchhammer et al., 1989). The SelB–GTP–Sec-tRNASec complex binds a hairpin adjacent to the UGA selenocysteine codon in the mRNA, forming a quaternary complex that directs the charged tRNASec to the A site of the ribosome. Regarding archaea, the isolation of the Methanococcus jannaschii SelB homolog was reported very recently (Rother et al., 2000). Much less is known about selenocysteine incorporation into animal selenoproteins. Two RNA partners are well characterized, the tRNASec and a hairpin structure called SECIS, which resides in the 3′-untranslated region (3′-UTR) of selenoprotein mRNAs. The SECIS element is mandatory for recognition of UGA as a selenocysteine codon (Berry et al., 1991; reviewed in Hubert et al., 1996a; Low and Berry, 1996). With structure–function studies, we have proposed an experimental model for the structure of the SECIS hairpin, which established the pivotal role of non-Watson–Crick base pairs in mediating selenoprotein translation (Walczak et al., 1996, 1998). More recently, it was discovered that SECIS hairpins can adopt two different apical structures, depending on the size of the apical loop in different SECIS RNAs (Grundner-Culeman et al., 1999; Fagegaltier et al., 2000a). Several SECIS-binding proteins have been described (Shen et al., 1995, 1998; Hubert et al., 1996b; Fujiwara et al., 1999; Copeland et al., 2000; Fagegaltier et al., 2000b). Among these, only the 120 kDa SBP2 was shown to be required effectively for mammalian selenoprotein translation (Copeland et al., 2000). Its sequence does not show any characteristic feature of elongation factors, in contrast to the eubacterial system where a single protein, SelB, possesses two functions: one as an elongation factor that binds the charged tRNASec, the other one as the mRNA hairpin-binding factor. The latter function is mediated by the C-terminal domain of SelB, which is not found in EF-Tu (Kromayer et al., 1996). Several proteins have been described to be associated with the tRNASec. Partially purified fractions containing SePF, a putative bovine SelB factor, protected the Sec-tRNASec ester bond against mild hydrolysis (Yamada et al., 1994). Autoantibodies from patients with a severe chronic active hepatitis precipitated a 48 kDa polypeptide associated with the tRNASec (Gelpi et al., 1992). Recently, it was shown that the mammalian protein SECp43 specifically binds the tRNASec (Ding and Grabowski, 1999). However, none of these proteins was reported to bear the SelB function, and the absence of a known mammalian homolog of the bacterial SelB prompted us to undertake experiments to isolate its cDNA. Here, we report the identification of the cDNA and the functional characterization of the elongation factor mSelB, a novel mouse protein of 583 amino acids specialized for selenoprotein translation. Results Identification of a partial human SelB sequence and obtaining the complete mouse SelB cDNA Two characteristic features of the Escherichia coli (Ec) and M.jannaschii (Mj) SelB sequences are: (i) the presence of deletions Δ1–Δ4 (Figure 1) with respect to the EF-Tu or EF1-α elongation factors (Hilgenfeld et al., 1996; Rother et al., 2000); and (ii) the FXI/VK sequence (the solid horizontal bar in Figure 1), which we found to constitute one of the hallmarks of the EcSelB and MjSelB sequences since the terminal lysine of the motif is replaced by threonine and glycine residues in EF-Tu and EF1-α, respectively (Figure 1). The tBLASTn program was used to identify the mammalian SelB homolog by querying expressed sequence tag (EST) databases at NCBI with the MjSelB amino acid sequence. Among the hits, one human EST (DDBJ/EMBL/GenBank accession No. R53658) showed the best match and was selected because its translation indicated that it indeed contained the FSIK sequence and the Δ4 deletion. Complete sequencing of this EST indicated the presence of a 1095 bp open reading frame (ORF), but sequence alignments with EcSelB, MjSelB, EF-Tu and EF1-α led to the conclusion that the EST lacked the region lying upstream of the FSIK sequence. 