Showing papers by "Marla J. Berry published in 2001"

Selective Inhibition of Selenocysteine tRNA Maturation and Selenoprotein Synthesis in Transgenic Mice Expressing Isopentenyladenosine-Deficient Selenocysteine tRNA

Abstract: Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.

In silico identification of novel selenoproteins in the Drosophila melanogaster genome.

Abstract: In selenoproteins, incorporation of the amino acid selenocysteine is specified by the UGA codon, usually a stop signal. The alternative decoding of UGA is conferred by an mRNA structure, the SECIS element, located in the 3′-untranslated region of the selenoprotein mRNA. Because of the non-standard use of the UGA codon, current computational gene prediction methods are unable to identify selenoproteins in the sequence of the eukaryotic genomes. Here we describe a method to predict selenoproteins in genomic sequences, which relies on the prediction of SECIS elements in coordination with the prediction of genes in which the strong codon bias characteristic of protein coding regions extends beyond a TGA codon interrupting the open reading frame. We applied the method to the Drosophila melanogaster genome, and predicted four potential selenoprotein genes. One of them belongs to a known family of selenoproteins, and we have tested experimentally two other predictions with positive results. Finally, we have characterized the expression pattern of these two novel selenoprotein genes.

Selenocysteine incorporation directed from the 3′UTR: Characterization of eukaryotic EFsec and mechanistic implications

Abstract: The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid. The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA. Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.

Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.

Abstract: Background Selenocysteine incorporation has been reported to be inefficient in all systems studied, including Escherichia coli, baculovirus-insect cell systems, rabbit reticulocyte in vitro translation systems, transiently transfected mammalian cells, and intact animals. Nonetheless, full-length selenoproteins containing up to 17 selenocysteine residues are produced in animals, indicating that the efficiency observed in manipulated systems might not accurately reflect the true efficiency of this process in nature. Results To begin to address this apparent discrepancy, we have examined the polysome profiles of endogenously expressed selenoprotein mRNAs in a mammalian cell line, and compared them with nonselenoprotein mRNAs. We report that three selenoprotein mRNAs, type 1 deiodinase, glutathione peroxidase and selenoprotein P, are under-loaded with ribosomes, based on their predicted open reading frame sizes. The average numbers of ribosomes per mRNA correspond to the sizes predicted by termination at the UGA selenocysteine codons. Appropriate loading on the type 1 deiodinase mRNA is seen following substitution of a cysteine codon for the selenocysteine codon, indicating that the UGA codon confers a translational penalty on the mRNA. Surprisingly, ribosomal loading is also increased by the expression of eukaryotic release factors eRF1 and eRF3. Conclusions These results suggest that the presence of a selenocysteine codon confers a translational penalty on selenoprotein mRNAs, and that increased levels of release factors may alter the kinetics of termination.

Towards a mechanism for selenocysteine incorporation in eukaryotes

Abstract: The recent identification and cloning of two key proteins involved in the cotranslational incorporation of selenocysteine in eukaryotes has allowed the investigation of the mechanism by which the internal UGA codons of selenoprotein mRNAs are recoded from a canonical reading as termination, to encode selenocysteine. These proteins, selenocysteine insertion sequence (SECIS) binding protein 2 (SBP2), and eukaryotic selenocysteyl-tRNA[Ser]Sec-specific elongation factor (eEFsec), represent the first trans-acting protein factors clearly demonstrated to be involved in selenocysteine incorporation in eukaryotes. This chapter will focus on the cloning of eEFsec, in addition to presenting a discussion of prevalent current models describing the mechanism of selenocysteine incorporation in eukaryotes.