Decoding apparatus for eukaryotic selenocysteine insertion

Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3′ untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor–SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA–elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.     

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

Decoding of UGA selenocysteine codons in eubacteria is mediated by the specialized elongation factor SelB, which conveys the charged tRNA(Sec) to the A site of the ribosome, through binding to the SECIS mRNA hairpin. In an attempt to isolate the eukaryotic homolog of SelB, a database search in this work identified a mouse expressed sequence tag containing the complete cDNA encoding a novel protein of 583 amino acids, which we called mSelB. Several lines of evidence enabled us to establish that mSelB is the bona fide mammalian elongation factor for selenoprotein translation: it binds GTP, recognizes the Sec-tRNA(Sec) in vitro and in vivo, and is required for efficient selenoprotein translation in vivo. In contrast to the eubacterial SelB, the recombinant mSelB alone is unable to bind specifically the eukaryotic SECIS RNA hairpin. However, complementation with HeLa cell extracts led to the formation of a SECIS-dependent complex containing mSelB and at least another factor. Therefore, the role carried out by a single elongation factor in eubacterial selenoprotein translation is devoted to two or more specialized proteins in eukaryotes.     

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.     

cDNA cloning,expression pattern and RNA binding analysis of human selenocysteine insertion sequence (SECIS) binding protein 2

Selenocysteine and selenoprotein synthesis require a complex molecular machinery in mammals. Among the key players is the RNA-protein complex formed by the selenocysteine insertion sequence (SECIS) binding protein (SBP2) and the SECIS element, an RNA hairpin in the 3' untranslated regions of selenoprotein messenger RNAs (mRNAs). We have isolated the DNA complementary to mRNA of the human SBP2, enabling us to establish that it differs from a previously reported human SBP2-like protein. Examination of the expression pattern revealed that the human SBP2 protein is encoded by a 4 kb long mRNA that is over-expressed in testis. Compared to the rat SBP2 sequence, the human SBP2 protein displays two highly conserved domains with 92 and 95% amino acid identity, the latter one containing the RNA binding domain. The inter-domain section carries 55% sequence identity, the remainder of the SBP2 sequences showing about 65% identity, values lower than expected for two mammalian proteins. Interestingly, we could show that the binding of human SBP2 to the SECIS RNA is stimulated by the selenoprotein-specialized elongation translation factor mSelB/eEFsec.     

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.     

Evolutionary history of selenocysteine incorporation from the perspective of SECIS binding proteins

Background  

The co-translational incorporation of selenocysteine into nascent polypeptides by recoding the UGA stop codon occurs in all domains of life. In eukaryotes, this event requires at least three specific factors: SECIS binding protein 2 (SBP2), a specific translation elongation factor (eEFSec), selenocysteinyl tRNA, and a cis -acting selenocysteine insertion sequence (SECIS) element in selenoprotein mRNAs. While the phylogenetic relationships of selenoprotein families and the evolution of selenocysteine usage are well documented, the evolutionary history of SECIS binding proteins has not been explored.     

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.     

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.     

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.     

The Selenocysteine-specific Elongation Factor Contains a Novel and Multi-functional Domain

The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNA^Sec) to the ribosome and suppresses UGA codons that are upstream of Sec insertion sequence (SECIS) elements bound by SECIS-binding protein 2 (SBP2). Multiple studies have highlighted the importance of SBP2 forming a complex with the SECIS element, but it is not clear how this regulates eEFSec during Sec incorporation. Compared with the canonical elongation factor eEF1A, eEFSec has a unique C-terminal extension called Domain IV. To understand the role of Domain IV in Sec incorporation, we examined a series of mutant proteins for all of the known molecular functions for eEFSec: GTP hydrolysis, Sec-tRNA^Sec binding, and SBP2/SECIS binding. In addition, wild-type and mutant versions of eEFSec were analyzed for Sec incorporation activity in a novel eEFSec-dependent translation extract. We have found that Domain IV is essential for both tRNA and SBP2 binding as well as regulating GTPase activity. We propose a model where the SBP2/SECIS complex activates eEFSec by directing functional interactions between Domain IV and the ribosome to promote Sec-tRNA^Sec binding and accommodation into the ribosomal A-site.     

Selenocysteine insertion sequence (SECIS)-binding protein 2 alters conformational dynamics of residues involved in tRNA accommodation in 80 S ribosomes

Sec-tRNA(Sec) is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine insertion sequence (SECIS) element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNA(Sec) is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNA(Sec) accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.     

Purification and characterization of hexahistidine-tagged elongation factor SelB

The cotranslational incorporation of selenocysteine into proteins is mediated by a specialized elongation factor, named SelB. Its amino-terminal three domains show homology to elongation factor EF-Tu and accordingly bind GTP and selenocysteyl-tRNASec. In addition, SelB exhibits a long carboxy-terminal extension that interacts with a secondary structure of selenoprotein mRNAs (SECIS element) positioned immediately downstream of the in-frame UGA codons specifying the sites of selenocysteine insertion. In this report, a fast and efficient method for the purification of large amounts of hexahistidine-tagged SelB is presented. After two chromatographic steps, 10 mg pure protein was isolated from 12 g wet cell pellet. Biochemical analysis of the purified protein showed that the tag does not influence the interaction of SelB with guanine nucleotides, SECIS elements, and selenocysteyl-tRNASec. In addition, the fusion protein is fully functional in mediating UGA read-through in vivo. It therefore represents an excellent model for studying the function of SelB and the mechanisms of selenocysteine incorporation.     

