Title: Selective Rescue of Selenoprotein Expression in Mice Lacking a Highly Specialized Methyl Group in Selenocysteine tRNA
Abstract: Selenocysteine (Sec) is the 21st amino acid in the genetic code. Its tRNA is variably methylated on the 2′-O-hydroxyl site of the ribosyl moiety at position 34 (Um34). Herein, we identified a role of Um34 in regulating the expression of some, but not all, selenoproteins. A strain of knock-out transgenic mice was generated, wherein the Sec tRNA gene was replaced with either wild type or mutant Sec tRNA transgenes. The mutant transgene yielded a tRNA that lacked two base modifications, N6-isopentenyladenosine at position 37 (i6A37) and Um34. Several selenoproteins, including glutathione peroxidases 1 and 3, SelR, and SelT, were not detected in mice rescued with the mutant transgene, whereas other selenoproteins, including thioredoxin reductases 1 and 3 and glutathione peroxidase 4, were expressed in normal or reduced levels. Northern blot analysis suggested that other selenoproteins (e.g. SelW) were also poorly expressed. This novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. The available data suggest that the Um34 modification has greater influence than the i6A37 modification in regulating the expression of various mammalian selenoproteins and Um34 is required for synthesis of several members of this protein class. Many proteins that were poorly rescued appear to be involved in responses to stress, and their expression is also highly dependent on selenium in the diet. Furthermore, their mRNA levels are regulated by selenium and are subject to nonsense-mediated decay. Overall, this study described a novel mechanism of regulation of protein expression by tRNA modification that is in turn regulated by levels of the trace element, selenium. Selenocysteine (Sec) is the 21st amino acid in the genetic code. Its tRNA is variably methylated on the 2′-O-hydroxyl site of the ribosyl moiety at position 34 (Um34). Herein, we identified a role of Um34 in regulating the expression of some, but not all, selenoproteins. A strain of knock-out transgenic mice was generated, wherein the Sec tRNA gene was replaced with either wild type or mutant Sec tRNA transgenes. The mutant transgene yielded a tRNA that lacked two base modifications, N6-isopentenyladenosine at position 37 (i6A37) and Um34. Several selenoproteins, including glutathione peroxidases 1 and 3, SelR, and SelT, were not detected in mice rescued with the mutant transgene, whereas other selenoproteins, including thioredoxin reductases 1 and 3 and glutathione peroxidase 4, were expressed in normal or reduced levels. Northern blot analysis suggested that other selenoproteins (e.g. SelW) were also poorly expressed. This novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. The available data suggest that the Um34 modification has greater influence than the i6A37 modification in regulating the expression of various mammalian selenoproteins and Um34 is required for synthesis of several members of this protein class. Many proteins that were poorly rescued appear to be involved in responses to stress, and their expression is also highly dependent on selenium in the diet. Furthermore, their mRNA levels are regulated by selenium and are subject to nonsense-mediated decay. Overall, this study described a novel mechanism of regulation of protein expression by tRNA modification that is in turn regulated by levels of the trace element, selenium. The mechanism of selenocysteine (Sec) 1The abbreviations used are: Sec, selenocysteine; SECIS, Sec insertion sequence; mcm5U, methylcarboxylmethyluridine; mcm5Um, methylcarboxymethyluridine-2′-O-methylribose; GPx1, -2, and -4, glutathione peroxidase 1, 2, and 4, respectively; TR1 and -3, thioredoxin reductase, 1 and 3, respectively; SelP, selenoprotein P; i6A37, N6-isopentenyladenosine at position 37. incorporation into protein as the 21st amino acid was elucidated in Escherichia coli by Böck (reviewed in Ref. 1Bo ̈ck A. Hatfield D.L. Selenium: Its Molecular Biology and Role in Human Health. Kluwer Academic Publishers, Norwell, MA2001: 7-22Crossref Google Scholar). In mammals, the mechanism of Sec insertion into protein is not as completely understood (reviewed in Refs. 2Hatfield D.L. Gladyshev V.N. Mol. Cell. Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (554) Google Scholar and 3Driscoll D.M. Copeland P.R. Annu. Rev. Nutr. 2003; 23: 17-40Crossref PubMed Scopus (325) Google Scholar). However, both prokaryotes and eukaryotes use the stop codon, UGA, to dictate the incorporation of Sec after the tRNA is initially aminoacylated with serine, and the biosynthesis of Sec occurs on its tRNA. The tRNA has therefore been designated Sec tRNA[Ser]Sec (2Hatfield D.L. Gladyshev V.N. Mol. Cell. Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (554) Google Scholar). The presence of a stem-loop structure that occurs downstream of UGA in selenoprotein mRNA, known as a Sec insertion sequence (SECIS) element (4Low S.C. Berry M.J. Trends Biochem. Sci. 1996; 21: 203-208Abstract Full Text PDF PubMed Scopus (393) Google Scholar), is responsible for dictating UGA as Sec instead of the cessation of protein synthesis. In mammals, a specific SECIS-binding protein, designated SECIS-binding protein 2 (5Copeland P.R. Fletcher J.E. Carlson B.A. Hatfield D.L. Driscoll D.M. EMBO J. 2000; 19: 306-314Crossref PubMed Scopus (315) Google Scholar), recognizes the SECIS element, and a specific elongation factor, designated EFsec (6Tujebajeva R.M. Copeland P.R. Xu X.M. Carlson B.A. Harney J.W. Driscoll D.M. Hatfield D.L. Berry M.J. EMBO Rep. 2000; 1: 158-163Crossref PubMed Scopus (245) Google Scholar, 7Fagegaltier D. Hubert N. Yamada K. Mizutani T. Carbon P. Krol A. EMBO J. 2000; 19: 4796-4805Crossref PubMed Scopus (245) Google Scholar), recognizes selenocysteyl-tRNA[Ser]Sec, and the resulting complex guides Sec into the nascent polypeptide in response to UGA (2Hatfield D.L. Gladyshev V.N. Mol. Cell. Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (554) Google Scholar, 3Driscoll D.M. Copeland P.R. Annu. Rev. Nutr. 2003; 23: 17-40Crossref PubMed Scopus (325) Google Scholar). In higher vertebrates, there are two Sec tRNA[Ser]Sec isoforms that differ from each other by a single nucleoside modification at position 34, which is the wobble position of the anticodon (2Hatfield D.L. Gladyshev V.N. Mol. Cell. Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (554) Google Scholar). One isoform contains methylcarboxylmethyluridine (mcm5U) at this site, and the other contains methylcarboxymethyluridine-2′-O-methylribose (mcm5Um). Several lines of evidence suggest that methylation of the ribosyl moiety at the 2′-O-hydroxyl site (designated Um34) is a highly specialized event. It is the last step in the maturation of Sec tRNA[Ser]Sec. This modification step is dependent on the prior synthesis of the four modified bases found in Sec tRNA[Ser]Sec and on an intact tertiary structure, whereas synthesis of the other modified nucleosides, including mcm5U, is less stringently connected to primary and tertiary structure (8Kim L.K. Matsufuji T. Matsufuji S. Carlson B.A. Kim S.S. Hatfield D.L. Lee B.J. RNA. 2000; 6: 1306-1315Crossref PubMed Scopus (56) Google Scholar). The methylation step is influenced by selenium status, whereby the levels of mcm5U are enriched and mcm5Um reduced under conditions of selenium deficiency, and the ratio of the two isoforms is reversed under conditions of selenium sufficiency (2Hatfield D.L. Gladyshev V.N. Mol. Cell. Biol. 2002; 22: 3565-3576Crossref PubMed Scopus (554) Google Scholar). The presence of Um34 dramatically affects Sec tRNA[Ser]Sec secondary and tertiary structure (9Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C.J. Hill K.E. Burk R.F. McCloskey J.A. Hatfield D.L. J. Biol. Chem. 1993; 268: 14215-14223Abstract Full Text PDF PubMed Google Scholar). The presence of Um34 on Sec tRNA[Ser]Sec correlated with the expression of certain selenoproteins (e.g. GPx1) (10Chittum H.S. Hill K.E. Carlson B.A. Lee B.J. Burk R.F. Hatfield D.L. Biochim. Biophys. Acta. 1997; 1359: 25-34Crossref