INTRODUCTION

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There are many examples of translational regulation that involve RNA binding proteins interacting with specific sequences in the 3' untranslated regions (UTRs) of various mRNAs (10, 18). A specialized case of this is found in the incorporation of selenocysteine (Sec) into a select group of eukaryotic proteins where a sequence specific 3' UTR binding protein is required for Sec insertion at its cognate UGA codon (6). The Sec-containing proteins that have been characterized perform myriad biological functions including oxidant defense and hormone maturation (reviewed in references 9 and 20). While mice deficient in the selenoprotein glutathione peroxidase are viable (11), one or more selenoproteins are apparently required for early development, as elimination of the tRNA^Sec gene in mice causes early embryo lethality (3).

While the incorporation of Sec into the Escherichia coli formate dehydrogenase isozymes is fairly well characterized (2), the system which governs mammalian Sec insertion has only been partially elucidated. Several essential components of what we here term the Sec insertion complex (SIC) have been identified. These include the UGA codon that encodes Sec (13), the Sec insertion sequence (SECIS) element found in all selenoprotein 3' UTRs (1), and the recently identified SECIS binding protein (SBP2), which we demonstrated to be required for Sec insertion in vitro (6). A fourth component of the SIC that is also likely to be required for Sec incorporation is the Sec-specific elongation factor (eEFsec) that has been recently shown to be functional in transfected cells (7, 21). Because Sec is encoded by what is ordinarily a stop codon, the mechanism of Sec insertion is likely to involve direct competition with translation termination. The SIC may therefore interact with or regulate the interactions of the release factors required for translation termination.

This work focuses on the RNA binding protein SBP2, a novel 94-kDa protein that shares a 32-amino acid motif with several ribosomal proteins and eukaryotic translation termination release factor 1 (eRF-1). eRF-1 functionally and structurally mimics a tRNA molecule that is specific for stop codons and in this fashion binds the ribosomal A site and terminates chain elongation (19). The fact that SBP2 and eRF-1 share this sequence suggests that they also share a common class of targets, which may shed light on the molecular basis for the competition between Sec insertion and termination. This conserved sequence has been proposed to be a novel RNA binding motif (12) and has recently been demonstrated to be involved in the binding of ribosomal protein L30 to its own mRNA (15). In this report, we establish that this motif, which we will refer to as the L30 RNA binding domain, is required for SBP2 RNA binding activity and function as measured by the ability to incorporate Sec in vitro. In addition, we have identified a nonoverlapping functional domain that is required for Sec insertion but not RNA binding. Further analysis indicates that SBP2 is stably associated with the ribosomal fraction in transfected cells and in vitro, and that this interaction may be mediated by 28S rRNA. We hypothesize that SBP2 may be involved in selecting a subset of ribosomes which are competent for Sec insertion.

	MATERIALS AND METHODS

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Mutagenesis. All truncated SBP2 constructs were derived from internal PCR primers (Table 1) which added a methionine to the N terminus. Annealing sites were chosen so that the optimal initiation sequence ATGG would be present for all truncation mutants except TM535-846, which used a naturally occurring ATG. PCR fragments were TA cloned into pCR3.1 (Invitrogen). Point mutations were made with an Altered Sites II Ex-1 mutagenesis kit (Promega). Mutant constructs were sequenced in their entirety by automated DNA sequencing. The human SBP2-like protein (hSLP) was derived from human hypothetical protein KIAA0256 (GenBank accession no. 6634006) obtained from the Kazusa DNA Research Institute (Kisarazu, Japan). This study made use of the C-terminal half of hSLP, which is similar to SBP2. It was obtained by PCR using primers hSLP-5' and hSLP-3' (Table 1).

RNA probes and binding assays. ³²P-labeled wild-type and mutant phospholipid hydroperoxide glutathione peroxidase (PHGPx) 3' UTR probes were synthesized exactly as described elsewhere (5). Electrophoretic mobility shift assays (EMSA) included 4.4 fmol of in vitro-translated SBP2 as indicated for the figures and 20 fmol of wild-type or mutant PHGPx 3' UTR in 1× phosphate-buffered saline (PBS) supplemented with 250 µg of E. coli tRNA per ml. 10 mM dithiothreitol (DTT), and 5 µg of soybean trypsin inhibitor (Sigma) per ml. Complexes were formed at 37°C for 30 min and then resolved on 4% nondenaturing polyacrylamide gels, which were dried and exposed to a PhosphorImager cassette (Molecular Dynamics).

