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Selenium Metabolism in Drosophila

1999; Elsevier BV; Volume: 274; Issue: 26 Linguagem: Inglês

10.1074/jbc.274.26.18729

1083-351X

Xuan Zhou, Sang Ick Park, Mohamed E. Moustafa, Bradley A. Carlson, Pamela F. Crain, Alan M. Diamond, Dolph L. Hatfield, Byeong Jae Lee,

RNA modifications and cancer

The selenocysteine (Sec) tRNA population inDrosophila melanogaster is aminoacylated with serine, forms selenocysteyl-tRNA, and decodes UGA. The K m of Sec tRNA and serine tRNA for seryl-tRNA synthetase is 6.67 and 9.45 nm, respectively. Two major bands of Sec tRNA were resolved by gel electrophoresis. Both tRNAs were sequenced, and their primary structures were indistinguishable and colinear with that of the corresponding single copy gene. They are 90 nucleotides in length and contain three modified nucleosides, 5-methylcarboxymethyluridine,N 6-isopentenyladenosine, and pseudouridine, at positions 34, 37, and 55, respectively. Neither form contains 1-methyladenosine at position 58 or 5-methylcarboxymethyl-2′-O-methyluridine, which are characteristically found in Sec tRNA of higher animals. We conclude that the primary structures of the two bands of Sec tRNA resolved by electrophoresis are indistinguishable by the techniques employed and that Sec tRNAs in Drosophila may exist in different conformational forms. The Sec tRNA gene maps to a single locus on chromosome 2 at position 47E or F. To our knowledge,Drosophila is the lowest eukaryote in which the Sec tRNA population has been characterized to date. The selenocysteine (Sec) tRNA population inDrosophila melanogaster is aminoacylated with serine, forms selenocysteyl-tRNA, and decodes UGA. The K m of Sec tRNA and serine tRNA for seryl-tRNA synthetase is 6.67 and 9.45 nm, respectively. Two major bands of Sec tRNA were resolved by gel electrophoresis. Both tRNAs were sequenced, and their primary structures were indistinguishable and colinear with that of the corresponding single copy gene. They are 90 nucleotides in length and contain three modified nucleosides, 5-methylcarboxymethyluridine,N 6-isopentenyladenosine, and pseudouridine, at positions 34, 37, and 55, respectively. Neither form contains 1-methyladenosine at position 58 or 5-methylcarboxymethyl-2′-O-methyluridine, which are characteristically found in Sec tRNA of higher animals. We conclude that the primary structures of the two bands of Sec tRNA resolved by electrophoresis are indistinguishable by the techniques employed and that Sec tRNAs in Drosophila may exist in different conformational forms. The Sec tRNA gene maps to a single locus on chromosome 2 at position 47E or F. To our knowledge,Drosophila is the lowest eukaryote in which the Sec tRNA population has been characterized to date. The use of UGA as a codon for selenocysteine (Sec) 1The abbreviations used are: Sec, selenocysteine; LC/MS, liquid chromatography/electrospray ionization mass spectrometry; mcm5U, 5-methylcarboxymethyluridine; mcm5Um, 5-methylcarboxymethyl-2′-O-methyluridine; i6A, isopentenyladenosine. has been well documented in higher vertebrates (see Refs. 1Low S.C. Berry M.J. Trends Biochem. Sci. 1996; 21: 203-207Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 2Lee B.J. Park S.I. Park J.M. Chittum H.J. Hatfield D.L. Mol. Cell. 1996; 6: 509-520Google Scholar, 3Hatfield D.L. Gladyshev V.N. Park J.M. Park S.I. Chittum H.S. Huh J.R. Carlson B.A. Kim M. Moustafa M.E. Lee B.J. Kelly J.W. Comprehensive Natural Products Chemistry. 4. Elsevier Science Ltd., Oxford, England1999: 353-380Google Scholar for reviews). The tRNAs that are responsible for inserting Sec into selenoprotein in response to UGA in protein biosynthesis have been characterized in several mammals and in Xenopus laevis (see Refs. 2Lee B.J. Park S.I. Park J.M. Chittum H.J. Hatfield D.L. Mol. Cell. 1996; 6: 509-520Google Scholar and 3Hatfield D.L. Gladyshev V.N. Park J.M. Park S.I. Chittum H.S. Huh J.R. Carlson B.A. Kim M. Moustafa M.E. Lee B.J. Kelly J.W. Comprehensive Natural Products Chemistry. 