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.     

An essential non-Watson-Crick base pair motif in 3'UTR to mediate selenoprotein translation.

The SECIS element is an RNA hairpin in the 3'UTR of selenoprotein mRNAs required for decoding UGA selenocysteine codons. Our experimentally derived 2D structure model for the SECIS RNA revealed the conservation of four consecutive non-Watson-Crick base pairs, with a central G.A/A.G tandem. The present study was dedicated to gaining insight into the role of this quartet of base pairs. The effects of mutations introduced into the SECIS quartet of the glutathione peroxidase (GPx) cDNA, an enzyme with selenocysteine in its active center, were reported in vivo by the GPx activity. The detrimental consequence of an all-Watson-Crick mutant quartet disclosed the paramount importance of the non-Watson-Crick base pairs for GPx activity. Next, structure probing established that base pair changes in the central G.A/A.G tandem, predicted by the model to be structurally unfavorable, effectively led to local opening of the helix at the quartet. A concomitant abolition of GPx activity was observed, arising from translational impairment of full-length GPx. In contrast, an isosteric base pair replacement in the tandem did not affect base pairing in the quartet, leading to an almost wt GPx activity. Collectively, the data provided conclusive evidence for the functional relevance of these non-Watson-Crick base pairs in vivo, thus identifying a noncanonical RNA motif crucial to SECIS function in mediating selenoprotein translation. Within the quartet, the prominent requirement for the central G.A/A.G tandem is highlighted, our previous structural model and the mutagenesis data presented here strongly arguing in favor of a sheared arrangement for the G.A base pairs. The SECIS RNA is therefore another member to be added to the growing list of RNAs containing building blocks of non-Watson-Crick base pairs, required for structure and/or function.     

Functional characterization of the eukaryotic SECIS elements which direct selenocysteine insertion at UGA codons.

We investigated the requirements for selenocysteine insertion at single or multiple UGA codons in eukaryotic selenoproteins. Two functional SECIS elements were identified in the 3' untranslated region of the rat selenoprotein P mRNA, with predicted stem-loops and critical nucleotides similar to those in the SECIS elements in the type I iodothyronine 5' deiodinase (5'DI) and glutathione peroxidase selenoprotein mRNAs. Site-directed mutational analyses of three SECIS elements confirmed that conserved nucleotides in the loop and in unpaired regions of the stem are critical for activity. This indicates that multiple contact sites are required for SECIS function. Stop codon function at any of five out-of-context UGA codons in the 5'DI mRNA was suppressed by SECIS elements from the 5'DI or selenoprotein P genes linked downstream. Thus, the presence of SECIS elements in eukaryotic selenoprotein mRNAs permits complete flexibility in UGA codon position.     

谷胱甘肽过氧化物酶的硒代半胱氨酸插入元件

真核生物将硒代半胱氨酸插入蛋白质必需硒代半胱氨酸插入元件（ＳＥＣＩＳ）的参与,后者位于硒蛋白ｍＲＮＡ的３′非翻译区．采用ＲＮＡ折叠程序对１５个谷胱甘肽过氧化物酶基因进行计算机处理发现,其可能的ＳＥＣＩＳ中都具有３段保守碱基ＡＵＧＡ－Ａ（Ｇ）ＡＡ－ＧＡ．根据Ａ（Ｇ）ＡＡ位于顶环或者顶环上游５′臂的突环上,可将ＳＥＣＩＳ分为Ⅰ型和Ⅱ型结构     

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.     

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.     

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.     

Two distinct SECIS structures capable of directing selenocysteine incorporation in eukaryotes.

Translation of UGA as selenocysteine requires specific RNA secondary structures in the mRNAs of selenoproteins. These elements differ in sequence, structure, and location in the mRNA, that is, coding versus 3' untranslated region, in prokaryotes, eukaryotes, and archaea. Analyses of eukaryotic selenocysteine insertion sequence (SECIS) elements via computer folding programs, mutagenesis studies, and chemical and enzymatic probing has led to the derivation of a predicted consensus structural model for these elements. This model consists of a stem-loop or hairpin, with conserved nucleotides in the loop and in a non-Watson-Crick motif at the base of the stem. However, the sequences of a number of SECIS elements predict that they would diverge from the consensus structure in the loop region. Using site-directed mutagenesis to introduce mutations predicted to either disrupt or restore structure, or to manipulate loop size or stem length, we show that eukaryotic SECIS elements fall into two distinct classes, termed forms 1 and 2. Form 2 elements have additional secondary structures not present in form 1 elements. By either insertion or deletion of the sequences and structures distinguishing the two classes of elements while maintaining appropriate loop size, conversion of a form 1 element to a functional form 2-like element and of a form 2 to a functional form 1-like element was achieved. These results suggest commonality of function of the two classes. The information obtained regarding the existence of two classes of SECIS elements and the tolerances for manipulations of stem length and loop size should facilitate designing RNA molecules for obtaining high-resolution structural information about these elements.     

