Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon

The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.     

A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs

In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.     

A sequence in the Escherichia coli fdhF "selenocysteine insertion Sequence" (SECIS) operates in the absence of selenium

The UGA codon context of the Escherichia coli fdhF mRNA includes an element called the selenocysteine insertion sequence (SECIS) that is responsible for the UGA-directed incorporation of the amino acid selenocysteine into a protein. Here, we describe an extended fdhF SECIS that includes the information for an additional function: the prevention of UGA readthrough under conditions of selenium deficiency. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively, and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Here, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium, the SECIS enables one of two alternatives for the translational machinery: either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.     

SBP,SECIS binding protein,binds to the RNA fragment upstream of the Sec UGA codon in glutathione peroxidase mRNA

In mammals, most of the selenium contained in the body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is typically recognized as a translation stop signal, it is intriguing how a cell recognizes and distinguishes a UGA Sec codon from a UGA stop codon. For eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated the Sec insertion sequence (SECIS) in the 3'-untranslated (3'-UTR) region is required for recognition of UGA as a Sec codon. Some proteins which bind to SECIS (SBP) have been reported. However, it is not clear how the SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA Sec codons. The idea that there must be a signal near the UGA Sec codon is still considered. Therefore, we searched for a protein which binds to an RNA sequence surrounding the UGA Sec codon on human glutathione peroxidase (GPx) mRNA. We found a protein which strongly bound to the RNA fragment upstream of the UGA Sec codon. However, this protein did not bind to the RNA sequence downstream of the UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human GPx, and this binding to SECIS was competed with the RNA fragment upstream of the UGA Sec codon. Comparison of the RNA fragment with the SECIS fragment identified the conserved regions, which appeared in the region upstream of the in-frame UGA Sec codon of Se-protein mRNAs. Thus, this study proposes a novel model to understand the mechanisms of Sec incorporation at the UGA Sec codon, especially the regions upstream of the UGA codon of mRNAs of mammalian selenoproteins. This model explains that the stem-loop structure covering the UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by eRF of the peptide releasing factor.     

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.     

Selenocysteine insertion directed by the 3'-UTR SECIS element in Escherichia coli

Co-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3′-untranslated region (3′-UTR) and adjacent to a sequence complementary to the region downstream of the Sec UGA codon. Although the major readthrough event at the UGA codon was insertion of tryptophan, Sec was also incorporated and its insertion was dependent on the functional SECIS element in the UTR, base-pairing potential of the SECIS flanking region and the Sec UGA codon. These data provide important implications into evolution of SECIS elements and development of a system for heterologous expression of selenoproteins and show that in addition to the primary sequence arrangement between UGA codons and SECIS elements, their proximity within the tertiary structure can support Sec insertion in bacteria.     

Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2

The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3' untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.     

Efficiency of mammalian selenocysteine incorporation

Five components have thus far been identified that are necessary for the incorporation of selenocysteine (Sec) into approximately 25 mammalian proteins. Two of these are cis sequences, a SECIS element in the 3'-untranslated region and a Sec codon (UGA) in the coding region. The three known trans-acting factors are a Sec-specific translation elongation factor (eEFSec), the Sec-tRNA(Sec), and a SECIS-binding protein, SBP2. Here we describe a system in which the efficiency of Sec incorporation was determined quantitatively both in vitro and in transfected cells, and in which the contribution of each of the known factors is examined. The efficiency of Sec incorporation into a luciferase reporter system in vitro is maximally 5-8%, which is 6-10 times higher than that in transfected rat hepatoma cells, McArdle 7777. In contrast, the efficiency of Sec incorporation into selenoprotein P in vitro is approximately 40%, suggesting that as yet unidentified cis-elements may regulate differential selenoprotein expression. In addition, we have found that SBP2 is the only limiting factor in rabbit reticulocyte lysate but not in transfected rat hepatoma cells where SBP2 is found to be mostly if not entirely cytoplasmic despite having a strong putative nuclear localization signal. The significance of these findings with regard to the function of known Sec incorporation factors is discussed.     