4. Elsevier Science Ltd., Oxford, England1999: 353-380Google Scholar for reviews). Sec tRNAs are initially aminoacylated with serine by seryl-tRNA synthetase and are therefore designated Sec tRNA[Ser]Sec. The tRNA[Ser]Sec population in higher vertebrates consists primarily of two isoacceptors that differ from each other by a single methyl group on the 2′-O-ribose of the nucleoside in the wobble position (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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, 5Amberg R. Urban C. Reuner B. Scharff P. Pomerantz S.C. McCloskey J.A. Gross H.J. Nucleic Acids Res. 1993; 21: 5583-5588Crossref PubMed Scopus (34) Google Scholar), resulting in either 5-methylcarboxymethyluridine (mcm5U) or 5-methylcarboxymethyl-2′-O-methyluridine (mcm5Um). Exogenous selenium alters the levels and distributions of these isoacceptors in mammalian cells grown in culture (6Hatfield D. Lee B.J. Hampton L. Diamond A.M. Nucleic Acids Res. 1991; 19: 939-943Crossref PubMed Scopus (83) Google Scholar), in the tissues of rats maintained on a selenium-deficient diet (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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), in the tissues of selenium-deficient rats supplemented intravenously with this element (7Chittum H.S. Hill K.E. Carlson B.A. Lee B.J. Burk R.F. Hatfield D. Biochim. Biophys. Acta. 1997; 1359: 25-34Crossref PubMed Scopus (48) Google Scholar), in Xenopus oocytes maintained in culture (8Choi I.S. Diamond A.M. Crain P.F. Kolker J.D. McCloskey J.A. Hatfield D.L. Biochemistry. 1994; 33: 601-605Crossref PubMed Scopus (34) Google Scholar), and in mouse embryonic stem cells harboring only one functional copy of the Sec tRNA[Ser]Sec gene (9Chittum H.S. Baek H.J. Diamond A.M. Fernandez-Salguero P. Gonzalez F. Ohama T. Hatfield D.L. Kuehn M. Lee B.J. Biochemistry. 1997; 36: 8634-8639Crossref PubMed Scopus (23) Google Scholar). The primary sequences of the major species of rat liver (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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) and bovine liver (5Amberg R. Urban C. Reuner B. Scharff P. Pomerantz S.C. McCloskey J.A. Gross H.J. Nucleic Acids Res. 1993; 21: 5583-5588Crossref PubMed Scopus (34) Google Scholar) Sec tRNA[Ser]Sec have been reported. They are colinear with the corresponding Sec tRNA genes from each animal (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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, 10Diamond A.M. Montero-Puerner Y. Lee B.J. Hatfield D. Nucleic Acids Res. 1990; 18: 6727Crossref PubMed Scopus (30) Google Scholar) and contain only four modified nucleosides. In addition to mcm5U or mcm5Um at position 34, both tRNAs contain N 6-isopentenyladenosine (i6A) at position 37, pseudouridine (ψ) at position 55, and 1-methyladenosine at position 58. A single copy gene for Sec tRNA has been shown to be present in the genomes of higher vertebrates as well as in other animals such as Drosophila andCaenorhabditis elegans (11Lee B.J. Rajagopalan M. Kim Y.S. You K.-H. Jacobson K.B. Hatfield D. Mol. Cell. Biol. 1990; 10: 1940-1949Crossref PubMed Scopus (106) Google Scholar). Several studies, in addition to the present one, are beginning to elucidate the role of selenium metabolism in Drosophila. For example, Perlaky et al. (12.Perlaky, S., Merritt, K., and Cavener, D. (1998) Abstracts of the 39th Annual Drosophila Research Conference, Washington, D. C. March 25–29, 1998, Abstract 414C.Google Scholar) have shown that UGA occurs in the opening reading frame of Gld mRNA from several species of Drosophila, and these investigators have provided strong evidence that this UGA is a Sec codon. Two groups have reported a gene encoding a homologue of selenophosphate synthetase inDrosophila (13Persson B.C. Böck A. Jäckle H. Vorbrűggen G. J. Mol. Biol. 1997; 274: 174-180Crossref PubMed Scopus (40) Google Scholar, 14Alsina B. Serras F. Baguna J. Corominas M. Mol. Gen. Genet. 1998; 257: 112-123Crossref Scopus (29) Google Scholar). The gene product did not show any selenophosphate synthetase activity (13Persson B.C. Böck A. Jäckle H. Vorbrűggen G. J. Mol. Biol. 1997; 274: 174-180Crossref PubMed Scopus (40) Google Scholar), although the gene has a role in imaginal disc morphogenesis (14Alsina B. Serras F. Baguna J. Corominas M. Mol. Gen. Genet. 1998; 257: 112-123Crossref Scopus (29) Google Scholar). The present study was initiated to expand our understanding of selenium metabolism and, in particular, selenoprotein biosynthesis inDrosophila melanogaster by analyzing the Sec tRNA population. Like its counterpart in higher vertebrates,Drosophila Sec tRNA is aminoacylated with serine, forms selenocysteyl-tRNA, decodes UGA and, therefore, is designated Sec tRNA[Ser]Sec (2Lee B.J. Park S.I. Park J.M. Chittum H.J. Hatfield D.L. Mol. Cell. 1996; 6: 509-520Google Scholar, 3Hatfield D.L. Gladyshev V.N. Park J.M. Park S.I. Chittum H.S. Huh J.R. Carlson B.A. Kim M. Moustafa M.E. Lee B.J. Kelly J.W. Comprehensive Natural Products Chemistry. 