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.     

The efficiency of Escherichia coli selenocysteine insertion is influenced by the immediate downstream nucleotide

Selenocysteine (Sec) incorporation requires the TGA opal codon and a downstream Sec insertion sequence (SECIS), which can be partially randomized and cloned into M13 pIII fusion constructs for phage display. This combinatorial approach provides a convenient non-radioactive assay that couples phage production to opal suppression. Two SECIS libraries were prepared, with the immediate downstream nucleotide either randomized (TGAN) or fixed as thymidine (TGAT). The TGAN library resulted in a majority of clones with a downstream purine and selenium-independent phage production, implicating the endogenous tryptophan-inserting opal suppression pathway. Although the addition of sodium selenite to the growth medium did not affect phage production, it did increase the level of Sec insertion, as shown by the chemical reactivity of the resulting phage. The TGAT phage library yielded clones with strictly selenium-dependent phage production and reactivity consistent with the presence of Sec. These clones were prone to spontaneous mutation upon further propagation, however, resulting in loss of the selenium-dependent phenotype. We conclude that the immediate downstream nucleotide determines whether the endogenous opal suppression pathway competes with co-translational Sec insertion.     

Revised Escherichia coli selenocysteine insertion requirements determined by in vivo screening of combinatorial libraries of SECIS variants

To investigate the stringency of the Escherichia coli selenocysteine insertion sequence (SECIS) requirements, libraries of SECIS variants were screened via a novel method in which suppression of the selenocysteine (Sec) opal codon was coupled to bacteriophage plaque formation. The SECIS variant libraries were designed with a mostly paired lower stem, so that randomization could be focused on the upper stem and loop regions. We identified 19 functional non-native SECIS sequences that violated the expected pairing requirements for the SECIS upper stem. All of the SECIS variants were shown to permit Sec insertion in phage (by chemical modification of the Sec residue) and fused to lacZα (by β-galactosidase assay). The diminished pairing of the upper stem appears to be mitigated by the overall stem stability; a given upper stem variant has significantly higher readthrough in the context of a paired, rather than unpaired, lower stem. These results suggest an unexpected downstream sequence flexibility in prokaryotic selenoprotein expression.     

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.     

Wobble decoding by the Escherichia coli selenocysteine insertion machinery

Selenoprotein expression in Escherichia coli redefines specific single UGA codons from translational termination to selenocysteine (Sec) insertion. This process requires the presence of a Sec Insertion Sequence (SECIS) in the mRNA, which forms a secondary structure that binds a unique Sec-specific elongation factor that catalyzes Sec insertion at the predefined UGA instead of release factor 2-mediated termination. During overproduction of recombinant selenoproteins, this process nonetheless typically results in expression of UGA-truncated products together with the production of recombinant selenoproteins. Here, we found that premature termination can be fully avoided through a SECIS-dependent Sec-mediated suppression of UGG, thereby yielding either tryptophan or Sec insertion without detectable premature truncation. The yield of recombinant selenoprotein produced with this method approached that obtained with a classical UGA codon for Sec insertion. Sec-mediated suppression of UGG thus provides a novel method for selenoprotein production, as here demonstrated with rat thioredoxin reductase. The results also reveal that the E. coli selenoprotein synthesis machinery has the inherent capability to promote wobble decoding.     

Functional analysis of the interplay between translation termination, selenocysteine codon context, and selenocysteine insertion sequence-binding protein 2

A selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region and an in-frame UGA codon are the requisite cis-acting elements for the incorporation of selenocysteine into selenoproteins. Equally important are the trans-acting factors SBP2, Sec-tRNA[Ser]Sec, and eEFSec. Multiple in-frame UGAs and two SECIS elements make the mRNA encoding selenoprotein P (Sel P) unique. To study the role of codon context in determining the efficiency of UGA readthrough at each of the 10 rat Sel P Sec codons, we individually cloned 27-nucleotide-long fragments representing each UGA codon context into a luciferase reporter construct harboring both Sel P SECIS elements. Significant differences, spanning an 8-fold range of UGA readthrough efficiency, were observed, but these differences were dramatically reduced in the presence of excess SBP2. Mutational analysis of the "fourth base" of contexts 1 and 5 revealed that only the latter followed the established rules for hierarchy of translation termination. In addition, mutations in either or both of the Sel P SECIS elements resulted in differential effects on UGA readthrough. Interestingly, even when both SECIS elements harbored a mutation of the core region required for Sec incorporation, context 5 retained a significantly higher level of readthrough than context 1. We also show that SBP2-dependent Sec incorporation is able to repress G418-induced UGA readthrough as well as eRF1-induced stimulation of termination. We conclude that a large codon context forms a cis-element that works together with Sec incorporation factors to determine readthrough efficiency.     

Supramolecular complexes mediate selenocysteine incorporation in vivo

Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA([Ser]Sec) and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA([Ser]Sec), termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA([Ser]Sec)-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.     

Selenoproteins and selenocysteine insertion system in the model plant cell system,Chlamydomonas reinhardtii

Known eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals.     

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.     

In vivo regulation of acetylcholinesterase insertion at the neuromuscular junction

The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse.     

Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome.

The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet). Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.     