PubMed Scopus (49) Google Scholar, 11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar), whereas enrichment of the isoform lacking Um34 correlated with the expression of other selenoproteins (e.g. TR3) (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar). In addition, a specialized role of the selenium-induced, Um34 tRNA[Ser]Sec in selenoprotein translation was recently reported (12Jameson R.R. Diamond A.M. RNA. 2004; 10: 1142-1152Crossref PubMed Scopus (38) Google Scholar). However, the specific role of this isoform in selenoprotein synthesis has not been elucidated. The selenoprotein population in rodents is composed of 24 members, and there are 25 members in humans (13Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigo R. Gladyshev V.N. Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1923) Google Scholar). The function of many of these selenoproteins is not known. However, several approaches have been used in assessing their function and to provide better insights into their possible roles in health. Direct assays of selenoproteins such as glutathione peroxidase 1 (GPx1) and thioredoxin reductase 1 (TR1) demonstrate that they can function as antioxidants (14Nordberg J. Arner E.S. Free Radic. Biol. Med. 2001; 31: 1287-1312Crossref PubMed Scopus (2263) Google Scholar, 15Cheng W.H. Quimby F.W. Lei X.G. Free Radic. Biol. Med. 2003; 34: 918-927Crossref PubMed Scopus (44) Google Scholar), whereas genetic and biochemical characterization of selenoproteins such as Sep15 (16Kumaraswamy E. Malykh A. Korotkov K.V. Kozyavkin S. Hu Y. Kwon S.Y. Moustafa M.E. Carlson B.A. Berry M.J. Lee B.J. Hatfield D.L. Diamond A.M. Gladyshev V.N. J. Biol. Chem. 2000; 275: 35540-35547Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Hu Y.J. Korotkov K.V. Mehta R. Hatfield D.L. Rotimi C.N. Luke A. Prewitt T.E. Cooper R.S. Stock W. Vokes E.E. Dolan M.E. Gladyshev V.N. Diamond A.M. Cancer Res. 2001; 61: 2307-2310PubMed Google Scholar) and GPx1 (18Hu Y.J. Diamond A.M. Cancer Res. 2003; 63: 3347-3351PubMed Google Scholar, 19Ratnasinghe D. Tangrea J.A. Andersen M.R. Barrett M.J. Virtamo J. Taylor P.R. Albanes D. Cancer Res. 2000; 60: 6381-6383PubMed Google Scholar) suggests that these members may in addition have roles in cancer prevention. Gene knock-out studies involving selenoproteins in mice show that some, such as glutathione peroxidase 4 (GPx4) (20Yant L.J. Ran Q. Rao L. Van Remmen H. Shibatani T. Belter J.G. Motta L. Richardson A. Prolla T.A. Free Radic. Biol. Med. 2003; 34: 496-502Crossref PubMed Scopus (578) Google Scholar), selenoprotein P (SelP) (21Hill K.E. Zhou J. McMahan W.J. Motley A.K. Atkins J.F. Gesteland R.F. Burk R.F. J. Biol. Chem. 2003; 278: 13640-13646Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 22Schomburg L. Schweizer U. Holtmann B. Flohe L. Sendtner M. Kohrle J. Biochem. J. 2003; 370: 397-402Crossref PubMed Scopus (360) Google Scholar), thyroid hormone deiodinase 2 (23Schneider M.J. Fiering S.N. Pallud S.E. Parlow A.F. St. Germain D.L. Galton V.A. Mol. Endocrinol. 2001; 15: 2137-2148Crossref PubMed Scopus (251) Google Scholar), and mitochondrial thioredoxin reductase (TR3) (24Conrad M. Jakupoglu C. Moreno S.G. Lippl S. Banjac A. Schneider M. Beck H. Hatzopoulos A.K. Just U. Sinowatz F. Schmahl W. Chien K.R. Wurst W. Bornkamm G.W. Brielmeier M. Mol. Cell. Biol. 2004; 24: 9414-9423Crossref PubMed Scopus (395) Google Scholar) have essential roles in cellular function, since their removal is lethal or results in an abnormal phenotypic change. Other selenoproteins, such as GPx1 (25Cheng W.H. Ho Y.S. Ross D.A. Valentine B.A. Combs G.F. Lei X.G. J. Nutr. 1997; 127: 1445-1450Crossref PubMed Scopus (132) Google Scholar) and glutathione peroxidase 2 (GPx2) (26Esworthy R.S. Aranda R. Martin M.G. Doroshow J.H. Binder S.W. Chu F.F. Am. J. Physiol. 2001; 281: G848-G855Crossref PubMed Google Scholar), probably have nonessential roles, since their removal manifests little or no phenotypic change. Exposing animals lacking a nonessential selenoprotein to stress, however, demonstrates that the animal may not cope with certain stresses compared with their wild type counterparts (27Lei X.G. Biofactors. 