4. Elsevier Science Ltd., Oxford, England1999: 353-380Google Scholar). The Sec tRNA[Ser]Secpopulation was resolved into two major bands on polyacrylamide gels that appear to be the result of conformational and not structural changes. Sequence analysis of the two major bands shows that they have indistinguishable structures, which are 90 nucleotides in length, and that they contain only three modified nucleosides, mcm5U, i6A, and ψ, at positions 34, 37, and 55, respectively. Wild type D. melanogaster adult flies, embryos, and the Drosophila cell line (Schneider Line 2 (designated SL2)) were obtained from Dr. C. Wu at the National Institutes of Health, and the 1st, 2nd, and 3rd instar larvae and pupae were obtained by growing Drosophila. 75SeO32− (specific activity 190 Ci/mmol as H2SeO3) was obtained from the University of Missouri Research Reactor Facility;l-[3H]serine (specific activity 29 Ci/mmol),l-[14C]serine (specific activity 171.6 mCi/mmol), and [α-32P]dCTP (specific activity >5000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. The probe used in northern hybridization assays was a 188-base pairDraIII-NdeI fragment encoding theDrosophila Sec tRNA gene and flanking sequences, and the probe used in in situ hybridization assays was a 5-kilobaseBamHI DNA fragment encoding the Drosophila Sec tRNA gene and flanking sequences (11Lee B.J. Rajagopalan M. Kim Y.S. You K.-H. Jacobson K.B. Hatfield D. Mol. Cell. Biol. 1990; 10: 1940-1949Crossref PubMed Scopus (106) Google Scholar). Mammalian seryl-tRNA synthetases were prepared as described (15Hatfield D. Mathews C.R. Rice M. Biochim. Biophys. Acta. 1979; 564: 414-423Crossref PubMed Scopus (59) Google Scholar), and an extract ofDrosophila embryos, normally used for transcription studies (16Becker P.B. Wu C. Mol. Cell. Biol. 1992; 12: 2241-2249Crossref PubMed Scopus (178) Google Scholar), was obtained from Drs. C. Wu and P. Georgel, NIH, Bethesda, MD and used as a source of Drosophila seryl-tRNA synthetase. An oligonucleotide, 5′-CTTCGACGCAAATCGACTAC-3′, was purchased from Bioneer, Inc. (Seoul, Korea) and used as primer for primer extension ofDrosophila Sec tRNA[Ser]Sec. Polyethyleneimine cellulose plates were obtained from J. T. Baker Inc. Total tRNA was extracted and isolated fromDrosophila as described (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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, 15Hatfield D. Mathews C.R. Rice M. Biochim. Biophys. Acta. 1979; 564: 414-423Crossref PubMed Scopus (59) Google Scholar). Transfer RNA was fractionated by gel electrophorsis (17Green M.R. Hatfield D. Miller M.J. Peacock A.C. Biochem. Biophys. Res. Commun. 1985; 129: 233-239Crossref PubMed Scopus (5) Google Scholar) with the exception that 17.5% gels containing 4 m urea were used. tRNA was aminoacylated with [14C]serine or [3H]serine in the presence of aminoacyl-tRNA synthetases from Drosophila or rabbit reticulocytes and fractionated on a RPC-5 column (18Kelmers A.D. Heatherly D.E. Anal. Biochem. 1971; 44: 486-495Crossref PubMed Scopus (152) Google Scholar) as given (15Hatfield D. Mathews C.R. Rice M. Biochim. Biophys. Acta. 1979; 564: 414-423Crossref PubMed Scopus (59) Google Scholar). Sec tRNA[Ser]Sec isoacceptors were purified in quantities sufficient for sequencing and for modified base analysis from 1 kg of frozen embryonic cells as described (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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, 19Diamond A.M. Dudock B. Hatfield D. Cell. 1981; 25: 497-506Abstract Full Text PDF PubMed Scopus (100) Google Scholar). Sec tRNA[Ser]Sec was purified from the 35,062A 260 units of total tRNA by BD-cellulose column chromatography, followed by two successive runs on a RPC-5 column (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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,19Diamond A.M. Dudock B. Hatfield D. Cell. 1981; 25: 497-506Abstract Full Text PDF PubMed Scopus (100) Google Scholar), and finally by gel electrophoreses as described above. Smaller quantities of Sec tRNAs were obtained from total tRNA for aminoacylation and coding studies by passing 3500A 260 units of tRNA over an RPC-5 column in 0.5m NaCl-buffer A, washing the column in this buffer until the A 260 units dropped below 1.0, and purifying Sec tRNA[Ser]Sec by two successive runs over the RPC-5 column as described (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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). Sec tRNA[Ser]Sec was identified in all column fractions by northern