In vitro translation. Plasmid DNAs containing wild-type and mutant SBP2 constructs were linearized with XbaI and used as templates for in vitro transcription with T7 RNA polymerase (Ribomax T7; Promega) in the presence of m⁷G(5')ppp(5')G (AP Biotech). A 50-ng aliquot of each SBP2 RNA was used in 12.5 or 25-µl rabbit reticulocyte lysate in vitro translation reactions in the presence of [³⁵S]Met as described by the manufacturer (Promega). Translation products were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel and quantitated by PhosphorImager analysis. The amount of each protein was determined by quantitation of known amounts of [³⁵S]Met spotted on 3-mm filter paper and calculated based on an endogenous concentration of 5 µM cold Met in the lysate (as specified by the manufacturer).

Expression of recombinant SBP2. For the construction of (Strep-tagged C-terminal SBP2, the original ATCC clone corresponding to expressed sequence tag H31811 was digested with EcoRI and XhoI and subcloned into the EcoRI and XhoI sites of pASK-IBA7 (Strep-tag II; Genosys). This vector added to the N terminus of the SBP2 clone the amino acid sequence MASWSHPQFEKIEGRRDRGP (the Strep tag sequence is underlined), which begins with amino acid 399.

Glycerol gradients. McArdle 7777 cells, a rat hepatoma cell line, were transiently transfected with wild-type and mutant SBP2 clones using LipofectAMINE (Life Technologies); 40 h posttransfection, the cells were washed and then harvested by scraping into PBSD (1× PBS, 2 mM DTT). The cells were disrupted by 15 strokes with a high-clearance Dounce homogenizer. The extracts were spun at 14,000 × g for 10 min at 4°C. The supernatants were diluted to 1 mg/ml, and 500 µl of each was loaded onto 10 to 30% linear glycerol gradients made in PBSD plus or minus NaCl or EDTA as indicated. For in vitro complex assembly, 5 µg of purified Strep-tagged SBP2 was incubated with or without 2.0 A₂₆₀ units of salt-washed ribosomes purified from rabbit reticulocyte lysate (generously provided by W. Merrick, Case Western Reserve University) in a total volume of 0.5 ml diluted with PBSD. The mixture was incubated at 37°C for 5 min, placed on ice for 10 to 15 min, and then loaded onto a 10 to 30% linear glycerol gradient made in PBSD. All gradients were spun at 210,000 × g in a SW41 rotor for 3.5 h (McArdle cell gradients) or 4.5 h (in vitro complexes) at 4°C. Fractions (0.6 ml) were pulled from the top of each gradient, and 200 µl from each fraction was subjected to trichloroacetic acid (TCA) precipitation (10% TCA, 0.05% Tween 20). Fractions from gradients that contained Strep-tagged recombinant SBP2 alone were supplemented with 5 µg of pure soybean trypsin inhibitor (Sigma) as a carrier. Precipitated proteins were resolved by SDS-PAGE (12% gel), blotted to nitrocellulose, blocked with 3% BSA in PBST (1× PBS plus 0.2% Tween 20), and, for Strep-tagged proteins, probed with a 1:4,000 dilution of alkaline phosphatase-conjugated streptavidin (AP Biotech). For untagged proteins, the blots were probed with a 1:2,000 dilution of anti-SBP2 polyclonal antibody followed by a 1:5,000 dilution of alkaline phosphatase-conjugated anti-rabbit immunoglobulin G secondary antibody. Blots were developed in nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (Promega).

PHGPx translation assay. PHGPx translation was monitored essentially as described previously (6), except that the protein was labeled with [³⁵S]Met instead of [⁷⁵Se]Sec. Our previous results showed that the incorporation of ⁷⁵Se-labeled Sec was both codon and SECIS element dependent (6). PHGPx translation can also be assayed by [³⁵S]Met labeling, which is also codon and SECIS element dependent and which yields exactly the same results as ⁷⁵Se labeling. Briefly, 4.4-fmol aliquots of in vitro-translated SBP2 proteins were added in a total volume of 2 µl (brought to volume with fresh rabbit reticulocyte lysate) to a standard 12.5-µl assay including 5 µCi of [³⁵S]Met. PHGPx translation products were purified from the entire reaction using bromosulfophthalein S-glutathione (BSP)-agarose, and the bound proteins were resolved by SDS-PAGE (15% gel) followed by autoradiography or PhosphorImager analysis.

