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. 2001 Jun;21(11):3840-52.
doi: 10.1128/MCB.21.11.3840-3852.2001.

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA

M E Moustafa  1 , B A Carlson, M A El-Saadani, G V Kryukov, Q A Sun, J W Harney, K E Hill, G F Combs, L Feigenbaum, D B Mansur, R F Burk, M J Berry, A M Diamond, B J Lee, V N Gladyshev, D L Hatfield
Affiliations

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA

M E Moustafa et al. Mol Cell Biol. 2001 Jun.

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.

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Figures

FIG. 1
FIG. 1
Secondary structure of tRNA[Ser]Sec and map of the construct used in making transgenic mice. (A) The cloverleaf model of tRNA[Ser]Sec is shown along with the sites of base changes at positions 9 and 37 used in this study and the sites of modified nucleosides (see the text). The numbering system for positions within tRNA[Ser]Sec is described in the text. (B) The map shows tandem 2.17-kb transgenic fragments containing 1.93 kb of mouse DNA (large open rectangles) encoding the tRNA[Ser]Sec gene (small dotted rectangles) and 0.24 kb of vector DNA (small solid rectangles). The AccI (located 48 bp upstream of the coding sequence of the tRNA[Ser]Sec gene) and XhoI (located in the multiple cloning site of the BlueScript II cloning vector) restriction sites are shown. The 3′ end of the tRNA[Ser]Sec gene is located 425 bp upstream of the 5′ end of the vector sequence. The small arrow inside the gene near the 5′ end shows the position of the T-to-C mutation at position 9 that distinguishes the two wild-type tRNA[Ser]Sec transgenes (see the text), and the other arrow inside the gene shows the position of the A-to-G mutation at position 37 that constitutes the i6A− mutant transgene.
FIG. 2
FIG. 2
Relative amounts of mcm5U and mcm5Um isoacceptors in livers of wild-type and heterozygous and homozygous transgenic mice bearing wild-type transgenes. Total tRNA was isolated from livers of littermates bearing +/+, +/+/TGWT2, and +/+/TGWT2/TGWT2 genotypes and aminoacylated with [3H]serine, and the resulting 3H-labeled tRNA was fractionated as described in Materials and Methods. The amounts of seryl-tRNA[Ser]Sec (mcm5U is the first eluting peak and mcm5Um is the second) found in livers of heterozygous and homozygous transgenic mice were standardized to that found in wild-type livers with the total [3H]seryl-tRNASer serving as an internal control. Sources of seryl-tRNA[Ser]Sec are shown in each graph.
FIG. 3
FIG. 3
Characterization of tRNA[Ser]Sec obtained from tissues of wild-type mice and heterozygous and homozygous transgenic mice bearing transgenes with a pyrimidine transition at position 9 by primer extension. Total tRNA (A), fractionated mcm5U (B), or fractionated mcm5Um (C) was used as a template for primer extension. Column fractions were selected to minimize the overlap of the peaks representing each isoacceptor. In each panel, the order of samples is as follows: lane 1, no template; lane 2, +/+ liver; lane 3, +/+/TG“WT”10 liver; lane 4, +/+/TG“WT”10/TG“WT”10 liver; lane 5, +/+ kidney; lane 6, +/+/TG“WT”10 kidney; and lane 7, +/+/TG“WT”10/TG“WT”10 kidney. Preparation, separation, and recovery of tRNA and tRNA fractions and primer extensions were done as described in Materials and Methods. The positions of the primer and the +3 (host tRNA[Ser]Sec) and +6 (transgene tRNA[Ser]Sec) extension products are indicated.
FIG. 4
FIG. 4
Protein and selenoprotein analysis in tissues of wild-type and sibling heterogeneous and homogenous i6A-deficient transgenic mice. Littermates were labeled with 75Se, and proteins were extracted from the different tissues, electrophoresed, and transblotted onto a membrane; the membrane was stained with Coomassie blue. Total protein of +/+, +/+/TGi6A−2, and +/+/TGi6A−2/TGi6A−2 (A), +/+, +/+/TGi6A−8, and +/+/TGi6A−8/TGi6A−8 (C), and +/+, +/+/TGi6A−20, and +/+/TGi6A−20/TGi6A−20 (E) mice and 75Se-labeled proteins of +/+, +/+/TGi6A−2, and +/+/TGi6A−2/TGi6A−2 (B), +/+, +/+/TGi6A−8, and +/+/TGi6A−8/TGi6A−8 (D), and +/+, +/+/TGi6A−20, and +/+/TGi6A−20/TGi6A−20 (F), mice were detected with a PhosphorImager as described in Materials and Methods. Protein marker sizes are shown on the left of each panel as indicated by the arrows.
FIG. 4
FIG. 4
Protein and selenoprotein analysis in tissues of wild-type and sibling heterogeneous and homogenous i6A-deficient transgenic mice. Littermates were labeled with 75Se, and proteins were extracted from the different tissues, electrophoresed, and transblotted onto a membrane; the membrane was stained with Coomassie blue. Total protein of +/+, +/+/TGi6A−2, and +/+/TGi6A−2/TGi6A−2 (A), +/+, +/+/TGi6A−8, and +/+/TGi6A−8/TGi6A−8 (C), and +/+, +/+/TGi6A−20, and +/+/TGi6A−20/TGi6A−20 (E) mice and 75Se-labeled proteins of +/+, +/+/TGi6A−2, and +/+/TGi6A−2/TGi6A−2 (B), +/+, +/+/TGi6A−8, and +/+/TGi6A−8/TGi6A−8 (D), and +/+, +/+/TGi6A−20, and +/+/TGi6A−20/TGi6A−20 (F), mice were detected with a PhosphorImager as described in Materials and Methods. Protein marker sizes are shown on the left of each panel as indicated by the arrows.
FIG. 5
FIG. 5
Northern analysis of several selenoprotein mRNAs. (A) mRNA levels of GPX1 in liver and kidney of heterozygous and homozygous transgenic mice carrying the highest number of mutant transgenes and their wild-type siblings are shown. (B) mRNA levels of Se1P, TR1, D1, SPS2, and GPX4 in liver tissue of homozygous transgenic mice carrying the highest number of mutant transgenes and their wild-type siblings are shown. mRNA was extracted from livers and kidneys of mice harboring 20 or 40 i6A− mutant tRNA[Ser]Sec transgenes and their wild-type siblings, electrophoresed, and transblotted onto membranes. The membranes were hybridized with 32P-labeled probes complementary to each mRNA shown in both panels, their levels were quantitated by phosphoimagery, and the filters were stripped and rehybridized with β-actin as described in Materials and Methods.
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