Structure of prokaryotic SECIS mRNA hairpin and its interaction with elongation factor SelB

In prokaryotes, the recoding of a UGA stop codon as a selenocysteine codon requires a special elongation factor (EF) SelB and a stem-loop structure within the mRNA called a selenocysteine insertion sequence (SECIS). Here, we used NMR spectroscopy to determine the solution structure of the SECIS mRNA hairpin and characterized its interaction with the mRNA-binding domain of SelB. Our structural and biochemical data identified the conserved structural features important for binding to EF SelB within different SECIS RNA sequences. In the free SECIS mRNA structure, conserved nucleotides are strongly exposed for recognition by SelB. Binding of the C-terminal domain of SelB stabilizes the RNA secondary structure. In the protein-RNA complex, a Watson-Crick loop base-pair leaves a GpU sequence accessible for SelB recognition. This GpU sequence at the tip of the capping tetraloop and a bulge uracil five Watson-Crick base-pairs apart from the GpU are essential for interaction with SelB.     

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.     

Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.     

The SBP2 and 15.5 kD/Snu13p proteins share the same RNA binding domain: identification of SBP2 amino acids important to SECIS RNA binding

Selenoprotein synthesis in eukaryotes requires the selenocysteine insertion sequence (SECIS) RNA, a hairpin in the 3' untranslated region of selenoprotein mRNAs. The SECIS RNA is recognized by the SECIS-binding protein 2 (SBP2), which is a key player in this specialized translation machinery. The objective of this work was to obtain structural insight into the SBP2-SECIS RNA complex. Multiple sequence alignment revealed that SBP2 and the U4 snRNA-binding protein 15.5 kD/Snu13p share the same RNA binding domain of the L7A/L30 family, also found in the box H/ACA snoRNP protein Nhp2p and several ribosomal proteins. In corollary, we have detected a similar secondary structure motif in the SECIS and U4 RNAs. Combining the data of the crystal structure of the 15.5 kD-U4 snRNA complex, and the SBP2/15.5 kD sequence similarities, we designed a structure-guided strategy predicting 12 SBP2 amino acids that should be critical for SECIS RNA binding. Alanine substitution of these amino acids followed by gel shift assays of the SBP2 mutant proteins identified four residues whose mutation severely diminished or abolished SECIS RNA binding, the other eight provoking intermediate down effects. In addition to identifying key amino acids for SECIS recognition by SBP2, our findings led to the proposal that some of the recognition principles governing the 15.5 kD-U4 snRNA interaction must be similar in the SBP2-SECIS RNA complex.     

Selenocysteine insertion sequence binding protein 2L is implicated as a novel post-transcriptional regulator of selenoprotein expression

The amino acid selenocysteine (Sec) is encoded by UGA codons. Recoding of UGA from stop to Sec requires a Sec insertion sequence (SECIS) element in the 3' UTR of selenoprotein mRNAs. SECIS binding protein 2 (SBP2) binds the SECIS element and is essential for Sec incorporation into the nascent peptide. SBP2-like (SBP2L) is a paralogue of SBP2 in vertebrates and is the only SECIS binding protein in some invertebrates where it likely directs Sec incorporation. However, vertebrate SBP2L does not promote Sec incorporation in in vitro assays. Here we present a comparative analysis of SBP2 and SBP2L SECIS binding properties and demonstrate that its inability to promote Sec incorporation is not due to lower SECIS affinity but likely due to lack of a SECIS dependent domain association that is found in SBP2. Interestingly, however, we find that an invertebrate version of SBP2L is fully competent for Sec incorporation in vitro. Additionally, we present the first evidence that SBP2L interacts with selenoprotein mRNAs in mammalian cells, thereby implying a role in selenoprotein expression.     

On elongation factor eEFSec,its role and mechanism during selenium incorporation into nascent selenoproteins

Background

Selenium, an essential dietary micronutrient, is incorporated into proteins as the amino acid selenocysteine (Sec) in response to in-frame UGA codons. Complex machinery ensures accurate recoding of Sec codons in higher organisms. A specialized elongation factor eEFSec is central to the process.

The three-dimensional structure of the Moorella thermoacetica selenocysteine insertion sequence RNA hairpin and its interaction with the elongation factor SelB

Incorporation of the amino acid selenocysteine into a growing protein chain involves the interaction between a hairpin in the mRNA termed the selenocysteine insertion sequence (SECIS) and the special elongation factor SelB. Here we present the structure of the SECIS from the thermophilic organism Moorella thermoacetica (SECIS-MT) determined using nuclear magnetic resonance (NMR) spectroscopy. The SECIS-MT hairpin structure contains a pentaloop with the first and fourth nucleotides of the loop forming a noncanonical GC base pair; the fifth loop nucleotide is bulged out and unstructured. The G and U in positions two and three are on opposite sides of the loop and solvent exposed. The backbone resonances of the SECIS-binding domain from the M. thermoacetica SelB protein were assigned, and the degree of chemical shift perturbations that occur upon SECIS binding were mapped onto the structure of the complex. We demonstrate that a region in the third winged-helix domain of SelB, not previously implicated in binding, is affected by SECIS binding.