	RESULTS

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Analysis of SBP2 truncation mutants. In earlier work, we showed that the N-terminal half of SBP2 is dispensable for both SECIS binding and selenoprotein synthesis (6). To gain more insight into the mechanism of SBP2 action, we constructed a series of truncation mutants in the C-terminal half of SBP2 in an effort to define domains necessary for RNA binding and functional activity. The truncation mutants (diagrammed in Fig. 2) are termed TMx-y, where x-y refers to the positions of the remaining amino acids derived from full-length SBP2. All constructs were efficiently translated in rabbit reticulocyte lysate (Fig. 1A) except for full-length SBP2 (lane 10). This is consistent with our difficulty in expressing full-length SBP2 in bacteria, but the basis for reduced translation is unknown. Excess SBP2 (4.4 fmol) protein was used in the following assays; the exception was the full-length protein, of which only 0.9 fmol was used. This amount of full-length SBP2 is still in excess for the functional assay but is within the lower portion of the linear range of the RNA binding assay (data not shown). As we previously reported, the apparent and predicted molecular masses of SBP2 are not in agreement and differ by approximately 26 kDa. The truncation mutants described above help to delineate the origin of this anomaly, as the difference in molecular mass between TM517-846 and TM585-846 is predicted to be only 7.4 kDa but is observed to be approximately 20 kDa by SDS-PAGE (Fig. 1). It should be noted that the major contaminating lower-molecular-mass band at 52 kDa is likely the result of internal initiation at Met 535. To test the contribution of this contaminant to SBP2 RNA binding and functional activity, TM535-846 (Fig. 1A, lane 11) was engineered to begin with Met 535 and runs at exactly the same molecular weight as the lower-molecular-weight band in lanes 1 to 3. 

View larger version (63K): [in this window] [in a new window]   FIG. 1.   Expression and activity of SBP2 truncation mutants. (A) [35S]Met-labeled in vitro-translated SBP2 resolved by SDS-PAGE. Mutants are identified by the amino acid positions indicated. (B) In vitro PHGPx translation assay in which 4.4 fmol of each SBP2 protein was added in the presence of [35S]Met. PHGPx was purified from the reaction using BSP-agarose. Lane 12 contains no SBP2. (C) EMSA for the mutants listed in panel A. 35S-labeled SBP2 (4.4 fmol) was incubated with 20 fmol of wild-type 32P-labeled PHGPx 3' UTR, and the complexes were resolved on a 4% nondenaturing gel. The asterisk indicates the position of a SECIS-specific complex in nonsupplemented reticulocyte lysate. (D) Same as panel C except that the probe was an AUGA deletion mutant of the PHGPx 3' UTR (14).

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Summary of SBP2 truncation mutant data. RNA binding and functional activity (Fig. 1) and ribosome binding activity (Fig. 6) are summarized to the right of the diagram of each SBP2 truncation mutant. The values for functional activity represent the percentage of the wild-type activity normalized to that found with the addition of C-terminal SBP2 (TM399-846); those for binding activity represent the percentage of wild-type probe shifted to slower-migrating species normalized to the shift observed with TM399-846. All quantitation was performed by PhosphorImager analysis. The putative functional domain as determined from the mutant data is indicated at the bottom. The hatched box on the full-length diagram represents the conserved L30 RNA binding domain. Constructs that were not tested in the ribosome binding assay are marked "ND."

Analysis of SBP2 point mutants. To further characterize the RNA binding domain, we introduced point mutations in the stretch of amino acids that is conserved among the sequences shown in the alignment of what we here term the L30 RNA binding motif (Fig. 3). No other significant similarity among these proteins exists. As shown in Fig. 3, SBP2 contains a Cys residue at position 25 (SBP2 position 684) that is unique among the group. To test if this unique Cys residue is required for binding, C684 was changed to the more commonly observed Leu or to a potentially disruptive Trp residue. As shown in Fig. 4B and C, neither mutant was significantly diminished in its ability to bind the PHGPx 3' UTR or enhance PHGPx translation. Quantitation of the effects indicates a slightly lower amount of both activities for the C684W mutant (Fig. 4). We suspect that the introduction of a bulky aromatic side group into the conserved RNA binding domain may cause some disruption of the local structure. Binding specificity is also not affected by these mutations, as shown in Fig. 4D, in which a PHGPx 3' UTR with a mutant SECIS element was used. These data indicate that the unique Cys residue found in SBP2 is not required for RNA binding activity.