hybridization (see “Experimental Procedures”). Ser tRNASer was identified from the BD-cellulose column (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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, 19Diamond A.M. Dudock B. Hatfield D. Cell. 1981; 25: 497-506Abstract Full Text PDF PubMed Scopus (100) Google Scholar) and further purified as described above for tRNA[Ser]Sec, and the single species of tRNASer that decoded UCG was used for kinetic studies. SL2 cells were grown in serum-free insect medium (HyQ-CCMTM3 from HyClone Laboratories) to which sodium selenite was added to 1 μm. Cells (1.7 g wet weight) were collected at midlog, washed several times in medium without added selenium, and resuspended in 50 ml of fresh medium (without added selenium). The cells were labeled with 5 mCi of75Se, and the resulting 75Se-labeled tRNA was extracted and chromatographed on a RPC-5 column; the75Se-selenocysteyl-tRNA[Ser]Sec was isolated as described (20Hatfield D.L. Choi I.S. Mischke S. Owens L.D. Biochem. Biophys. Res. Commun. 1992; 184: 254-259Crossref PubMed Scopus (50) Google Scholar). Labeled aminoacyl-tRNAs were chromatographed on a RPC-5 column and responses of fractionated aminoacyl-tRNAs to trinucleoside diphosphate code words were examined by the ribosomal binding assay of Nirenberg and Leder (21Nirenberg M. Leder P. Science. 1964; 145: 1399-1407Crossref PubMed Scopus (653) Google Scholar) as described (15Hatfield D. Mathews C.R. Rice M. Biochim. Biophys. Acta. 1979; 564: 414-423Crossref PubMed Scopus (59) Google Scholar). 75Se-Sec attached to tRNA was identified by the procedure of Forshhammeret al. (22Forshhammer K. Leinfelder W. Boesmiller K. Veprek B. Bőck A. J. Biol. Chem. 1991; 266: 6318-6323PubMed Google Scholar) as described (20Hatfield D.L. Choi I.S. Mischke S. Owens L.D. Biochem. Biophys. Res. Commun. 1992; 184: 254-259Crossref PubMed Scopus (50) Google Scholar). Samples were dot- or slot-blotted onto nitrocellulose filters or electrotransfered onto Hybond-N+ membranes in 0.3× 40 mmTris-acetate, 1 mm EDTA at 50 V for 18 h at 4 °C; the tRNA was cross-linked to filters or membranes with a UV StratalinkerTM 1800 cross-linker and was hybridized with QuikHybRHybridization Solution from Stratagene. Labeled probe was prepared by the protocol of Prime-ItRRmT Random Primer Labeling kit from Stratagene. After hybridization, membranes were washed 2× for 15 min each at room temperature in 0.2× SSC with 0.1% SDS and then 1× for 30 min at room temperature in 0.1× SSC with 0.1% SDS. The relative amounts of probe attached to filters or membranes in response to tRNA in northern hybridization assays were determined using a PhosphorImager. Polytene chromosomes from salivary glands of late, 3rd instar larvae of wild type D. melanogaster (Oregon R) were prepared for in situhybridization as described (23Pardue M.L. Gall J.G. Methods Cell Biol. 1975; 10: 1-16Crossref PubMed Scopus (229) Google Scholar) and probed with the 5-kilobaseBamHI fragment encoding the Sec tRNA gene. The nonradioactive digoxigenin-labeled DNA probe was prepared according to the method of random-primed labeling (24Feinberg A.P. Vogelstein B. Anal. Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5190) Google Scholar), and hybridization and detection of the hybridized fragment were carried out by the procedures of Langer-Safer et al. (25Langer-Safer P.R. Levine M. Ward D.C. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4381-4385Crossref PubMed Scopus (555) Google Scholar) and Schmidt et al.(26Schmidt E.R. Keyl H.-G. Hankeln T. Chromosoma (Berlin). 1988; 96: 353-359Crossref Scopus (34) Google Scholar). Purified Sec tRNAs were sequenced as described previously (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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, 19Diamond A.M. Dudock B. Hatfield D. Cell. 1981; 25: 497-506Abstract Full Text PDF PubMed Scopus (100) Google Scholar), and modified nucleosides were determined by combined liquid chromatography/electrospray ionization mass spectrometry (LC/MS) as follows: tRNAs were digested totally to nucleosides using nuclease P1, phosphodiesterase I, and bacterial alkaline phosphatase as described (27Crain P.F. Methods Enzymol. 1990; 193: 782-790Crossref PubMed Scopus (242) Google Scholar), and the digest was injected directly into the liquid chromatograph without prior cleanup. Electrospray ionization LC/MS was conducted on a Fisons Quattro II mass spectrometer (Micromass, Inc., Beverly, MA) interfaced to a Hewlett-Packard 1090 liquid chromatograph. Chromatography was conducted on a 250 × 2 mm LC-18S column with 20 × 2 mm LC-18 precolumn (Supelco, Inc., Bellefonte, PA). The nucleoside mixture was fractionated using an ammonium acetate-acetonitrile gradient as described (28Pomerantz S.C. McCloskey J.A. Methods Enzymol. 