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.

Identification and functional characterization of the candidate tumor suppressor gene TRIT1 in human lung cancer

“…The ability to decode UGA as a Sec codon requires specific mRNA secondary structures and specific translational factors (Low and Berry, 1996;Berry et al, 2001) and is affected when tRNA [Ser]Sec lacks i 6 A at position 37, as demonstrated by Warner et al, 2000). Similarly, transgenic mice overexpressing i 6 A-deficient tRNA [Ser]Sec show defective levels of numerous selenoproteins due to problems occurring during translation and not transcription since mRNA levels remain unchanged (Moustafa et al, 2001). Many studies have correlated deregulation of protein biosynthesis with cancer, but it remains to be ascertained whether this process is necessary and/or sufficient for neoplastic transformation.…”

Selenoprotein deficiency and high levels of selenium compounds can effectively inhibit hepatocarcinogenesis in transgenic mice

“…TR1 (cytosolic enzyme) and TR3 (this mitochondrial enzyme is also called TnxRd2 and TrxR2) expression was assessed by immunoblot assays with antibodies specific for each isozyme, after the enzymes were enriched by binding to ADP-Sepharose as described previously (Moustafa et al, 2001). Expression of SelR, GPx1, and Sep15 was analysed by immunoblot assays with antibodies specific for these proteins.…”

Identification and characterization of phosphoseryl-tRNA [Ser]Sec kinase

“…PSTK or thioredoxin containing a His-tag, which was used as a control protein, was added in a total volume of 100 l containing reaction mixture (see above) with or without 50 M unlabeled ATP and 500 ng of a highly purified sample of a mixture of both tRNA [ After washing three times with 1 ml of 1ϫ TBS͞0.1% Tween, the beads were suspended in 40 l of TBE-urea loading buffer (90 mM Tris͞64.6 mM boric acid͞2.5 mM EDTA͞3.5 M urea, pH 8.3), and 5 l of each sample was loaded onto a 15% TBE-urea gel. After electrophoresis and transfer of the RNA to a nylon membrane, RNA was detected by Northern blotting with the appropriate probe (17).…”