5′ RACE PCR and library screening provided information for a new 1991 bp human sequence harboring an ORF with a coding capacity of 526 amino acids for the putative human SelB (hSelB). As the ORF was still incomplete, its sequence was used for a second round of EST database searches, leading to the identification of a mouse EST (DDBJ/EMBL/Genbank accession No. AI317100). After complete sequencing, its translation and sequence comparisons with MjSelB, EcSelB and EF1-α indicated that it contained the complete cDNA for the putative mouse SelB (mSelB), with a 1749 bp ORF encoding a 583 amino acid protein of 63.5 kDa predicted molecular mass. The deduced amino acid sequences of the putative hSelB and mSelB are shown in Figure 1. Analysis of their sequences over the common region indicated that they share 88% amino acid identity. Figure 1.Alignment of SelB sequences from mouse (mSelB), human (hSelB), C.elegans (CeSelB), Drosophila (dSelB), M.jannaschii (MjSelB), E.coli (EcSelB) and of the human hEF1-α sequence. The alignment was made with ClustalW (Thompson et al., 1994) and manually refined with MegAlign (DNASTAR). Identical amino acids are in reverse, similar residues are shaded in gray. The G1–G4 GTP-binding domains are indicated, as well as the Δ1–Δ5 deletions mentioned in the text. The open bar depicts the mSelB G503–I519 block of homology, and the solid bar maps the hSelB FSIK sequence (positions 162–165). The closed circles and the asterisk position residues that are mentioned in the Discussion. Download figure Download PowerPoint Sequence features of the human and mouse SelB Figure 1 shows the presence of significant blocks of homology between amino acid positions V8 and V290 in mSelB, and I8 and V325 in hEF1-α, yielding 23% amino acid identity. Sequence alignments between the mammalian SelB, MjSelB and EcSelB indicated that the blocks of homology extend to positions F337 in mSelB, Y319 in MjSelB and L307 in EcSelB. In the latter region, the amino acid identity between mSelB and EcSelB is 24% but reaches 36% between mSelB and MjSelB. The eubacterial and MjSelB proteins contain the GTP-binding domains but possess two features that distinguish them from EF-Tu and EF1-α, respectively: the presence of a C-terminal extension and the lack of most of the contact domains (Δ1–Δ4 in Figure 1) with the guanine nucleotide exchange factor(s) (Hilgenfeld et al., 1996; Rother et al., 2000). Four blocks of sequence similarity to the G1–G4 GTP-binding domains of EcSelB, MjSelB and hEF1-α were detected in mSelB (only G3 and G4 in hSelB since the sequence is incomplete). Other stretches of sequence identity or similarity with EcSelB and hEF1-α were found flanking the G2 and G3 domains, or corresponding to amino acid positions D215–I236, V253–M256, L272–I274 and R286–G287 in the mSelB sequence. Amino acids at these positions in EF-Tu were described to contact the T or acceptor stems, or the aminoacyl group, in the crystal structures of the Thermus aquaticus tRNAPhe–EF-Tu and E.coli tRNACys–EF-Tu complexes (Nissen et al., 1995, 1999). Also showing up in mSelB were the four deletion regions Δ1–Δ4 (Δ2–Δ4 in hSelB) that occur in EcSelB and MjSelB. Observation of the C-terminal parts of the mSelB, hSelB, MjSelB and EcSelB SelB sequences revealed the presence of sequence similarities corresponding to mSelB positions F403–P583, creating a C-terminal extension with respect to hEF1-α (Figure 1). The mammalian extension is longer than that of MjSelB, but shorter than in EcSelB. mSelB is a guanine nucleotide-binding protein The amino acid sequence conservation in the G1–G4 domains (Figure 1) predicted that mSelB is a guanine nucleotide-binding protein. To investigate whether this correlates with GTP binding in vitro, binding experiments were performed with a His-tagged recombinant protein that was overexpressed in E.coli and purified in two chromatographic steps as shown in Figure 2. The binding activities were characterized by adding a fixed amount of mSelB to various concentrations of [3H]GTP or the non-hydrolyzable [γ-35S]GTP analog. The experiments in Figure 3 established that mSelB is actually a guanine nucleotide-binding protein. From the saturation curve in Figure 3A, we calculated that the apparent dissociation constant Kd was 0.3 μM for GTP. Similar experiments with [3H]GDP gave an apparent Kd of 0.6 μM for the binding of GDP (Figure 3B). Figure 2.Purification steps of the recombinant mouse SelB. Supernatants (SN) of E.coli transformed with the His-tagged mSelB expression vector, induced (i in lane 2) or non-induced (Ni in lane 1) by IPTG, were fractionated by affinity chromatography on an Ni-NTA column (lane 3). The eluted fractions were pooled and loaded onto a carboxymethyl-Sephadex C50 column (lane 4). Samples were run on a 10% SDS–polyacrylamide gel and the proteins revealed by Coomassie staining. Download figure Download PowerPoint Figure 3.Determination of the apparent dissociation constants of mSelB for GTP and GDP. Binding assays were performed as described in Materials and methods. (A) The concentration-dependent binding of [3H]GTP to 0.5 (circles) or 0.3 μM (squares) mSelB. (B) Concentration- dependent binding of [3H]GDP to 0.3 (squares), 0.6 (triangles) or 0.9 μM (circles) mSelB. Download figure Download PowerPoint mSelB binds specifically the selenocysteyl-tRNASec in vitro To provide additional evidence that mSelB is indeed the selenoprotein translation factor, we used a protection assay to ask whether it could specifically recognize the tRNASec. This experiment takes advantage of the ability of elongation factors to protect the aminoacyl-tRNA ester bond against mild alkaline hydrolysis when bound to the charged tRNA (Forchhammer et al., 1989; Yamada et al., 1994). A T7 tRNASec transcript was selenocysteylated in vitro with a 75Se selenol group and then submitted to hydrolysis. Figure 4A shows that an 80 min incubation led to 40% of residual selenocysteyl-tRNASec in the absence of mSelB. In contrast, the presence of the recombinant mSelB prevented substantial hydrolysis from occurring since the percentage of protected Sec-tRNASec did not fall below 78%. To attest that the protection was afforded only to the tRNASec, the seryl-tRNASer was submitted to the same treatment. Under similar conditions, mSelB did not preclude hydrolysis of that ester bond (Figure 4B). Furthermore, and most importantly, the ester bond of the seryl-tRNASec, the precursor in the selenocysteine biosynthesis pathway, was clearly susceptible to mild alkaline hydrolysis (Figure 4B). This established that the protection was ascribed to the selenocysteyl moiety. Figure 4.mSelB specifically protects the selenocysteyl-tRNASec ester bond against mild alkaline hydrolysis. (A) Hydrolysis of the selenocysteyl-tRNASec in the presence (closed circles) or absence (open circles) of mSelB. (B) Hydrolysis of the seryl-tRNASec in the absence (open triangles) or presence (closed triangles) of mSelB. Hydrolysis of the seryl-tRNASer was performed likewise in the absence (open circles) or presence (closed circles) of mSelB. Download figure Download PowerPoint From this set of experiments, we conclude that mSelB specifically recognizes the selenocysteyl-tRNASec, but neither the seryl-tRNASec nor the seryl-tRNASer. The tRNASec is associated with mSelB in vivo We wished to determine whether the protection detected in vitro reflects an association between mSelB and the tRNASec in vivo. To this end, a hemagglutinin (HA)-tagged mSelB cDNA was transiently transfected into COS-7 cells and the total cell extracts (Figure 5A, lane 3) were submitted to an immunoprecipitation with anti-HA antibodies to recover the tagged mSelB present in the total extract. In the first place, anti-HA antibodies verified that the HA-tagged mSelB was immunoprecipitated effectively from the extract (Figure 5A, lane 4) and that the signal did not show up in immunoprecipitated mock-transfected cell extracts (lane 2). The band observed in the control lane 1 (but not in the immunoprecipitate in lane 2) results from cross-reaction of the polyclonal anti-HA antibody with a cellular protein, as previously reported (Lescure et al., 1999). Figure 5.The tRNASec is associated with mSelB in vivo. (A) Extracts of COS-7 cells transfected with the HA-tagged mSelB expression vector, or mock-transfected, were blotted with the anti-HA antibody before (lanes 3 and 1, respectively) or after immunoprecipitation (IPP in lanes 4 and 2, respectively). The signal in lane 1 results from cross-reaction of the anti-HA with a cellular protein. Samples were run on a 10% SDS–polyacrylamide gel and revealed by chemiluminescence. (B) Enzymatic determination of the tRNASec sequence with RNase T1 (G), RNase U2 (A), RNase PhyM (A/U) and RNase CL3 (C>U). L is an alkaline ladder; control is a lane that received no enzyme. Shown on the left are sequencing gels (two separate migrations) for the immunoprecipitated tRNASec arising from the experiment in (A). The T7 tRNASec transcript was sequenced in parallel for band assignments. The two-dimensional structure of the mammalian tRNASec (Sturchler et al., 1993) is represented with the modified nucleotides. Download figure Download PowerPoint In a second step, we tested whether the tRNASec was associated with mSelB. A fraction of the immunoprecipitated mSelB was phenol extracted. After mild treatment of the RNA moiety with phosphodiesterase and phosphatase to remove the 3′-terminal CCA, the 3′ end was labeled by action of the tRNA nucleotidyl transferase in the presence of [α-32P]ATP. The sequencing gel in Figure 5B identified the tRNASec, indicating that it was co-immunoprecipitated with the HA-tagged mSelB. In contrast