1990; 193: 796-824Crossref PubMed Scopus (211) Google Scholar), except that the ammonium acetate concentration was decreased to 5 mm and the flow rate to 300 μl/min for compatibility with electrospray mass spectrometry. The entire effluent was conducted into the mass spectrometer without splitting; the interface temperature was 180 °C. Capillary and lens voltages were 2.8 and 0.25 kV, respectively. The mass range 105–400 was scanned in 0.4 s with a 0.1-s interscan delay. The cone voltage was ramped from 25 to 5 volts during the scan. Positive ions were detected. The 5′-ends of both Sec tRNA[Ser]Sec forms were examined by primer extending them to their 5′-termini using a 20-mer as template (see above) and determining the resulting length of the extended product by gel electrophoresis. To identify the Sec tRNA population within total tRNA, tRNA from Drosophila embryos was aminoacylated with [3H]serine in the presence of Drosophilaaminoacyl-tRNA synthetases. The resulting [3H]seryl-tRNA was fractionated on a RPC-5 column as shown in Fig.1. The bulk of the labeled tRNA eluted from the column in fractions 36–60, whereas a minor peak eluted in fractions 72–75. Because vertebrate Sec tRNAs are more hydrophobic than the corresponding Ser tRNAs and they represent from about 1 to 5% of the Ser tRNA population (reviewed in Refs. 2Lee B.J. Park S.I. Park J.M. Chittum H.J. Hatfield D.L. Mol. Cell. 1996; 6: 509-520Google Scholar and 3Hatfield D.L. Gladyshev V.N. Park J.M. Park S.I. Chittum H.S. Huh J.R. Carlson B.A. Kim M. Moustafa M.E. Lee B.J. Kelly J.W. Comprehensive Natural Products Chemistry. 4. Elsevier Science Ltd., Oxford, England1999: 353-380Google Scholar), we suspected that the minor later eluting peak represented the Sec tRNA population. To determine if this latter peak is Sec tRNA, column fractions were tested for their ability to hybridize to a 188-base pair fragment encoding the Drosophila Sec tRNA gene (11Lee B.J. Rajagopalan M. Kim Y.S. You K.-H. Jacobson K.B. Hatfield D. Mol. Cell. Biol. 1990; 10: 1940-1949Crossref PubMed Scopus (106) Google Scholar) by northern hybridization. The analysis confirmed that the minor later eluting peak represented the Sec tRNA population in Drosophila (see Fig.1). The Sec tRNA[Ser]Sec population represents 0.54% of the Ser tRNA population in Drosophila, which is lower than that found in mammalian cells and tissues (2Lee B.J. Park S.I. Park J.M. Chittum H.J. Hatfield D.L. Mol. Cell. 1996; 6: 509-520Google Scholar, 3Hatfield D.L. Gladyshev V.N. Park J.M. Park S.I. Chittum H.S. Huh J.R. Carlson B.A. Kim M. Moustafa M.E. Lee B.J. Kelly J.W. Comprehensive Natural Products Chemistry. 4. Elsevier Science Ltd., Oxford, England1999: 353-380Google Scholar). To obtain sufficient quantities of Sec tRNA[Ser]Sec for determining its codon recognition properties, it was separated from the bulk of other tRNAs, including tRNASer, as described under “Experimental Procedures.” Sec tRNA[Ser]Sec was aminoacylated with [3H]serine and chromatographed on a RPC-5 column, and the resulting single, homogeneous peak of [3H]seryl-tRNA[Ser]Sec, recognized UGA but not UGU, UGC, or UGG, which are codons with a degenerate base in the 3′-position, or the serine codons UCU, UCG, or AGU (data not shown). The data presented above show that Drosophila embryos contain a tRNA that is aminoacylated with serine, that specifically and efficiently decodes UGA in a ribosomal binding assay, and that hybridizes to the previously identified gene for Sec tRNA[Ser]Sec from this organism (11Lee B.J. Rajagopalan M. Kim Y.S. You K.-H. Jacobson K.B. Hatfield D. Mol. Cell. Biol. 1990; 10: 1940-1949Crossref PubMed Scopus (106) Google Scholar). To obtain direct evidence that Drosophila contain selenocysteyl-tRNA[Ser}Sec, we utilized theDrosophila cell line, SL2 (29Schneider I. J. Embryol. Exp. Morphol. 