Selenocysteine incorporation in eukaryotes: insights into mechanism and efficiency from sequence, structure, and spacing proximity studies of the type 1 deiodinase SECIS element.

SECIS elements are stem-loop structures located in the 3' untranslated regions (UTRs) of eukaryotic selenoprotein mRNAs that are required for directing cotranslational selenocysteine incorporation at UGA codons. In prokaryotes, stem-loops mediating selenocysteine incorporation are located immediately downstream of the UGA selenocysteine codon, in the coding region. Previous characterization studies of the mammalian SECIS elements of type 1 deiodinase, glutathione peroxidase, and selenoprotein P showed that conserved nucleotides in the loops and unpaired bulges, and base pairing in the stems are required for SECIS function. These initial studies utilized approximately 175-230-nt segments of the 3'UTRs of the selenoprotein mRNAs. Here we define the minimal functional rat type 1 deiodinase SECIS element, a 45-nt segment, the 5' boundary of which corresponds precisely to the 5'-most critical conserved nucleotide identified previously. We also define base pairing requirements in the stem of this element. In view of the presence of SECIS elements in the open reading frames (ORFs) of bacterial selenoproteins, we examine the effects in the type 1 deiodinase of extending the ORF into the SECIS element, and find that this dramatically inhibits SECIS function. Finally, we define a minimal spacing requirement of 51-111 nt between a eukaryotic UGA selenocysteine codon and SECIS element.     

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.     

Structural analysis of new local features in SECIS RNA hairpins

Decoding of the UGA selenocysteine codon for selenoprotein translation requires the SECIS element, a stem-loop motif in the 3'-UTR of the mRNA carrying short or large apical loops. In previous structural studies, we derived a secondary structure model for SECIS RNAs with short apical loops. Work from others proposed that intra-apical loop base pairing can occur in those SECIS that possess large apical loops, yielding form 2 SECIS versus the form 1 with short loops. In this work, SECIS elements arising from eight different selenoprotein mRNAs were assayed by enzymatic and/or chemical probing showing that seven can adopt form 2. Further, database searches led to the discovery in drosophila and zebrafish of SECIS elements in the selenophosphate synthetase 2, type 1 deiodinase and SelW mRNAs. Alignment of SECIS sequences not only highlighted the predominance of form 2 but also made it possible to classify the SECIS elements according to the type of selenoprotein mRNA they belong to. Interestingly, the alignment revealed that an unpaired adenine, previously thought to be invariant, is replaced by a guanine in four SECIS elements. Tested in vivo, neither the A to G nor the A to U changes at this position greatly affected the activity while the most detrimental effect was provided by a C. The putative contribution of the various SECIS motifs to function and ligand binding is discussed.     

An improved definition of the RNA-binding specificity of SECIS-binding protein 2, an essential component of the selenocysteine incorporation machinery

By binding to SECIS elements located in the 3′-UTR of selenoprotein mRNAs, the protein SBP2 plays a key role in the assembly of the selenocysteine incorporation machinery. SBP2 contains an L7Ae/L30 RNA-binding domain similar to that of protein 15.5K/Snu13p, which binds K-turn motifs with a 3-nt bulge loop closed by a tandem of G.A and A.G pairs. Here, by SELEX experiments, we demonstrate the capacity of SBP2 to bind such K-turn motifs with a protruding U residue. However, we show that conversion of the bulge loop into an internal loop reinforces SBP2 affinity and to a greater extent RNP stability. Opposite variations were found for Snu13p. Accordingly, footprinting assays revealed strong contacts of SBP2 with helices I and II and the 5′-strand of the internal loop, as opposed to the loose interaction of Snu13p. Our data also identifies new determinants for SBP2 binding which are located in helix II. Among the L7Ae/L30 family members, these determinants are unique to SBP2. Finally, in accordance with functional data on SECIS elements, the identity of residues at positions 2 and 3 in the loop influences SBP2 affinity. Altogether, the data provide a very precise definition of the SBP2 RNA specificity.     