2001; 14: 93-99Crossref PubMed Scopus (47) Google Scholar, 28Chu F.F. Esworthy R.S. Chu P.G. Longmate J.A. Huycke M.M. Wilczynski S. Doroshow J.H. Cancer Res. 2004; 64: 962-968Crossref PubMed Scopus (265) Google Scholar). Thus, some selenoproteins that are nonessential to life of the organism probably provide protection from environmental stress. Several studies have examined the effect of altering Sec tRNA[Ser]Sec expression on selenoprotein biosynthesis, which provided another means of elucidating the function of selenoproteins and their roles in health. Removal of trsp in knock-out mice is embryonic lethal, demonstrating that selenoproteins are essential to mammalian development (29Bo ̈sl M.R. Takaku K. Oshima M. Nishimura S. Taketo M.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5531-5534Crossref PubMed Scopus (285) Google Scholar, 30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar). Generation of a conditional knock-out of trsp using loxP-Cre technology has shown that Sec tRNA[Ser]Sec levels can be reduced in mammary tissue by about 80%, resulting in an altered selenoprotein expression in a selenoprotein-specific manner (30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar), whereas complete removal of trsp in liver demonstrated that selenoprotein expression is required for proper function of this organ (31Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Overexpression of Sec tRNA[Ser]Sec in transgenic mice carrying extra copies of the wild type transgene resulted in little or no effect on selenoprotein expression (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar). However, transgenic mice carrying extra copies of a Sec tRNA[Ser]Sec mutant transgene that produces a tRNA gene product lacking N6-isopentenyladenosine (i6A37) at this site and Um34 (8Kim L.K. Matsufuji T. Matsufuji S. Carlson B.A. Kim S.S. Hatfield D.L. Lee B.J. RNA. 2000; 6: 1306-1315Crossref PubMed Scopus (56) Google Scholar) affect selenoprotein synthesis in a selenoprotein- and tissue-specific manner (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar). In the present study, transgenic mice possessing trsp wild type or mutant transgenes (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar) were used to rescue trsp null mice (30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar). Mice dependent on the wild type transgene for survival manifested little or no change in selenoprotein expression, whereas mice dependent on the i6A mutant transgene rescued only some selenoproteins. Rescued selenoproteins included TR1 and TR3, whereas those that were poorly rescued included GPx1, GPx3, SelR, SelT, and SelW. Other selenoproteins, such as GPx2, GPx4, SelP, and Sep15, appeared to be partially rescued. These studies not only show that expression of some selenoproteins in mammals is highly dependent on Um34, but generating genetically altered mice that express reduced levels of a particular subclass of selenoproteins provides a unique model for examining the function of certain selenoproteins and their possible roles in health. Materials and Animals—Selenium-75 (specific activity 1000 Ci/mmol) was obtained from the Research Reactor Facility, University of Missouri (Columbia, MO); [α-32P]dCTP (specific activity ∼6000 Ci/mmol) was from PerkinElmer Life Sciences; and [3H]serine (specific activity 29 Ci/mmol) and Hybond Nylon N+ membranes were from Amersham Biosciences. NuPage 10% polyacrylamide gels, polyvinylidene difluoride membranes, Superscript II reverse transcriptase, See-Blue Plus2-protein markers, and the expressed sequence tag clone used in making the GPx2 probe (IMAGE Consortium Clone ID 6395265 (AN BQ961211)) were purchased from Invitrogen, and Universal Reference RNA was from Stratagene. SuperSignal West Dura extended duration substrate was obtained from Pierce, PCI-Neo vector was from Promega, and anti-rabbit horseradish peroxidase- and anti-chicken horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma. All other reagents were obtained commercially and were products of the highest grade available. Mice (strain C57BL/6) that were heterozygous for the Sec tRNA[Ser]Sec gene (wild type gene is designated as trsp and the deleted gene as Δtrsp) were from a previous study in our laboratory (30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar), as were the transgenic mice that were either homozygous for the wild type transgenes (strain FVB/N) wherein each allele carried 10 copies of the Sec tRNA[Ser]Sec transgene (designated trspt) or for the mutant Sec tRNA[Ser]Sec transgenes whose product lacked i6A37 and Um34, wherein each allele carried 20 copies of the mutant transgene (designated trspti6A–) (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar). Antibodies against GPx1 were obtained from Qichang Shen, antibodies against GPx2 were from Regina Brigelius-Flohé, and antibodies against GPx4 were from Donna Driscoll, whereas antibodies against TR1, TR3, SelR, SelT, and Sep15 were from our laboratories (13Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigo R. Gladyshev V.N. Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1923) Google Scholar, 32Gladyshev V.N. Jeang K.T. Wootton J.C. Hatfield D.L. J. Biol. Chem. 1998; 273: 8910-8915Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). 2B. A. Carlson, X.-M. Xu, V. N. Gladyshev, and D. L. Hatfield, unpublished observations. The care of animals was in accordance with the National Institutes of Health institutional guidelines under the expert direction of Dr. Kyle Stump (NCI, National Institutes of Health, Bethesda, MD). Genotyping Mice—DNA was extracted from mouse tail clippings and the presence or absence of trsp, trspt, and trspti6A– determined by PCR with the appropriate primers. The forward and reverse primers designated 5FPROA and CKNOR12, respectively, that complement bases beginning at –2137 bp and –255 bp upstream of the gene (30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar, 31Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) were used to detect trsp (yielding a 1.9-kb PCR fragment). CKNO2, a forward primer that complements bases beginning at –442 bp upstream of trsp (30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar), was used to detect trsp. Two new reverse primers, designated RES1 (5′-ccttgtgagacgaccttctatg-3′) and VP1 (5′-tgtggaattgtgagcggata-3′), where RES1 complements bases beginning at +538 bp downstream of trsp (the 5′-end of trsp is base +1) and VP1 corresponds to that portion of the vector sequence (pBluescript II) retained in the transgene for monitoring its insertion into the genome (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar), were used to monitor, along with the forward primer, CKNO2, the presence or loss of trsp (CKNO2-RES1) or trspt and trspti6A– (CKNO2-VP1). CKNO2-RES1 yields a 980-bp PCR trsp fragment and a 500-bp PCR Δtrsp fragment, and CKNO2-VP1 yields a 1072-bp PCR trspt or trspti6A– fragment. Isolation, Aminoacylation, Fractionation, and Quantification of tRNA and Primer Extension—Total tRNA was isolated from mouse liver, aminoacylated with [3H]serine and 19 unlabeled amino acids in the presence of rabbit reticulocyte synthetases (33Hatfield D. Matthews C.R. Rice M. Biochim. Biophys. Acta. 1979; 564: 414-423Crossref PubMed Scopus (59) Google Scholar), the resulting aminoacylated tRNA fractionated on a RPC-5 column (34Kelmers A.D. Heatherly D.E. Anal. Biochem. 