1972; 27: 353-365PubMed Google Scholar). SL2 cells were labeled with75Se, and the resulting labeled tRNA was extracted and chromatographed on a RPC-5 column (Fig.2). Two 75Se-labeled tRNA isoacceptors were observed, a minor front-running peak followed by a major later eluting peak. Both 75Se-labeled tRNAs recognized UGA in a ribosomal binding assay as shown in the figure. The major eluting peak was deacylated, and the 75Se-labeled material was identified as Sec (data not shown). Because this tRNA is aminoacylated with serine and forms selenocysteyl-tRNA, it can be designated Sec tRNA[Ser]Sec like its counterpart in higher vertebrates. Sec tRNA[Ser]Sec purified by RPC-5 chromatography (see “Experimental Procedures”) was used for aminoacylation and kinetic studies. The extent of aminoacylation of tRNA[Ser]Sec in the presence of either Drosophila or mammalian aminoacyl-tRNA synthetases was determined over a wide range of tRNA[Ser]Sec concentrations (data not shown). After 25 min of incubation, the extent of attachment of serine to tRNA[Ser]Sec was indistinguishable with homologous or heterologous synthetase. However, the rate of aminoacylation of Sec tRNA[Ser]Sec with serine was much faster withDrosophila than with mammalian synthetase (see Fig.3). The K m ofDrosophila Sec tRNA[Ser]Sec and Ser tRNASer for the homologous seryl-tRNA synthetase was determined. The K m for Sec tRNA[Ser]Sec was 6.67 nm and for Ser tRNASer it was 9.45 nm. TheK m for Ser tRNASer, which decodes UCU, UCC, and UCA, from bovine liver for rabbit reticulocyte seryl-tRNA synthetase was determined as a control and found to be 3.03 nm. Supplementation of the medium of cultured mammalian cells (6Hatfield D. Lee B.J. Hampton L. Diamond A.M. Nucleic Acids Res. 1991; 19: 939-943Crossref PubMed Scopus (83) Google Scholar), of Xenopus oocytes (8Choi I.S. Diamond A.M. Crain P.F. Kolker J.D. McCloskey J.A. Hatfield D.L. Biochemistry. 1994; 33: 601-605Crossref PubMed Scopus (34) Google Scholar), or the diets of rats (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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) with selenium enhances the level of the Sec tRNA[Ser]Secpopulation and causes a redistribution of the major Sec isoacceptors in cells and tissues. We therefore examined the effects of selenium on the Sec tRNA[Ser]Sec population in SL2 cells grown in culture (Fig. 4). SL2 cells were grown in varying levels of supplemented selenium, and the tRNA was extracted and examined by gel electrophoresis. Lane 1, which was included as a control, shows the resolution of two Sec tRNA[Ser]Sec forms by gel electrophoresis from adult flies. Lane 2 shows a single band of Sec tRNA[Ser]Sec from SL2 cells that corresponds to the lower running band in adult flies, indicating that cells grown in culture appeared to have little or none of the slower migrating band of Sec tRNA[Ser]Sec. These cells were grown in medium supplemented with nontoxic levels of sodium selenite (1 μm). Interestingly, higher levels of selenium that inhibited growth rate, and therefore are probably toxic to the cells, resulted in the appearance of the slower migrating band (lanes 3 and 4). Total tRNA isolated fromDrosophila at various stages of development, including embryonic, 1st, 2nd, and 3rd larval stages, pupae, and adult flies, manifested two bands of Sec tRNA[Ser]Sec as determined by gel electrophoresis (see Fig. 4 for adult flies). The amounts of these two bands did not appear to vary throughout development (data not shown). Transfer RNAs from both bands were isolated and purified from embryos as described under “Experimental Procedures,” and their primary structures were analyzed. Initially, both purified Sec tRNAs[Ser]Sec were analyzed by the partial formamide hydrolysis method previously used to determine the structure of bovine (19Diamond A.M. Dudock B. Hatfield D. Cell. 1981; 25: 497-506Abstract Full Text PDF PubMed Scopus (100) Google Scholar) and rat Sec tRNAs[Ser]Sec(4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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). In short, this procedure involves partial hydrolysis of purified tRNA, 5′-end labeling of the resulting 5′-hydroxyl termini, and resolution of the labeled “ladder” on a denaturing polyacrylamide gel. A representative autoradiogram of such a gel is presented in Fig.5. Examination of the radioactive ladders indicated that the patterns are very similar, differing mainly in the higher molecular weight region. In addition to the bands seen in the faster migrating tRNA species, the pattern seen for the slower migrating species has a large radioactive band separated by a gap in the ladder. Furthermore, several bands within the patterns show different relative intensities that are indicated by anasterisk in the figure. This signifies differences in accessibility to cleavage by formamide and typically is an indication of different conformational states of the compared molecules. It is