Ribosomal protein L30 is a component of the UGA-selenocysteine recoding machinery in eukaryotes

The translational recoding of UGA as selenocysteine (Sec) is directed by a SECIS element in the 3' untranslated region (UTR) of eukaryotic selenoprotein mRNAs. The selenocysteine insertion sequence (SECIS) contains two essential tandem sheared G.A pairs that bind SECIS-binding protein 2 (SBP2), which recruits a selenocysteine-specific elongation factor and Sec-tRNA(Sec) to the ribosome. Here we show that ribosomal protein L30 is a component of the eukaryotic selenocysteine recoding machinery. L30 binds SECIS elements in vitro and in vivo, stimulates UGA recoding in transfected cells and competes with SBP2 for SECIS binding. Magnesium, known to induce a kink-turn in RNAs that contain two tandem G.A pairs, decreases the SBP2-SECIS complex in favor of the L30-SECIS interaction. We propose a model in which SBP2 and L30 carry out different functions in the UGA recoding mechanism, with the SECIS acting as a molecular switch upon protein binding.     

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 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.     

Eukaryotic selenoprotein synthesis: mechanistic insight incorporating new factors and new functions for old factors

Selenium is an essential micronutrient that has been linked to various aspects of human health. Selenium exerts its biological activity through the incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationally at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of specific secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Herein, we discuss the various proteins known to function in eukaryotic selenoprotein biosynthesis, including several players whose roles have only been elucidated very recently.     

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.     

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.     

Nuclease sensitive element binding protein 1 associates with the selenocysteine insertion sequence and functions in mammalian selenoprotein translation

Biosynthesis of selenium-containing proteins requires insertion of the unusual amino acid selenocysteine by alternative translation of a UGA codon, which ordinarily serves as a stop codon. In eukaryotes, selenoprotein translation depends upon one or more selenocysteine insertion sequence (SECIS) elements located in the 3'-untranslated region of the mRNA, as well as several SECIS-binding proteins. Our laboratory has previously identified nuclease sensitive element binding protein 1 (NSEP1) as another SECIS-binding protein, but evidence has been presented both for and against its role in SECIS binding in vivo and in selenoprotein translation. Our current studies sought to resolve this controversy, first by investigating whether NSEP1 interacts closely with SECIS elements within intact cells. After reversible in vivo cross-linking and ribonucleoprotein immunoprecipitation, mRNAs encoding two glutathione peroxidase family members co-precipitated with NSEP1 in both human and rat cell lines. Co-immunoprecipitation of an epitope-tagged GPX1 construct depended upon an intact SECIS element in its 3'-untranslated region. To test the functional importance of this interaction on selenoprotein translation, we used small inhibitory RNAs to reduce the NSEP1 content of tissue culture cells and then examined the effect of that reduction on the activity of a SECIS-dependent luciferase reporter gene for which expression depends upon readthrough of a UGA codon. Co-transfection of small inhibitory RNAs directed against NSEP1 decreased its expression by approximately 50% and significantly reduced luciferase activity. These studies demonstrate that NSEP1 is an authentic SECIS binding protein that is structurally associated with the selenoprotein translation complex and functionally involved in the translation of selenoproteins in mammalian cells.     

SECIS-SBP2 interactions dictate selenocysteine incorporation efficiency and selenoprotein hierarchy

Selenocysteine incorporation at UGA codons requires cis-acting mRNA secondary structures and several specialized trans-acting factors. The latter include a selenocysteine-specific tRNA, an elongation factor specific for this tRNA and a SECIS-binding protein, SBP2, which recruits the elongation factor to the selenoprotein mRNA. Overexpression of selenoprotein mRNAs in transfected cells results in inefficient selenocysteine incorporation due to limitation of one or more of these factors. Using a transfection-based competition assay employing overexpression of selenoprotein mRNAs to compete for selenoprotein synthesis, we investigated the ability of the trans-acting factors to overcome competition and restore selenocysteine incorporation. We report that co-expression of SBP2 overcomes the limitation produced by selenoprotein mRNA overexpression, whereas selenocysteyl-tRNA and the selenocysteine-specific elongation factor do not. Competition studies indicate that once bound to SECIS elements, SBP2 does not readily exchange between them. Finally, we show that SBP2 preferentially stimulates incorporation directed by the seleno protein P and phospholipid hydroperoxide glutathione peroxidase SECIS elements over those of other selenoproteins. The mechanistic implications of these findings for the hierarchy of selenoprotein synthesis and nonsense-mediated decay are discussed.