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Molecular and Cellular Biology, June 2001, p. 3840-3852, Vol. 21, No. 11
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.11.3840-3852.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Mohamed E. Moustafa,1 Bradley A. Carlson,1 Muhammad A. El-Saadani,2 Gregory V. Kryukov,3 Qi-An Sun,3 John W. Harney,4 Kristina E. Hill,5 Gerald F. Combs,6 Lionel Feigenbaum,7 David B. Mansur,8 Raymond F. Burk,5 Marla J. Berry,4 Alan M. Diamond,9 Byeong Jae Lee,10 Vadim N. Gladyshev,3 and Dolph L. Hatfield1,*

Section on the Molecular Biology of Selenium, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208921; Department of Biochemistry, Faculty of Science, Alexandria University, Alexandria, Egypt2; Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 685883; Thyroid Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 0211154; Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 372325; Department of Nutrition, Division of Nutritional Science, Cornell University, Ithaca, New York 148536; Science Applications International Corporation, Frederick Cancer Research and Development Center, Frederick, Maryland 217027; Radiation Oncology Center, Washington University, St. Louis, Missouri 631108; Department of Human Nutrition, University of Illinois at Chicago, Chicago, Illinois 606129; and Laboratory of Molecular Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea10

Received 23 January 2001/Returned for modification 12 March 2001/Accepted 20 March 2001

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 (i6A-) 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 i6A- 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 i6A- 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 i6A- tRNA[Ser]Sec transgenic mice will be useful in assessing the biological roles of selenoproteins.

* Corresponding author. Mailing address: National Cancer Institute, National Institutes of Health, Building 37, Room 2D09, Bethesda, MD 20892. Phone: (301) 496-2797. Fax: (301) 435-4957. E-mail: hatfield{at}dc37a.nci.nih.gov.

Molecular and Cellular Biology, June 2001, p. 3840-3852, Vol. 21, No. 11
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.11.3840-3852.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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