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J. Biol. Chem., Vol. 282, Issue 45, 32591-32602, November 9, 2007

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Selective Restoration of the Selenoprotein Population in a Mouse Hepatocyte Selenoproteinless Background with Different Mutant Selenocysteine tRNAs Lacking Um34^*

Bradley A. Carlson¹, Mohamed E. Moustafa¹², Aniruddha Sengupta, Ulrich Schweizer, Rajeev Shrimali, Mahadev Rao³, Nianxin Zhong, Shulin Wang⁴, Lionel Feigenbaum^¶, Byeong Jae Lee^||, Vadim N. Gladyshev^**, and Dolph L. Hatfield⁵

From the Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, Neurobiology of Selenium, Neuroscience Research Center, Institute for Experimental Endocrinology, Charité Universitätsmedizin Berlin, 10117 Berlin, Germany, ^¶Science Applications International Corporation, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, the ^||School of Biological Sciences and Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea, and the ^**Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA^[Ser]Sec gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34TA) or 37 (37AG) in tRNA^[Ser]Sec. The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N⁶-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with ⁷⁵Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.

Received for publication, August 22, 2007

^* This work was supported by the Intramural Research Program of the Center for Cancer Research, NCI, National Institutes of Health; by National Institutes of Health grants (to V. N. G.); and by Korea Research Foundation Grant C00086 (to B. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Present address: Faculty of Science, Alexandria University, Alexandria, Egypt.

³ Present address: CRC-Hatfield Clinical Research Center, Dept. of Surgery, NCI, National Institutes of Health, Bethesda, MD 20892.

⁴ Present address: Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China.

⁵ To whom correspondence should be addressed: MBSS, LCB, CCR, NCI, Bldg. 37, Rm. 6032A, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-2797; Fax: 301-435-4957; E-mail: hatfield{at}mail.nih.gov.

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