Nematode selenoproteome: the use of the selenocysteine insertion system to decode one codon in an animal genome?

Selenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly.     

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.     

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.     

UGA codon position-dependent incorporation of selenocysteine into mammalian selenoproteins

It is thought that the SelenoCysteine Insertion Sequence (SECIS) element and UGA codon are sufficient for selenocysteine (Sec) insertion. However, we found that UGA supported Sec insertion only at its natural position or in its close proximity in mammalian thioredoxin reductase 1 (TR1). In contrast, Sec could be inserted at any tested position in mammalian TR3. Replacement of the 3′-UTR of TR3 with the corresponding segment of a Euplotes crassus TR restricted Sec insertion into the C-terminal region, whereas the 3′-UTR of TR3 conferred unrestricted Sec insertion into E. crassus TR, in which Sec insertion is normally limited to the C-terminal region. Exchanges of 3′-UTRs between mammalian TR1 and E. crassus TR had no effect, as both proteins restricted Sec insertion. We further found that these effects could be explained by the use of selenoprotein-specific SECIS elements. Examination of Sec insertion into other selenoproteins was consistent with this model. The data indicate that mammals evolved the ability to limit Sec insertion into natural positions within selenoproteins, but do so in a selenoprotein-specific manner, and that this process is controlled by the SECIS element in the 3′-UTR.     

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.     

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.     

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.     

硒蛋白的分子生物学研究进展

已有35种硒蛋白被分离和表征,但许多硒蛋白及其功能仍未完全阐明.硒半胱氨酸(Sec)作为参入蛋白质的第21种氨基酸,由硒蛋白mRNA上的UGA编码.在原核生物,Sec参入硒蛋白的复杂机制已经较为明确,需要四种基因产物（SELA、SELB、SELC和SELD）和一个存在于硒蛋白mRNA上的被称为Sec插入序列(SECIS)的茎环(stem loop)样二级结构.在真核生物,硒蛋白生物合成途径可能在SECIS的结构和位置、特异的延伸因子及其他RNA-RNA或RNA-蛋白质因子之间的相互作用等方面与原核生物不同.另外,哺乳动物硒蛋白mRNA上的UGA翻译为Sec的过程低效,特定位点的UGA密码子不同功能(终止密码和Sec密码)的调控可能是硒蛋白表达低效的关键.     

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     

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.     

New selenoproteins identified in silico from the genome of Anopheles gambiae

Selenoprotein is biosynthesized by the incorporation of selenocysteine into proteins, where the TGA codon in the open reading frame does not act as a stop signal but is translated into selenocysteine. The dual functions of TGA result in mis-annotation or lack of selenoproteins in the sequenced genomes of many species. Available computational tools fail to correctly predict selenoproteins. Thus, we developed a new method to identify selenoproteins from the genome of Anopheles gambiae computationally.Based on released genomic information, several programs were edited with PERL language to identify selenocysteine insertion sequence (SECIS) element, the coding potential of TGA codons, and cysteine-containing homologs of selenoprotein genes. Our results showed that 11365 genes were terminated with TGA codons, 918 of which contained SECIS elements. Similarity search revealed that 58genes contained Sec/Cys pairs and similar flanking regions around in-frame TGA codons. Finally, 7genes were found to fully meet requirements for selenoproteins, although they have not been annotated as selenoproteins in NCBI databases. Deduced from their basic properties, the newly found selenoproteins in the genome of Anopheles gambiae are possibly related to in vivo oxidation tolerance and protein regulation in order to interfere with anopheles' vectorial capacity of Plasmodium. This study may also provide theoretical bases for the prevention of malaria from anopheles transmission.     

New selenoproteins identified in silico from the genome of Anopheles gambiae

Selenoprotein is biosynthesized by the incorporation of selenocysteine into proteins,where the TGA codon in the open reading frame does not act as a stop signal but is translated into selenocysteine.The dual functions of TGA result in mis-annotation or lack of selenoproteins in the sequenced genomes of many species.Available computational tools fail to correctly predict selenoproteins.Thus,we devel-oped a new method to identify selenoproteins from the genome of Anopheles gambiae computationally.Based on released genomic information,several programs were edited with PERL language to identify selenocysteine insertion sequence(SECIS)element,the coding potential of TGA codons,and cys-teine-containing homologs of selenoprotein genes.Our results showed that 11365 genes were termi-nated with TGA codons,918 of which contained SECIS elements.Similarity search revealed that 58 genes contained Sec/Cys pairs and similar flanking regions around in-frame TGA codons.Finally,7 genes were found to fully meet requirements for selenoproteins,although they have not been anno-tated as selenoproteins in NCBI databases.Deduced from their basic properties,the newly found se-lenoproteins in the genome of Anopheles gambiae are possibly related to in vivo oxidation tolerance and protein regulation in order to interfere with anopheles' vectorial capacity of Plasmodium.This study may also provide theoretical bases for the prevention of malaria from anopheles transmission.     

A 4-selenocysteine, 2-selenocysteine insertion sequence (SECIS) element methionine sulfoxide reductase from Metridium senile reveals a non-catalytic function of selenocysteines

Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic (75)Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions.     

Selenium metabolism in Trypanosoma: characterization of selenoproteomes and identification of a Kinetoplastida-specific selenoprotein

Proteins containing the 21st amino acid selenocysteine (Sec) are present in the three domains of life. However, within lower eukaryotes, particularly parasitic protists, the dependence on the trace element selenium is variable as many organisms lost the ability to utilize Sec. Herein, we analyzed the genomes of Trypanosoma and Leishmania for the presence of genes coding for Sec-containing proteins. The selenoproteomes of these flagellated protozoa have three selenoproteins, including distant homologs of mammalian SelK and SelT, and a novel multidomain selenoprotein designated SelTryp. In SelK and SelTryp, Sec is near the C-terminus, and in all three selenoproteins, it is within predicted redox motifs. SelTryp has neither Sec- nor cysteine-containing homologs in the human host and appears to be a Kinetoplastida-specific protein. The use of selenium for protein synthesis was verified by metabolically labeling Trypanosoma cells with ⁷⁵Se. In addition, genes coding for components of the Sec insertion machinery were identified in the Kinetoplastida genomes. Finally, we found that Trypanosoma brucei brucei cells were highly sensitive to auranofin, a compound that specifically targets selenoproteins. Overall, these data establish that Trypanosoma, Leishmania and likely other Kinetoplastida utilize and depend on the trace element selenium, and this dependence is due to occurrence of selenium in at least three selenoproteins.     

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.     

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.     

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

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

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.