also noteworthy that neither pattern has a single gap at the position representing the wobble nucleoside within the anticodon, as would be expected if these tRNAs contained the modified residue mcm5Um in this position that was previously observed with one of the mammalian Sec tRNA[Ser]Sec isoacceptors (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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). Each of the radioactive bands from the acrylamide gel was excised, as were others obtained from the same digest/labeling following electrophoresis for different times to optimally resolve the ladder and finally digested to 5′,3′-nucleotide diphosphates. Digested products were resolved by ascending chromatography on polyethyleneimine cellulose to obtain the primary sequence by comparison to the known mobilities of modified and nonmodified residues (30Silberklang M. Gillum A.M. RajBhandary U.L. Methods Enzymol. 1979; 59: 58-109Crossref PubMed Scopus (352) Google Scholar). This analysis indicated that the primary sequence of both tRNAs was colinear with that reported for the Drosophila Sec tRNA[Ser]Sec gene (11Lee B.J. Rajagopalan M. Kim Y.S. You K.-H. Jacobson K.B. Hatfield D. Mol. Cell. Biol. 1990; 10: 1940-1949Crossref PubMed Scopus (106) Google Scholar). Only three modified residues were identified in this manner, mcm5U at position 34, i6A at position 37, and ψ at position 55, in both characterized molecules (Fig. 6). Autoradiograms of representative polyethyleneimine plates are shown for the TψCG loop, variable region (Fig. 6 A), and the region including the anticodon (Fig. 6 B). The identities of modified nucleoside diphosphates were confirmed by further digestion with nuclease P1 and two dimensional chromatography as described previously for the same modified residues (data not shown) (see Refs. 4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. Hill K.E. Burk R.F. McCloskey J.A. Hatfield D.L. J. Biol. Chem. 1993; 268: 14215-14223Abstract Full Text PDF PubMed Google Scholarand 19Diamond A.M. Dudock B. Hatfield D. Cell. 1981; 25: 497-506Abstract Full Text PDF PubMed Scopus (100) Google Scholar). The modified residue content of both tRNA[Ser]Sec forms was verified by electrospray ionization LC/MS (Fig. 7). Modified nucleoside identities were established from mass spectra and characteristic relative retention times (5Amberg R. Urban C. Reuner B. Scharff P. Pomerantz S.C. McCloskey J.A. Gross H.J. Nucleic Acids Res. 1993; 21: 5583-5588Crossref PubMed Scopus (34) Google Scholar, 28Pomerantz S.C. McCloskey J.A. Methods Enzymol. 1990; 193: 796-824Crossref PubMed Scopus (211) Google Scholar). In agreement with results from direct sequencing of both tRNAs, only three modified nucleosides are present in each tRNA: ψ, mcm5U, and i6A.Figure 7Chromatogram (UV absorbance at 260 nm) from LC/MS analysis of modified nucleosides in electrophoretically separated tRNA [Ser] Sec species. A, faster migrating tRNA; B, slower migrating tRNA.View Large Image Figure ViewerDownload (PPT) The analyses described above failed to determine a structural explanation for the different mobilities of the two Sec tRNA species detected by electrophoresis of Drosophila embryo tRNA. Clearly, the molecules are identical for the 90 nucleotides resolved by our techniques. Furthermore, 5′-end labeling of both tRNAs and complete digestion with ribonuclease T1 resulted in labeled fragments of indistinguishable mobility, consistent with the previously reported sequence of the Drosophila Sec tRNA[Ser]Sec gene. Elongation of both tRNA[Ser]Sec forms to their 5′-ends by primer extension (see “Experimental Procedures”) revealed that each contained the same number of nucleotides in this region, suggesting that these ends of both molecules were identical. Based on the collective data, we propose that the observed differences in mobility of the Sec tRNA[Ser]Sec species in embryos and in SL2 cells when they are exposed to high concentrations of selenium (Fig. 4) are because of altered conformational states of these molecules. This conclusion is supported by the observed differences in formamide cleavage susceptibility reported above. However, determination of the factors involved in the stabilization of the molecules between the conformational states will require further investigation. The Sec tRNA[Ser]Sec gene occurs in single copy in the Drosophila genome (11Lee B.J. Rajagopalan M. Kim Y.S. You K.