Knowing when not to stop: selenocysteine incorporation in eukaryotes

The regulation of translation frequently involves protein-RNA interactions. An intriguing example of this is the alternative decoding of UGA, typically a stop codon, as selenocysteine. Two RNA structures, the mRNA selenocysteine insertion sequence (SECIS element) and a unique selenocysteyl-tRNA, are required for this process. In prokaryotes, a single RNA-binding protein, a selenocysteine-specific elongation factor, interacts with both the tRNA and mRNA to confer decoding. Whether eukaryotes use a similar mechanism is currently the subject of intense investigation.     

Rabbit beta-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps

Translational reading gaps occur when genetic information encoded in mRNA is not translated during the normal course of protein synthesis. This phenomenon has been observed thus far only in prokaryotes and is a mechanism for extending the reading frame by circumventing the normal stop codon. Reading frames of proteins may also be extended by suppression of the stop codon mediated by a suppressor tRNA. The rabbit beta-globin read-through protein, the only known, naturally occurring read-through protein in eukaryotes, was sequenced by ion trap mass spectrometry to determine how the reading frame is extended. Seven different proteolytic peptide fragments decoded by the same sequence that spans the UGA stop codon of rabbit beta-globin mRNA were detected. Three of these peptides contain translational reading gaps of one to three amino acids that correspond to the UGA stop codon site and/or one or two of the immediate downstream codons. To our knowledge, this is the first reported example of the occurrence of reading gaps in protein synthesis in eukaryotes. This event is unique in that it is associated with bypasses involving staggered lengths of untranslated information. Four of the seven peptides contain serine, tryptophan, cysteine, and arginine decoded by UGA and thus arise by suppression. Serine is donated by selenocysteine tRNA, and it, like the other tRNAs, has previously been shown to suppress UGA in vitro in mammals, but not in vivo.     

UGA codon position affects the efficiency of selenocysteine incorporation into glutathione peroxidase-1

A UGA codon and a selenocysteine insertion sequence in the 3'-untranslated region are the only established mRNA elements necessary for selenocysteine (Sec or U) incorporation during translation. These two elements, however, do not universally confer efficient Sec incorporation. The objective of this study was to systematically examine the effect of UGA codon position on efficiency of Sec insertion. In a glutathione peroxidase-1 (F-GPX1) expression vector, the UGA at the native position (U47) was mutated to a cysteine codon, and codons for Ser-7, Ser-12, Ser-18, Ser-29, Ser-45, Ser-93, Cys-154, Val-172, Ser-178, and Ser-195 were individually mutated to UGA and transiently expressed in COS-7 cells. 75Se incorporation at the 11 positions was 31, 72, 54, 105, 90, 100, 146, 135, 13, 11, and 43%, respectively, of 75Se incorporation at U47, suggesting that Sec is more efficiently incorporated at UGA codons positioned in the middle of the coding region rather than close to the 5' or 3' ends. Ribonuclease protection showed that these differences were not due to differences in mRNA level. When the green fluorescence protein (GFP) coding region was placed in-frame at the 5' or 3' ends of the coding region in F-GPX1 to produce chimeric 50-51-kDa GFP/GPX1 proteins, Sec incorporation at UGA codons, formerly close to the 5' or 3' ends, was increased to levels comparable to the UGA at U47. Insertion of GFP after the UAA-stop was just as effective in increasing Sec insertion efficiency as GFP inserted before the stop. These studies used a recombinant expression model that incorporated Sec at non-native UGA codons at rates equal to those of endogenous glutathione peroxidase-1 and showed that the efficiency of Sec incorporation can be modulated by UGA position; Sec incorporation at high efficiency appears to require that the UGA be >21 nucleotides from the AUG-start and >204 nucleotides from the selenocysteine insertion sequence element.     

Epidemiology of IgA nephropathy in central and eastern Kentucky for the period 1975 through 1994. Central Kentucky Region of the Southeastern United States IgA Nephropathy DATABANK Project

Bacillus subtilis has been thought to have a high readthrough rate at the UGA stop codon because no opal suppressor tRNA has been isolated so far [Lovett et al. (1991) J. Bacteriol. 173, 1810-1812]. To examine whether a tRNATrp which we have characterized [Matsugi et al. (1992) Nucleic Acids Res. 20, 3514] has the ability to read the UGA codon, in vitro translation was performed with a synthetic mRNA containing a test codon, UGA, UAG, UAA, or UGG, in a reading frame. Addition of Trp-tRNATrp to the system significantly increased the readthrough rate only in the case of UGA. This suggests that this tRNATrp has a dual recognition pattern in B. subtilis, i.e., for the canonical tryptophan codon and for readthrough at the UGA stop codon.     

Confidence intervals for median survival times under a piecewise exponential model with proportional hazards covariate effects

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.     

Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue

We have determined the sequence of 23 peptides from bovine thioredoxin reductase covering 364 amino acid residues. The result was used to identify a rat cDNA clone (2.19 kilobase pairs), which contained an open reading frame of 1496 base pairs encoding a protein with 498 residues. The bovine and rat thioredoxin reductase sequences revealed a close homology to glutathione reductase including the conserved active site sequence (Cys-Val-Asn-Val-Gly-Cys). This also confirmed the identity of a previously published putative human thioredoxin reductase cDNA clone. Moreover, one peptide of the bovine enzyme contained a selenocysteine residue in the motif Gly-Cys-SeCys-Gly (where SeCys represents selenocysteine). This motif was conserved at the carboxyl terminus of the rat and human enzymes, provided that TGA in the sequence GGC TGC TGA GGT TAA, being identical in both cDNA clones, is translated as selenocysteine and that TAA confers termination of translation. The 3'-untranslated region of both cDNA clones contained a selenocysteine insertion sequence that may form potential stem loop structures typical of eukaryotic selenocysteine insertion sequence elements required for the decoding of UGA as selenocysteine. Carboxypeptidase Y treatment of bovine thioredoxin reductase after reduction by NADPH released selenocysteine from the enzyme with a concomitant loss of enzyme activity measured as reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid). This showed that the carboxyl-terminal motif was essential for the catalytic activity of the enzyme.     

Barriers to heterologous expression of a selenoprotein gene in bacteria

The specificity parameters counteracting the heterologous expression in Escherichia coli of the Desulfomicrobium baculatum gene (hydV) coding for the large subunit of the periplasmic hydrogenase which is a selenoprotein have been studied. hydV'-'lacZ fusions were constructed, and it was shown that they do not direct the incorporation of selenocysteine in E. coli. Rather, the UGA codon is efficiently suppressed by some other aminoacyl-tRNA in an E. coli strain possessing a ribosomal ambiguity mutation. The suppression is decreased by the strA1 allele, indicating that the hydV selenocysteine UGA codon has the properties of a "normal" and suppressible nonsense codon. The SelB protein from D. baculatum was purified; in gel shift experiments, D. baculatum SelB displayed a lower affinity for the E. coli fdhF selenoprotein mRNA than E. coli SelB did and vice versa. Coexpression of the hydV'-'lacZ fusion and of the selB and tRNA(Sec) genes from D. baculatum, however, did not lead to selenocysteine insertion into the protein, although the formation of the quaternary complex between SelB, selenocysteyl-tRNA(Sec), and the hydV mRNA recognition sequence took place. The results demonstrate (i) that the selenocysteine-specific UGA codon is readily suppressed under conditions where the homologous SelB protein is absent and (ii) that apart from the specificity of the SelB-mRNA interaction, a structural compatibility of the quaternary complex with the ribosome is required.     

Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons

Prokaryotic translational release factors, RF1 and RF2, catalyze polypeptide release at UAG/UAA and UGA/UAA stop codons, respectively. In this study, we isolated a bacterial RF2 mutant (RF2*) containing an E167K substitution that restored the growth of a temperature-sensitive RF1 strain of Escherichia coli and the viability of a chromosomal RF1/RF2 double knockout. In both in vivo and in vitro polypeptide termination assays, RF2* catalyzed UAG/UAA termination, as does RF1, as well as UGA termination, showing that RF2* acquired omnipotent release activity. This result suggests that the E167K mutation abolished the putative third-base discriminator function of RF2. These findings are interpreted as indicating that prokaryotic and eukaryotic release factors share the same anticodon moiety and that only one omnipotent release factor is sufficient for bacterial growth, similar to the eukaryotic single omnipotent factor.     

The organization of angiography suites for endovascular treatments and combined interventional-surgical procedures

The path of unspecific selenium incorporation into proteins was studied in Escherichia coli mutants blocked in the biosynthesis of cysteine and methionine or altered in its regulation. Selenium incorporation required all enzymatic steps of cysteine biosynthesis except sulfite reduction, indicating that intracellular reduction of selenite occurs nonenzymatically. Cysteine (but not methionine) supplementation prevented unspecific incorporation of selenium by repressing cysteine biosynthesis. On the other hand, when the biosynthesis of cysteine was derepressed in regulatory mutants, selenium was incorporated to high levels. These findings and the fact that methionine auxotrophic strains still displayed unspecific incorporation show that selenium incorporation into proteins in E. coli occurs mainly as selenocysteine. These findings also provide information on the labeling conditions for incorporating 75Se only and specifically into selenoproteins.     

Anxiolytic activity of NPY receptor agonists in the conflict test

Selenium is an important trace element that was considered toxic for humans and animals for a long time. The best known biochemical role of selenium is, as a selenocysteine residue, to be a part of the active site of the enzyme glutathione peroxidase (GSH-Px). The highest values of selenium have been found in protein foods (meat and fish); although selenium from vegetables sources are more available than from the other foods. Nowadays are not known exactly the recommended dietary allowances for humans, mainly for children. The selenium intake in Spain is 221 micrograms/person/day and the plasmatic values of Spanish people (87 +/- 14 micrograms/L) are within the European average (85 micrograms/L).     

Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.     

Translation in plants--rules and exceptions

Translation processes in plants are very similar to those in other eukaryotic organisms and can in general be explained with the scanning model. Particularly among plant viruses, unconventional mRNAs are frequent, which use modulated translation processes for their expression: leaky scanning, translational stop codon readthrough or frameshifting, and transactivation by virus-encoded proteins are used to translate polycistronic mRNAs; leader and trailer sequences confer (cap-independent) efficient ribosome binding, usually in an end-dependent mechanism, but true internal ribosome entry may occur as well; in a ribosome shunt, sequences within an RNA can be bypassed by scanning ribosomes. Translation in plant cells is regulated under conditions of stress and during development, but the underlying molecular mechanisms have not yet been determined. Only a small number of plant mRNAs, whose structure suggests that they might require some unusual translation mechanisms, have been described.