to the T7 tRNASec, the CUUCAAA sequence (positions 32–38) in the anticodon of the immunoprecipitated tRNASec could not be resolved because of missing bands and aberrant migration on the gel, presumably provoked by the mcm5U and i6A modified bases. The same phenomenon was reported by Gelpi et al. (1992) in the course of sequencing the tRNASec isolated from an immunoprecipitated ribonucleoparticle in HeLa cells extracts. Collectively, these experiments showed that the tRNASec is associated with mSelB in vivo. mSelB is required for efficient selenoprotein translation To investigate whether mSelB is involved in the translation of the selenocysteine codon in vivo, we used the following strategy. COS-7 cells were transfected with the cDNA encoding the HA-tagged selenoprotein SelX (Lescure et al., 1999) under experimental conditions (co-transfection of the tRNASec gene) allowing predominant translation of the 17 kDa full-length protein (Figure 6A, lane 2). The weaker intensity signal at 15 kDa arose from premature termination of translation at the selenocysteine codon read as a stop (Lescure et al., 1999). This is supported by the control using the SelX cDNA lacking the SECIS element, which should not lead to the 17 kDa protein (Figure 6B, lane 6). In that lane, the residual full-length SelX very probably arose from unspecific UGA suppression, as previously reported (Walczak et al., 1998; Lescure et al., 1999). If cells were transfected with an excess of SelX cDNA, the intensity of the 15 kDa band was on a par with that of the full-length protein (Figure 6B, lane 2). Co-transfection of the tRNASec gene did not significantly alter the pattern (Figure 6B, compare lanes 2 and 3). This could be interpreted to mean that one (or several) component(s) of the selenoprotein translation machinery, other than the tRNASec, was limiting in the cells to obtain efficient selenoprotein translation when higher amounts of SelX cDNA were transfected. Assuming that the specialized translation factor SelB was one such limiting component, the cells were co-transfected with the mSelB-encoding cDNA. Figure 6B, lane 4 shows that the translation pattern was affected dramatically since the intensity of the 15 kDa product dropped to the level observed in Figure 6A, lane 2, with a concomitant increase in the intensity of the 17 kDa protein (compare lanes 2 and 3 with lane 4 in Figure 6B). The effect was observed regardless of the presence or absence of the tRNASec gene (Figure 6B, lanes 5 and 4, respectively). A control with an anti-mSelB anti-peptide antibody established that mSelB was actually translated in the cells (lanes 4 and 5 in the bottom panel of Figure 6B). This experiment established that mSelB was limiting under our conditions and that its subsequent expression in transfected cells led to stimulation of selenoprotein translation by efficient readthrough of the selenocysteine codon. Figure 6.mSelB is required for efficient selenoprotein translation. (A) Separation of the 17 and 15 kDa SelX polypeptides (lane 2, with 3 μg of SelX expression vector). (B) Fractionation of the SelX polypeptides arising from cells transfected with an excess (10 μg) of SelX expression vector (lane 2), and co-transfected with the tRNASec gene (lane 3). Under the same conditions, co-transfection of the mSelB expression vector is shown, in the absence (lane 4) or presence (lane 5) of the tRNASec gene. Lane 6: expression of SelX in cells transfected with a cDNA lacking the SECIS element. Lanes 1 in (A) and (B) are controls with mock-transfected cells. Proteins were fractionated by 12% SDS–PAGE, blotted with the anti-HA antibody and revealed with the ECL kit. Bottom panel: same extracts as above on a separate (10%) gel where mSelB in lanes 4 and 5 was revealed with the anti-mSelB anti-peptide antibody. Download figure Download PowerPoint In contrast to eubacteria, the eukaryotic SelB does not bind the SECIS element specifically Eubacterial SelB factors recognize and bind specifically the bacterial SECIS RNA, even in the absence of Sec-tRNASec (Kromayer et al., 1996). To investigate whether mSelB possesses the same property, bandshift assays were performed with the SECIS element of the glutathione peroxidase (GPx) mRNA. Figure 7A shows that a retarded complex (marked by the arrow) appeared with 0.4 μg (lane 2) of recombinant mSelB. The intensity of the complex did not increase with higher amounts of mSelB (Figure 7A, 1 and 2 μg in lanes 3 and 4, respectively), and it even disappeared in the presence of a 2-fold molar excess (versus mSelB) of bulk yeast tRNA (lane 