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.     

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.     

A single homozygous point mutation in a 3'untranslated region motif of selenoprotein N mRNA causes SEPN1-related myopathy

Mutations in the SEPN1 gene encoding the selenoprotein N (SelN) have been described in different congenital myopathies. Here, we report the first mutation in the selenocysteine insertion sequence (SECIS) of SelN messenger RNA, a hairpin structure located in the 3' untranslated region, in a patient presenting a classical although mild form of rigid spine muscular dystrophy. We detected a significant reduction in both mRNA and protein levels in the patient's skin fibroblasts. The SECIS element is crucial for the insertion of selenocysteine at the reprogrammed UGA codon by recruiting the SECIS-binding protein 2 (SBP2), and we demonstrated that this mutation abolishes SBP2 binding to SECIS in vitro, thereby preventing co-translational incorporation of selenocysteine and SelN synthesis. The identification of this mutation affecting a conserved base in the SECIS functional motif thereby reveals the structural basis for a novel pathological mechanism leading to SEPN1-related myopathy.     

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.     

Heterologous expression of archaeal selenoprotein genes directed by the SECIS element located in the 3' non-translated region

Previous in silico analysis of selenoprotein genes in Archaea revealed that the selenocysteine insertion (SECIS) motif necessary to recode UGA with selenocysteine was not adjacent to the UGA codon as is found in Bacteria. Rather, paralogous stem-loop structures are located in the 3' untranslated region (3' UTR), reminiscent of the situation in Eukarya. To assess the function of such putative SECIS elements, the Methanococcus jannaschii MJ0029 (fruA, which encodes the A subunit of the coenzyme F420-reducing hydrogenase) mRNA was mapped in vivo and probed enzymatically in vitro. It was shown that the SECIS element is indeed transcribed as part of the respective mRNA and that its secondary structure corresponds to that predicted by RNA folding programs. Its ability to direct selenocysteine insertion in vivo was demonstrated by the heterologous expression of MJ0029 in Methanococcus maripaludis, resulting in the synthesis of an additional selenoprotein, as analysed by 75Se labelling. The selective advantage of moving the SECIS element in the untranslated region may confer the ability to insert more than one selenocysteine into a single polypeptide. Evidence for this assumption was provided by the finding that the M. maripaludis genome contains an open reading frame with two in frame TGA codons, followed by a stem-loop structure in the 3' UTR of the mRNA that corresponds to the archaeal SECIS element.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

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.     

The selenocysteine insertion sequence binding protein SBP is different from the Y-box protein dbpB

In eukaryotes, translation of internal UGA selenocysteine codons requires the SECIS stem-loop structure in the 3'UTR of selenoprotein mRNAs. In an earlier work, we identified SBP as a selenocysteine insertion sequence (SECIS)-binding protein. Here, the yeast three-hybrid screen was employed to capture the cDNA of SBP. One candidate, satisfying the genetic screens, was identified as the already known dbpB protein. Although it was also found by another group, but with a different strategy, to carry SECIS-binding activity, further experiments enabled us to show that dbpB was unable to bind the SECIS element in vitro. Altogether, our findings led us to conclude that, under our conditions, dbpB and SBP are two distinct proteins.     

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.     

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.     

Analysis of eukaryotic mRNA structures directing cotranslational incorporation of selenocysteine.

Translation of an mRNA encoding a selenoprotein requires that at least one UGA codon in the reading frame is recoded as a site for the insertion of selenocysteine. In eukaryotes, the termination codon recoding event is directed by a cis-acting signal element located in the 3' untranslated region of the gene. This 'selenocysteine insertion sequence' (SECIS) comprises conserved sequences in a region of extensive base-pairing. In order to study the structure-function relationships of the SECIS structure, we have applied a newly developed reporter gene system which allows analysis of stop codon suppression in animal cell lines. This system obviates the need for enzymatic or immunological estimation of selenoprotein synthesis, relying instead on the simple quantification of translational readthrough from the lacZ gene into the luciferase gene. The 3'-UTR of the phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene was shown to contain a highly active SECIS element. Mutations in the base-paired sequences of other SECIS elements were used to analyse the significance of primary structure, secondary structure and pairing stability in the stem regions. The results demonstrate that the exact sequences of the paired nucleotides are comparatively unimportant, provided that a consensus combination of length and thermodynamic stability of the base-paired structures is maintained.     

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.     

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.     

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.