1971; 44: 486-495Crossref PubMed Scopus (152) Google Scholar) in the absence and subsequently in the presence of Mg2+ as given (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar, 30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar). The amount of Sec tRNA[Ser]Sec expressed from the host trsp or from wild type (trspt) or mutant (trspti6A–) transgenes relative to the total Ser tRNA population and the distributions of mcm5U and mcm5Um has been detailed elsewhere (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar, 30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar). The presence of U at position 9 in host Sec tRNA[Ser]Sec and of a C at position 9 in the transgene Sec tRNA[Ser]Sec provided a means of distinguishing host from transgene-generated tRNA[Ser]Sec by primer extension using either ddG or ddA in the reaction and the appropriate primer as given (see Ref. 11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar and references therein). Labeling of Selenoproteins and GPx Activity Assays and Selenium Assay—Mice were injected intraperitoneally with 50 μCi of 75Se/g and sacrificed 48 h after injection. Tissues and organs were excised, immediately frozen in liquid nitrogen and stored at –80 °C until ready for use. Tissues were homogenized, the extracts were electrophoresed along with molecular weight markers, and the developed gel was stained with Coomassie Blue, dried, and exposed to a PhosphorImager as described (see Ref. 31Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar and references therein). GPx1–3 activities were measured using a standard assay with hydrogen peroxide as substrate as described previously (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar, 30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar, 31Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The amount of selenium in extracts of liver, heart, brain, and testes was determined by Oscar E. Olsen Biochemistry Laboratories at South Dakota State University as described (31Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Northern and Western Blot Analyses—Total RNA was isolated from liver, kidney, intestine, brain and testes, quantified and loaded onto gels, and transblotted onto a nylon membrane; the membrane was hybridized with 32P-labeled probe; and the Northern blot was analyzed with a PhosphorImager as given (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar). GPx1, GPx4, SPS2, D1, TR1, and SelP probes were prepared and used as described (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar). The SelV 733-bp probe was obtained from James Weaver, who generated it by digestion of a mouse SelV 1.16-bp XhoI-NotI fragment with BamHI. The remaining probes were generated by RT-PCR using Superscript II reverse transcriptase and universal reference RNA or mouse liver RNA (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar) with the exception of GPx2, which was generated from an expressed sequence tag clone (see "Materials and Animals"). Sizes of all RT-PCR-generated probes were in the 500–1200-bp range. Protein extracts were prepared from liver, kidney, brain, testes, and intestine, electrophoresed on NuPage 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and immunoblotted as given (11Moustafa M.E. Carlson B.A. El Saadani M.A. Kryukov G.V. Sun Q.A. Harney J.W. Hill K.E. Combs G.F. Feigenbaum L. Mansur D.B. Burk R.F. Berry M.J. Diamond A.M. Lee B.J. Gladyshev V.N. Hatfield D.L. Mol. Cell. Biol. 2001; 21: 3840-3852Crossref PubMed Scopus (116) Google Scholar, 30Kumaraswamy E. Carlson B.A. Morgan F. Miyoshi K. Robinson G.W. Su D. Wang S. Southon E. Tessarollo L. Lee B.J. Gladyshev V.N. Hennighausen L. Hatfield D.L. Mol. Cell. Biol. 2003; 23: 1477-1488Crossref PubMed Scopus (94) Google Scholar, 31Carlson B.A. Novoselov S.V. Kumaraswamy E. Lee B.J. Anver M.R. Gladyshev V.N. Hatfield D.L. J. Biol. Chem. 2004; 279: 8011-8017Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) with antibodies against Gpx1 (1:1000 dilution), Gpx2 (1:1000), Gpx4 (1:2000), SelR (1:1000), SelT (1:400), Sep15 (1:1000), TR1 (1:1000), and TR3 (1:1000). Anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:30,000) was used in all Western blots with the exception of that with TR3, in which anti-chicken horseradish peroxidase-conjugated secondary antibody (1:10,000) was used. Following the attachment of the secondary antibody, membranes were washed with 0.1% TBS-T, incubated in SuperSignal West Dura Extended Duration Substrate and exposed to x-ray film. Generation of Rescued Mice—To determine the role of the mutant Sec tRNA[Ser]Sec, mice were generated wherein the wild type Sec t