View larger version (70K): [in this window] [in a new window]   FIG. 3.   Alignment of amino acids contained within the L30 RNA binding motif. Sequences are grouped according to similarity. The top group consists of the omnipotent suppressors of translation termination (SUP1) which have since been identified as eRF-1. The second group consists of the ribosomal protein L30 sequences. The third group consists of SBP2 and hSLP. The remaining groups consist of ribosomal proteins and RimK. Conserved amino acids are in boldface. Species noted include Trypanosoma cruzi, Methanococcus vannielii, Suefolobus acidocaldarius, Haloarcula marismortui, Bacillus subtilis, and Trypanosoma brucei.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Analysis of SBP2 point mutants and hSLP. (A) [35S]Met-labeled SBP2 proteins were resolved by SDS-PAGE. (B) Assay of PHGPx expression as describe for Fig. 1B. Lane 8 contains no SBP2. (C) EMSA for SBP2 mutant proteins as described for Fig. 1C. (D) Same as panel C except that the probe was an AUGA deletion mutant of the PHGPx 3' UTR. Lane 7 contains 2 µl of rabbit reticulocyte lysate without SBP2; lane 8 contains probe in the absence of rabbit reticulocyte lysate. (E) Quantitation of the results in panels B and C expressed as percentage of activity (%Act) or percentage of binding (%Binding) as described for Fig. 2.

Interaction of SBP2 with ribosomes. Our current model of Sec incorporation employs SBP2 in preventing termination while perhaps simultaneously delivering a functional SBP2-eEFsec-Sec-tRNA^Sec complex to the ribosomal A site. To test whether SBP2 is stably associated with ribosomes, we used glycerol gradients to study SBP2 sedimentation after transient transfection of wild-type and mutant SBP2 constructs. Our initial attempts to use standard sucrose-based polysome gradients failed due to the sensitivity of the SBP2 complex to the artificially high magnesium concentration necessary to stabilize polysomes (data not shown). Therefore, the conditions used for this experiment do not show an analysis of mRNAs stably associated with the ribosomal fraction. Strep-tagged C-terminal SBP2 (TM399-846) was transiently transfected into the rat hepatoma cell line McArdle 7777. After 40 h, extracts from these cells were fractionated on 10 to 30% glycerol gradients. Fractions were collected and precipitated with TCA followed by Western blot analysis using an anti-Strep tag probe. Figure 5 shows the results of three gradients run under various conditions. Figure 5A indicates that the vast majority of Strep-tagged SBP2 sediments with the heavier fractions (numbers 8 to 10) under low-stringency conditions (PBS-2 mM DTT). The addition of 0.5 M NaCl to the extraction and gradient buffers resulted in complete disruption of the complex, as shown in Fig. 5B. Complex formation was not sensitive to 5 mM EDTA (Fig. 5C), suggesting that divalent metal ions are not necessary for this interaction. Reprobing of these blots with anti-SBP2 antibody indicates that all detectable endogenous SBP2 is found to cofractionate with fractions 8 to 10 (data not shown). It is unlikely that transfected SBP2 is "pulling" endogenous SBP2 onto the ribosomes because our analysis of cells transfected with the N-terminal half of SBP2, which is not ribosome associated, also shows that the endogenous protein sediments with ribosomes (data not shown). Staining of the blot in Fig. 5A for total protein demonstrated that most protein sedimented in the first five fractions of the gradient (Fig. 5F). To ascertain whether or not a SECIS element was involved in this interaction, we extracted RNA from the EDTA-containing gradient fractions and analyzed the distribution of PHGPx mRNA by Northern blot analysis. PHGPx mRNA was chosen because it is expressed to relatively high levels in McArdle cells (8) and because GPx mRNA was not detectable in this experiment, which contains limited amounts of mRNA. As shown in Fig. 5D, the bulk of PHGPx mRNA does not cosediment with SBP2, suggesting that SBP2 is not stably associated with SECIS elements and the ribosome simultaneously. This was a surprising result and may reflect an effect of the methods used, but it may suggest that SBP2 preferentially binds to ribosomes and interacts with SECIS elements only after translation initiation. PHGPx mRNA sedimentation was the same as that of nonselenoprotein (glyceraldehyde-3-phosphate dehydrogenase) mRNA (data not shown). To analyze the positions of the ribosomal subunits, the Northern gel described above was stained with ethidium bromide to allow visualization of the positions of the 18S and 28S rRNAs in the gradient, which indicates the positions of the 40 and 60/80S ribosomal subunits (Fig. 5E). These results suggest that SBP2 is stably associated with either 60S or 80S, but not isolated 40S, ribosomal subunits.