An extended Escherichia coli "selenocysteine insertion sequence" (SECIS) as a multifunctional RNA structure

The genetic code, once thought to be rigid, has been found to permit several alternatives in its reading. Interesting alternative relates to the function of the UGA codon. Usually, it acts as a stop codon, but it can also direct the incorporation of the amino acid selenocysteine into a polypeptide. UGA-directed selenocysteine incorporation requires a cis-acting mRNA element called the "selenocysteine insertion sequence" (SECIS) that can form a stem-loop RNA structure. Here we discuss our investigation on the E. coli SECIS. This includes the follows: 1) The nature of the minimal E. coli SECIS. We found that in E. coli only the upper-stem and loop of 17 nucleotides of the SECIS is necessary for selenocysteine incorporation on the condition that it is located in the proper distance from the UGA [34]; 2) The upper stem and loop structure carries a bulged U residue that is required for selenocysteine incorporation [34] because of its interaction with SelB; and 3) We described an extended fdhF SECIS that includes the information for an additional function: The prevention of UGA readthrough under conditions of selenium deficiency [35]. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Finally, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium the SECIS enables one of two alternatives for the translational machinery: Either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.     

Dynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome

The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo. A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10%. This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS. When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced. When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression. The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present. The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs.     

A model for Sec incorporation with the regions upstream of the UGA Sec codon to play a key role

For eukaryotic selenoprotein mRNAs, it has been proposed that the SECIS element in the 3'-UTR is required for recognition of UGA as a Sec codon. Some proteins which bind to SECIS (SBP) have been reported. However, it is not clear how the SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA Sec codons. The idea that there must be a signal near the UGA Sec codon is still being considered. Therefore, we searched for a protein which binds to an RNA sequence surrounding the UGA Sec codon on human GPx mRNA. We found a protein, prepared from bovine brain microsomes, which strongly bound to the RNA fragment upstream of the UGA Sec codon but not to the RNA sequence downstream of the UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human GPx, and this binding to SECIS was competed with the RNA fragment upstream of the UGA Sec codon. We also obtained the similar results with the RNA fragments of type I iodothyronine 5'-deiodinase (5'DI) mRNAs. Comparison of such RNA fragments with SECIS fragments revealed similarities in the region upstream of the in-frame UGA Sec codon of several Se-protein mRNAs. The study thus favors a novel model of Sec incorporation at the UGA Sec codon that involves the regions upstream of the UGA codon of mRNAs of mammalian selenoproteins. This model explains that the stem-loop structure covering the UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by eRF.     

SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

Expression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3'-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins.     

A recoding element that stimulates decoding of UGA codons by Sec tRNA[Ser]Sec

Selenocysteine insertion during decoding of eukaryotic selenoprotein mRNA requires several trans-acting factors and a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 3' UTR. A second cis-acting selenocysteine codon redefinition element (SRE) has recently been described that resides near the UGA-Sec codon of selenoprotein N (SEPN1). Similar phylogenetically conserved elements can be predicted in a subset of eukaryotic selenoprotein mRNAs. Previous experimental analysis of the SEPN1 SRE revealed it to have a stimulatory effect on readthrough of the UGA-Sec codon, which was not dependent upon the presence of a SECIS element in the 3' UTR; although, as expected, readthrough efficiency was further elevated by inclusion of a SECIS. In order to examine the nature of the redefinition event stimulated by the SEPN1 SRE, we have modified an experimentally tractable in vitro translation system that recapitulates efficient selenocysteine insertion. The results presented here illustrate that the SRE element has a stimulatory effect on decoding of the UGA-Sec codon by both the methylated and unmethylated isoforms of Sec tRNA([Ser]Sec), and confirm that efficient selenocysteine insertion is dependent on the presence of a 3'-UTR SECIS. The variation in recoding elements predicted near UGA-Sec codons implies that these elements may play a differential role in determining the amount of selenoprotein produced by acting as controllers of UGA decoding efficiency.     