Dynamics of in vivo power output and efficiency of Nasonia asynchronous flight muscle

By simultaneously measuring aerodynamic performance, wing kinematics, and metabolic activity, we have estimated the in vivo limits of mechanical power production and efficiency of the asynchronous flight muscle (IFM) in three species of ectoparasitoid wasps genus Nasonia (N. giraulti, N. longicornis, and N. vitripennis). The 0.6 mg animals were flown under tethered flight conditions in a flight simulator that allowed modulation of power production by employing an open-loop visual stimulation technique. At maximum locomotor capacity, flight muscles of Nasonia are capable to sustain 72.2 +/- 18.3 W kg(-1) muscle mechanical power at a chemo-mechanical conversion efficiency of approximately 9.8 +/- 0.9%. Within the working range of the locomotor system, profile power requirement for flight dominates induced power requirement suggesting that the cost to overcome wing drag places the primary limit on overall flight performance. Since inertial power is only approximately 25% of the sum of induced and profile power requirements, Nasonia spp. may not benefit from elastic energy storage during wing deceleration phases. A comparison between wing size-polymorphic males revealed that wing size reduction is accompanied by a decrease in total flight muscle volume, muscle mass-specific mechanical power production, and total flight efficiency. In animals with small wings maximum total flight efficiency is below 0.5%. The aerodynamic and power estimates reported here for Nasonia are comparable to values reported previously for the fruit fly Drosophila flying under similar experimental conditions, while muscle efficiency of the tiny wasp is more at the lower end of values published for various other insects.     

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.     

A novel RNA structural motif in the selenocysteine insertion element of eukaryotic selenoprotein mRNAs.

In eukaryotes, co-translational insertion of selenocysteine into selenoproteins necessitates the participation of the selenocysteine insertion sequence (SECIS), an element lying in the 3'-untranslated region of selenoprotein mRNAs. We report a detailed experimental study of the secondary structures of the SECIS elements of three selenoprotein mRNAs, the rat and human type I iodothyronine deiodinase (5'DI) and rat glutathione peroxidase (GPx). Based on RNase and chemical probing, a new secondary structure model is established. It is characterized by a stem-loop structure, comprising two helices (I and II) separated by an internal loop, with an apical loop surmounting helix II. Sequence comparisons of 20 SECIS elements, arising from 2 5'DI, 13 GPx, 2 selenoprotein P, and 1 selenoprotein W mRNAs, confirm the secondary structure model. The most striking finding of the experimental study concerns a set of conserved sequences in helix II that interact to form a novel RNA structural motif consisting of a quartet composed of non-Watson-Crick base pairs 5'UGAY3': 5'UGAU3'. The potential for forming the quartet is preserved in 15 SECIS elements, but three consecutive non-Watson-Crick base pairs can nevertheless form in the other five SECIS, the central G.A tandem being invariant in all cases. A 3D model, derived by computer modeling with the use of the solution data, suggests that the base pairing interactions in the G.A tandem are of the type found in GNRA loops. The 3D model displays the quartet lying in an accessible position at the foot of helix II, which is bent at the internal loop, suggesting that the non-Watson-Crick base pair arrangement provides an unusual pattern of chemical groups for putative ligand interaction.     

An algorithm for identification of bacterial selenocysteine insertion sequence elements and selenoprotein genes

MOTIVATION: Incorporation of selenocysteine (Sec) into proteins in response to UGA codons requires a cis-acting RNA structure, Sec insertion sequence (SECIS) element. Whereas SECIS elements in Escherichia coli are well characterized, a bacterial SECIS consensus structure is lacking. RESULTS: We developed a bacterial SECIS consensus model, the key feature of which is a conserved guanosine in a small apical loop of the properly positioned structure. This consensus was used to build a computational tool, bSECISearch, for detection of bacterial SECIS elements and selenoprotein genes in sequence databases. The program identified 96.5% of known selenoprotein genes in completely sequenced bacterial genomes and predicted several new selenoprotein genes. Further analysis revealed that the size of bacterial selenoproteomes varied from 1 to 11 selenoproteins. Formate dehydrogenase was present in most selenoproteomes, often as the only selenoprotein family, whereas the occurrence of other selenoproteins was limited. The availability of the bacterial SECIS consensus and the tool for identification of these structures should help in correct annotation of selenoprotein genes and characterization of bacterial selenoproteomes.     

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.     

Characterization of the SECIS binding protein 2 complex required for the co-translational insertion of selenocysteine in mammals

Selenocysteine is incorporated into at least 25 human proteins by a complex mechanism that is a unique modification of canonical translation elongation. Selenocysteine incorporation requires the concerted action of a kink-turn structural RNA (SECIS) element in the 3′ untranslated region of each selenoprotein mRNA, a selenocysteine-specific translation elongation factor (eEFSec) and a SECIS binding protein (SBP2). Here, we analyze the molecular context in which SBP2 functions. Contrary to previous findings, a combination of gel filtration chromatography and co-purification studies demonstrates that SBP2 does not self-associate. However, SBP2 is found to be quantitatively associated with ribosomes. Interestingly, a wild-type but not mutant SECIS element is able to effectively compete with the SBP2 ribosome interaction, indicating that SBP2 cannot simultaneously interact with the ribosome and the SECIS element. This data also supports the hypothesis that SBP2 interacts with one or more kink turns on 28S rRNA. Based on these results, we propose a revised model for selenocysteine incorporation where SBP2 remains ribosome bound except during selenocysteine delivery to the ribosomal A-site.     

The efficiency of a cis-cleaving ribozyme in an mRNA coding region is influenced by the translating ribosome in vivo.

A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene. The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension. It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA. Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein. Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product. However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein. Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.