View larger version (65K): [in this window] [in a new window]   FIG. 5.   Sedimentation of SBP2 in glycerol gradients. Strep-tagged TM399-846 was transiently transfected into McArdle 7777 cells, and 500-µg aliquots of extracted proteins were loaded onto 10 to 30% linear glycerol gradients. Fractions (0.6 ml) were collected from the top of each gradient. Proteins were TCA precipitated, resolved by SDS-PAGE, blotted to nitrocellulose, and probed with alkaline phosphatase-conjugated streptavidin, which recognizes the Strep tag. (A to C) Gradients run in PBSD, PBSD plus 0.5 M NaCl, and PBSD plus 5 mM EDTA respectively. (D) RNA was extracted from the fractions run in 5 mM EDTA, resolved on a 1% denaturing agarose gel, blotted to nylon, and probed for PHGPx mRNA. (E) Gel in panel D stained with ethidium bromide. (F) Blot in panel A stained with India ink.

View larger version (35K): [in this window] [in a new window]   FIG. 6.   Sedimentation of SBP2 mutants in glycerol gradients. McArdle 7777 cell extracts containing the mutant SBP2 proteins indicated to the right were loaded onto glycerol gradients as described for Fig. 5. TM399-846, G669R, and TM1-397 are Strep-tagged proteins detected with alkaline phosphatase-conjugated streptavidin. The remainder of the proteins were untagged and detected with anti-SBP2 antibodies.

View larger version (8K): [in this window] [in a new window]   FIG. 7.   Sedimentation of recombinant SBP2 and purified ribosomes. (A) Purified Strep-tagged C-terminal SBP2 (TM399-846; 5 µg) was layered onto a 10 to 30% glycerol gradient. Fractions processed as described for Fig. 5. (B) Purified SBP2 (TM399-846; 5 µg) was incubated with 2.0 A260 units of purified salt-washed ribosomes from rabbit reticulocyte lysate and then layered onto a glycerol gradient and processed as for panel A. (C) Same as panel B except that 0.5 M NaCl was added to the reaction mix.

SBP2 interacts with rRNA. Because the RNA binding domain of SBP2 is required for ribosome binding and because ribosomal protein L30 uses a similar domain to interact with its own mRNA as well as 28S rRNA (15, 22), we decided to test for the ability of SBP2 to bind rRNA in vitro. Toward this end, Strep-tagged TM399-846 was incubated with rRNA extracted from the purified salt-washed ribosomes used for Fig. 7 and subjected to glycerol gradient fractionation. As shown in Fig. 8A, SBP2 is not found in the early fractions (numbers 3 and 4) where the 18S rRNA alone is present. However, SBP2 does form a complex with 28S rRNA or the combination of 28S and 18S rRNAs which, as shown in Fig. 8B, is present in fractions 6 to 9. While we cannot rule out that this interaction requires the presence of both rRNA species, it is likely that the SBP2-ribosome interaction depends on SBP2 binding to 28S rRNA.

View larger version (38K): [in this window] [in a new window]   FIG. 8.   Cosedimentation of SBP2 and purified rRNA. Purified Strep-tagged C-terminal SBP2 (TM399-846; 5 µg) was incubated with purified total rRNA and fractionated on a 10 to 30% glycerol gradient. Gradient fractions were analyzed for SBP2 content by Western blotting (A) and rRNA content by agarose gel electrophoresis and ethidium bromide staining (B).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The recent discovery of two novel factors involved in mammalian selenoprotein synthesis, SBP2 and eEFSec, has shed significant light on the mechanism of Sec incorporation. The latter of these factors has been described as the Sec-specific elongation factor which is required for delivering the Sec-tRNA^Sec to the ribosomal A site. The role of SBP2 is much less clear, and we have previously hypothesized that it is involved in the other major dynamic presumed to be required for Sec insertion, competition with translation termination. Here we describe the beginning of a detailed investigation of the structure and function of SBP2 in order to elucidate its role in Sec insertion. From this and previous work, we know that SBP2 is a multifunctional protein that binds to SECIS elements, interacts with eEFSec, and also binds stably to ribosomes.