Efficient incorporation of multiple selenocysteines involves an inefficient decoding step serving as a potential translational checkpoint and ribosome bottleneck

Selenocysteine is incorporated into proteins via "recoding" of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3' untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a "bottleneck," slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.     

A protein binds the selenocysteine insertion element in the 3'-UTR of mammalian selenoprotein mRNAs.

Several gene products are involved in co-translational insertion of selenocysteine by the tRNA(Sec). In addition, a stem-loop structure in the mRNAs coding for selenoproteins is essential to mediate the selection of the proper selenocysteine UGA codon. Interestingly, in eukaryotic selenoprotein mRNAs, this stem-loop structure, the selenocysteine insertion sequence (SECIS) element, resides in the 3'-untranslated region, far downstream of the UGA codon. In view of unravelling the underlying complex mechanism, we have attempted to detect RNA-binding proteins with specificity for the SECIS element. Using mobility shift assays, we could show that a protein, present in different types of mammalian cell extracts, possesses the capacity of binding the SECIS element of the selenoprotein glutathione peroxidase (GPx) mRNA. We have termed this protein SBP, for Secis Binding Protein. Competition experiments attested that the binding is highly specific and UV cross-linking indicated that the protein has an apparent molecular weight in the range of 60-65 kDa. Finally, some data suggest that the SECIS elements in the mRNAs of GPx and another selenoprotein, type I iodothyronine 5' deiodinase, recognize the same SBP protein. This constitutes the first report of the existence of a 3' UTR binding protein possibly involved in the eukaryotic selenocysteine insertion mechanism.     

A SECIS binding protein (SBP) is distinct from selenocysteyl-tRNA protecting factor (SePF).

In mammals, most of the selenium contained in their body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is normally recognized as a translational stop signal, it is intriguing how cells recognize and distinguish the UGA Sec codon from the UGA stop codon. In eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated Sec insertion sequence (SECIS) located in the 3'-untranslated regions is required for recognition of UGA as a Sec codon. Although some proteins (SBPs) have been reported to bind to SECIS, it is not clear how the SECIS element can mediate Sec insertion at UGA. Eukaryotic Sec-tRNA(Sec) is not recognized by elongation factor EF-1alpha, but is recognized specifically by a Sec-tRNA(Sec) protecting factor, SePF, in bovine liver extracts. In this study, we provide evidence that SePF is distinct from SBP by chromatography. Upon UV irradiation, the SECIS RNA was cross-linked to a 47.5 kDa protein, a likely candidate of SBP, that is contained in the complex with a molecular mass of 150 kDa. These results suggest that SBP and SePF play different roles for the Sec incorporation. To our knowledge, this is the first demonstration that SBP is discriminated from the factor which directly recognizes Sec-tRNA(Sec), providing a novel clue to the mechanism of selenocysteine decoding in eukaryotes.     

Novel structural determinants in human SECIS elements modulate the translational recoding of UGA as selenocysteine

The selenocysteine insertion sequence (SECIS) element directs the translational recoding of UGA as selenocysteine. In eukaryotes, the SECIS is located downstream of the UGA codon in the 3′-UTR of the selenoprotein mRNA. Despite poor sequence conservation, all SECIS elements form a similar stem-loop structure containing a putative kink-turn motif. We functionally characterized the 26 SECIS elements encoded in the human genome. Surprisingly, the SECIS elements displayed a wide range of UGA recoding activities, spanning several 1000-fold in vivo and several 100-fold in vitro. The difference in activity between a representative strong and weak SECIS element was not explained by differential binding affinity of SECIS binding Protein 2, a limiting factor for selenocysteine incorporation. Using chimeric SECIS molecules, we identified the internal loop and helix 2, which flank the kink-turn motif, as critical determinants of UGA recoding activity. The simultaneous presence of a GC base pair in helix 2 and a U in the 5′-side of the internal loop was a statistically significant predictor of weak recoding activity. Thus, the SECIS contains intrinsic information that modulates selenocysteine incorporation efficiency.     

