Co-translational Insertion Of Selenocysteine Research Articles

In vivo probing of SECIS-dependent selenocysteine translation in Archaea.

Cotranslational insertion of selenocysteine (Sec) proceeds by recoding UGA to a sense codon. This recoding is governed by the Sec insertion sequence (SECIS) element, an RNA structure on the mRNA, but size, location, structure determinants, and mechanism differ for Bacteria, Eukarya, and Archaea. For Archaea, the structure-function relation of the SECIS is poorly understood, as only rather laborious experimental approaches are established. Furthermore, these methods do not allow for quantitative probing of Sec insertion. In order to overcome these limitations, we engineered bacterial β-lactamase into an archaeal selenoprotein, thereby establishing a reporter system, which correlates enzyme activity to Sec insertion. Using this system, in vivo Sec insertion depending on the availability of selenium and the presence of a SECIS element was assessed in Methanococcus maripaludis Furthermore, a minimal SECIS element required for Sec insertion in M. maripaludis was defined and a conserved structural motif shown to be essential for function. Besides developing a convenient tool for selenium research, converting a bacterial enzyme into an archaeal selenoprotein provides proof of concept that novel selenoproteins can be engineered in Archaea.

Aminoglycoside-driven biosynthesis of selenium-deficient Selenoprotein P

Selenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro, SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects.

Update on selenoprotein biosynthesis.

Selenium is an essential trace element that is incorporated in the small but vital family of proteins, namely the selenoproteins, as the selenocysteine amino acid residue. In humans, 25 selenoprotein genes have been characterized. The most remarkable trait of selenoprotein biosynthesis is the cotranslational insertion of selenocysteine by the recoding of a UGA codon, normally decoded as a stop signal. In eukaryotes, a set of dedicated cis- and trans-acting factors have been identified as well as a variety of regulatory mechanisms, factors, or elements that control the selenoprotein expression at the level of the UGA-selenocysteine recoding process, offering a fascinating playground in the field of translational control. It appeared that the central players are two RNA molecules: the selenocysteine insertion sequence (SECIS) element within selenoprotein mRNA and the selenocysteine-tRNA([Ser]Sec); and their interacting partners. After a couple of decades, despite many advances in the field and the discovery of many essential and regulatory components, the precise mechanism of UGA-selenocysteine recoding remains elusive and more complex than anticipated, with many layers of control. This review offers an update of selenoproteome biosynthesis and regulation in eukaryotes. The regulation of selenoproteins in response to a variety of pathophysiological conditions and cellular stressors, including selenium levels, oxidative stress, replicative senescence, or cancer, awaits further detailed investigation. Clearly, the efficiency of UGA-selenocysteine recoding is the limiting stage of selenoprotein synthesis. The sequence of events leading Sec-tRNA([Ser]Sec) delivery to ribosomal A site awaits further analysis, notably at the level of a three-dimensional structure.

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.

Eukaryotic Selenocysteine Incorporation: Mechanistic Insights

Cotranslational insertion of selenocysteine occurs in both prokaryotic and eukaryotic organisms. For the most part, what is known about this process in eukaryotes has been derived from extensive analysis of the RNA requirements for selenocysteine incorporation. Studies which have defined the selenocysteine insertion sequence (SECIS) element, identified its critical sequence and structural motifs, and established constraints on its spatial relationship with the UGA selenocysteine codon, have given mechanistic insights into this critical process.

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.

The function of selenocysteine synthase and SELB in the synthesis and incorporation of selenocysteine

The selAB operon codes for the proteins selenocysteine synthase and SELB which catalyse the synthesis and co-translational insertion of selenocysteine into protein. This communication deals with the biochemical characterisation of these proteins and in particular with their specific interaction with the selenocysteine-incorporating tRNA Sec. Selenocysteine synthase catalyses the synthesis of selenocysteyl-tRNA Sec from seryl-tRNA Sec in a pyridoxal phosphate-dependent reaction mechanism. The enzyme specifically recognizes the tRNA Sec molecule; a cooperative interaction between the tRNA binding site and the catalytically active pyridoxal phosphate site is suggested. SELB is an EF-Tu-like protein which specifically complexes selenocysteyl-tRNA Sec. Interaction with the selenol group of the side chain of the aminoacylated residue is a prerequisite for the formation of a stable SELB·tRNA complex. Mechanistically, this provides the biochemical basis for the exclusive selection of selenocysteyl-tRNA Sec in the decoding step of a selenocysteine-specific UGA triplet.

Specific occurrence of selenium in enzymes and amino acid tRNAs 1

In contrast to the widespread ability of bacteria, plants, and animals to incorporate selenium nonspecifically into proteins in the form of selenomethionine residues, the selenoamino acid selenocysteine occurs as a highly specific component of a few selenium-dependent enzymes. Selenocysteine has been identified in glycine reductase, formate dehydrogenase, and hydrogenase of bacterial origin and glutathione peroxidase from mammalian and avian sources. In these enzymes there is evidence that the selenol group, which is largely ionized at physiological pH, functions as a redox center. It now seems clear, from studies with both prokaryotes and eukaryotes, that the UGA opal stop codon is used to specify the cotranslational insertion of selenocysteine into proteins. The factors that allow this unusual use of the stop codon are, however, unknown. The occurrence of selenium as a normal constituent of several bacterial tRNA species has been established. The presence of a selenonucleoside, 5-methylaminomethyl-2-selenouridine, in the first or wobble position of the anticodons of certain glutamate and lysine iso-acceptor species influences codon-anticodon interaction and thus may serve to regulate translational processes. The biosynthesis of the selenonucleoside appears to involve the ATP-dependent activation of the sulfur in a preformed 5-methylaminomethyl-2-thiouridine residue in tRNA and replacement of the sulfur with selenium.