5). With the G24 SECIS mutant, which carries four Watson–Crick base pairs instead of the non-Watson–Crick base pair quartet (Walczak et al., 1998), a faint but nevertheless observable retarded complex was still obtained at high mSelB concentrations (Figure 7A, lanes 7–9). Collectively, the data showed that the complex formed between mSelB and the SECIS element was unspecific. We next wished to determine whether an ancillary factor, contained in fractionated HeLa whole-cell extracts, could promote the binding of mSelB to the SECIS element. Figure 7B, lane 3 showed that incubation of the wild-type SECIS with the extract led to three retarded bands: complex B marked by the arrow, and the other two denoted by the asterisks. Of the three, only complex B was specific since it was totally abrogated by the G24 SECIS mutant (Figure 7B, lane 8). Since the SECIS-binding protein SBP2 also responded to point mutations in the non-Watson–Crick base pair quartet of the SECIS element (Copeland and Driscoll, 1999), it is very likely that complex B contains SBP2. Surprisingly, complementation of 1 μg of the recombinant mSelB with the extract led to formation of the retarded complex A (Figure 7B, lane 4), with a much lower electrophoretic mobility than the unspecific complex formed with mSelB alone (Figure 7B, lane 2). Formation of complex A is dependent on mSelB since it appeared only in its presence (compare lanes 4 and 3, which contained whole-cell extract in the presence or absence of mSelB, respectively). Pre-incubation of mSelB with the anti-peptide anti-mSelB antibody was inhibitory to complex A formation (Figure 7B, lane 5), whereas this effect was not observed with the pre-immune IgGs (Figure 7B, lane 6), arguing that mSelB is actually contained in complex A. Finally, we could determine that the retarded complex A contained at least one component binding the SECIS element, because utilization of the G24 SECIS mutant abolished its formation (Figure 7B, lane 9). Figure 7.A protein from HeLa whole-cell extracts forms with mSelB a SECIS-dependent complex. (A) Gel retardation assays obtained with the wild-type GPx SECIS RNA alone (lane 1) or in the presence of increasing amounts of recombinant mSelB protein (400-, 1000- and 2000-fold molar excess mSelB/SECIS RNA, corresponding to 0.4, 1 and 2 μg in lanes 2–4, respectively). Lane 5 contained 700 ng of total yeast tRNA (2-fold molar excess/mSelB). Lanes 6–9 used the G24 GPx SECIS mutant depicted at the bottom. The non-Watson–Crick base pair quartet of the SECIS element is displayed. (B) The wild-type SECIS was incubated alone (lane 1), or in the presence of a 1000-fold molar excess of mSelB alone or complemented with 600 ng of fractionated whole-cell extract (lanes 3 and 4, respectively). The IgG fraction containing the anti-mSelB anti-peptide antiboby or the pre-immune IgGs (PI) were added in lanes 5 and 6, respectively. The G24 SECIS mutant was used in lanes 7–9 under the same conditions as in lanes 1, 3 and 4. Asterisks denote unspecific complexes. WCE, whole-cell extract. Download figure Download PowerPoint Therefore, the recombinant mSelB alone is unable to recognize the SECIS element specifically. However, mSelB forms a SECIS-dependent complex with at least one component contained in the HeLa whole-cell extract, likely to be SBP2. Putative SelB homologs in the Caenorhabditis elegans and Drosophila genomes A query of the C.elegans and Drosophila genomes with the mSelB amino acid sequence identified two genes (DDBJ/EMBL/GenBank accession Nos Z99709 and AC004434, respectively) that yielded 501 (C.elegans) and 494 (Drosophila) amino acid polypeptides bearing 26 and 38% sequence identity with mSelB, respectively (Figure 1). The sequence alignment in Figure 1 showed that both sequences carry the G1–G4 homology blocks and the Δ1–Δ4 deletions. It is worth noting the presence of the other deletion Δ5 in the C.elegans and Drosophila sequences that partially amputates the region corresponding to amino acid positions T373–Q397 in mSelB and A317–Q340 in hSelB. This deletion also occurs in MjSelB where it extends towards the N-terminus. The C.elegans and Drosophila proteins also carry C-terminal extensions with respect to EF1-α. Although shorter, they harbor significant blocks of homology with the mammalian and MjSelB. Taking into account the occurrence of the GTP-binding domain sequence similarities, the SelB-specific Δ1–Δ4 deletion domains and the presence of sequence similarities in the C-terminal extensions, it is very likely that the C.elegans and Drosophila genes encode the CeSelB and dSe