The selenocysteine incorporation machinery: interactions between the SECIS RNA and the SECIS-binding protein SBP2.

The decoding of UGA as a selenocysteine (Sec) codon in mammalian selenoprotein mRNAs requires a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region. The SECIS is a hairpin structure that contains a non-Watson-Crick base-pair quartet with a conserved G.A/A.G tandem in the core of the upper helix. Another essential component of the Sec insertion machinery is SECIS-binding protein 2 (SBP2). In this study, we define the binding site of SBP2 on six different SECIS RNAs using enzymatic and hydroxyl radical footprinting, gel mobility shift analysis, and phosphate-ethylation binding interference. We show that SBP2 binds to a variety of mammalian SECIS elements with similar affinity and that the SBP2 binding site is conserved across species. Based on footprinting studies, SBP2 protects the proximal part of the hairpin and both strands of the lower half of the upper helix that contains the non-Watson-Crick base pair quartet. Gel mobility shift assays showed that the G.A/A.G tandem and internal loop are critical for the binding of SBP2. Modification of phosphates by ethylnitrosourea along both strands of the non-Watson-Crick base pair quartet, on the 5' strand of the lower helix and part of the 5' strand of the internal loop, prevented binding of SBP2. We propose a model in which SBP2 covers the central part of the SECIS RNA, binding to the non-Watson-Crick base pair quartet and to the 5' strands of the lower helix and internal loop. Our results suggest that the affinity of SBP2 for different SECIS elements is not responsible for the hierarchy of selenoprotein expression that is observed in vivo.     

Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination

The synthesis of eukaryotic selenoproteins involves the recoding of an internal UGA codon as a site for selenocysteine incorporation. This recoding event is directed by a selenocysteine insertion sequence in the 3'-untranslated region. Because UGA also functions as a signal for peptidyl-tRNA hydrolysis, we have investigated how the rates of translational termination and selenocysteine incorporation relate to cis-acting elements in the mRNA as well as to trans-acting factors in the cytoplasm. We used cis-elements from the phospholipid glutathione peroxidase gene as the basis for this work because of its relatively high efficiency of selenocysteine incorporation. The last two codons preceding the UGA were found to exert a far greater influence on selenocysteine incorporation than nucleotides downstream of it. The efficiency of selenocysteine incorporation was generally much less than 100% but could be partially enhanced by concomitant overexpression of the tRNA(Sec) gene. The combination of two or three UGA codons in one reading frame led to a dramatic reduction in the yield of full-length protein. It is therefore unlikely that multiple incorporations of selenocysteine are processive with respect to the mode of action of the ribosomal complex binding to the UGA site. These observations are discussed in terms of the mechanism of selenoprotein synthesis and its ability to compete with termination at UGA codons.     

Interplay between termination and translation machinery in eukaryotic selenoprotein synthesis.

Termination of translation in eukaryotes is catalyzed by eRF1, the stop codon recognition factor, and eRF3, an eRF1 and ribosome-dependent GTPase. In selenoprotein mRNAs, UGA codons, which typically specify termination, serve an alternate function as sense codons. Selenocysteine incorporation involves a unique tRNA with an anticodon complementary to UGA, a unique elongation factor specific for this tRNA, and cis-acting secondary structures in selenoprotein mRNAs, termed SECIS elements. To gain insight into the interplay between the selenocysteine insertion and termination machinery, we investigated the effects of overexpressing eRF1 and eRF3, and of altering UGA codon context, on the efficiency of selenoprotein synthesis in a transient transfection system. Overexpression of eRF1 does not increase termination at naturally occurring selenocysteine codons. Surprisingly, selenocysteine incorporation is enhanced. Overexpression of eRF3 did not affect incorporation efficiency. Coexpression of both factors reproduced the effects with eRF1 alone. Finally, we show that the nucleotide context immediately upstream and downstream of the UGA codon significantly affects termination to incorporation ratios and the response to eRF overexpression. Implications for the mechanisms of selenocysteine incorporation and termination are discussed.