As shown in Fig. 3, SBP2 shares a sequence motif with a variety of eukaryotic and prokaryotic proteins. The best-studied member of this family is ribosomal protein L30 which is involved in its own splicing and translational regulation by means of binding to its own pre-mRNA and mRNA, respectively. The fact that the L30 RNA binding domain is involved in RNA binding has been established by extensive nuclear magnetic resonance analysis on the L30 protein and mRNA target (15). While there is no sequence similarity between the L30 mRNA and a SECIS element, the overall structures of both SBP2 and L30 binding sites can be described as a bulge flanked on both sides by paired sequences (5, 15), and both sequences may form the GA quartet that is known to be essential for Sec insertion. Interestingly, the L30 target site on 25S rRNA from Saccharomyces cerevisiae has also recently been determined and found to be very similar to the L30 mRNA binding site in both primary and secondary structures (22). The truncation and point mutants described in this report establish that the L30 RNA binding domain is required for SBP2 RNA binding and functional activity. However, mutations which eliminate SECIS binding do not eliminate ribosome association, suggesting that this motif is not strictly relegated to ribosome-rRNA interactions. In addition, the conserved motif is not sufficient for sequence specific binding, as demonstrated by the truncation mutants which still retain the motif but are unable to sustain binding activity. Furthermore, the SBP2-related protein that was tested here was barely able to bind the PHGPx 3' UTR, suggesting that even a related sequence is not sufficient for specific binding. This point is further supported by the fact that the L30 nuclear magnetic resonance data indicate that residues C terminal to the conserved region are involved in protein-RNA contacts (15). These residues are not conserved in SBP2.

In terms of SBP2 function, it is clear from the data presented here that RNA binding activity is required for function and that a functional domain can be identified as a discrete stretch of amino acids. This region, however, is not a member of any known sequence families and thus does not shed light on its mechanism of action. It is likely that this region is involved in the interactions necessary for Sec insertion, and so our current efforts are involved in identifying the critical residues in this region and testing mutants for their ability to interact with eEFSec. It is possible, for example, that this region is involved directly with preventing termination either by binding to the release factor(s) directly or by blocking access of eRF-1 to the ribosomal A site. We have thus far been unable to demonstrate any interaction between SBP2 and either of the eukaryotic release factors (eRF-1 or eRF-3), but work is still in progress on that front.

Perhaps the most significant finding from this work is that SBP2 is stably associated with the ribosomal fraction of glycerol gradients both in vivo and in vitro. This interaction appears to be direct, as purified SBP2 is able to interact with salt-washed purified ribosomes. It is striking that the point mutant that fails to bind the SECIS element still associates with the ribosome, but that deletions which encroach on the RNA binding domain greatly reduce ribosome association. In light of these results, we propose that the ribosome binding domain and SECIS binding domain overlap but are not identical. Considering that SBP2 appears to form a homomeric complex based on its sedimentation in glycerol gradients in the absence of any other factors, it is tempting to suggest that an SBP2 dimer in a head-to-head orientation uses one RNA binding domain to interact with the ribosome and the other to interact with the SECIS element.

These results lead us to speculate that SBP2 may be involved in selecting ribosomes for Sec insertion. That is, by binding to a subset of ribosomes without discrimination, SBP2 is making that pool of ribosomes competent for Sec insertion. This model would predict, therefore, that selenoproteins translated on ribosomes lacking SBP2 will terminate at the Sec codon, while those being translated on SBP2-containing ribosomes will bypass termination and insert Sec. While the data presented here do not directly support the idea that SBP2 is involved in preventing termination, we anticipate that future work which will define the site of the SBP2-ribosome interaction should shed light on potential interactions with the termination mechanism. The binding of SBP2 to the ribosome being the first step, a second step will likely consist of quaternary complex formation (SBP2, SECIS element, eEFSec, and Sec-tRNA^Sec), and that this step will also be regulated by SBP2 by means of differential affinity for the various SECIS elements. Thus, it is conceivable that SBP2 is the major determinant of the efficiency of selenoprotein synthesis, and that it may be involved in the differential stability of selenoprotein mRNA that is known to be highly regulated during selenium deficiency (4, 17). In reticulocyte lysates SBP2 is the clearly limiting factor, and this may also be true in tissues that have limited selenoprotein synthesis capacity.