-H. Jacobson K.B. Hatfield D. Mol. Cell. Biol. 1990; 10: 1940-1949Crossref PubMed Scopus (106) Google Scholar). A 5-kilobase BamHI fragment encoding Sec tRNA[Ser]Sec was used as a probe to determine the location of the gene within the genome of Drosophila. As shown in Fig. 8, the gene maps to a single site on chromosome 2 in region 47E or F. Species representative of the three primary domains, archaea, bacteria, and eukaryotes (31Woese C.R. Kandler O. Wheelis M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4576-4579Crossref PubMed Scopus (4535) Google Scholar), recognize the UGA triplet as the codon for Sec (32Hatfield D.L. Diamond A.M. Trends Genet. 1993; 9: 69-70Abstract Full Text PDF PubMed Scopus (40) Google Scholar). Because Sec tRNAs[Ser]Sec are essential for both Sec biosynthesis and the subsequent incorporation of this amino acid into selenoproteins, we have chosen to examine tRNA[Ser]Sec in an eukaryotic organism that is both evolutionarily distant from mammals yet amenable to study either as a whole organism or as an established cell line. Herein, we demonstrate that Drosophila contains a Sec tRNA[Ser]Sec, which is aminoacylated with serine, and that the seryl-tRNA[Ser]Sec is converted to selenocysteyl-tRNA[Ser]Sec, which is capable of specifically recognizing UGA in a ribosomal binding assay. In this respect, this process is similar to that observed in bacteria and mammals. Drosophila represents the lowest eukaryote from which the direct sequence analysis of a Sec tRNA[Ser]Sec has been determined. The sequence presented within this study is colinear with the gene sequence previously reported (11Lee B.J. Rajagopalan M. Kim Y.S. You K.-H. Jacobson K.B. Hatfield D. Mol. Cell. Biol. 1990; 10: 1940-1949Crossref PubMed Scopus (106) Google Scholar). We have detected only three modified nucleosides, ψ, i6A, and mcm5U, although it remains possible that other modifications occur in a fraction of the tRNA population that is below the limits of our detection. Although we detect two distinct Sec tRNAs by electrophoresis of Drosophila tRNA derived from embryos and numerous other stages of development, only one species was detected in this manner from cultured cells. On the other hand, two peaks of selenocysteyl-tRNA[Ser]Sec from SL2 cells were resolved by RPC-5 chromatography when the cells were labeled with75Se. This observation suggests the existence of multiple species of Sec tRNA[Ser]Sec in SL2 cells, which may differ either in base modification or in their primary sequences that might also occur in developing tissues but are below our limits of detection during sequencing procedures. It is possible that the multiple forms of Sec tRNA[Ser]Sec analyzed in embryos are present in different cell types, whereas only one form is seen in SL2 cells by electrophoresis because of its clonagenic origin. Our analyses of these two forms clearly demonstrate that they do not differ because of 2′-O-methylation of mcm5U, as seen in mammalian cells and tissues. This would have been evident from the partial formamide hydrolysis procedure (Fig. 5) or from modified nucleoside analysis by LC/MS (Fig. 7). As we have been unable to detect either a difference in primary sequence or base modification of these Sec tRNA[Ser]Sec forms, we hypothesize that the distinct electrophoretic mobilities are because of a conformational difference between these molecules. Although the basis for the stabilization might occur during the initial folding of the molecules or by association with as yet undetectable molecules, we note that incubation of SL2 cells at toxic concentrations of selenium appears to stimulate the conversion of the single species of Sec tRNA[Ser]Secin cells to one with the mobility observed for the slower migrating species detected in embryos. Because mammalian Sec tRNAs respond to selenium with a shift in the distribution of the mcm5U species to that containing mcm5Um and an accompanying change in conformation (4Diamond A.M. Choi I.S. Crain P.F. Hashizume T. Pomerantz S.C. Cruz R. Steer C. 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), our observation on the SL2 Sec tRNA response to selenium may represent a primordial response, which accomplishes the same end. Future studies are directed toward the determination of the biological roles for distinct Sec tRNAs[Ser]Sec observed in eukaryotes. In summary, the investigation of the selenoprotein biosynthesis machinery in Drosophila has revealed features both in common with and distinct from that already described for other eukaryotes. The general principle that this process requires a dedicated, relatively undermodified tRNA that is the site of selenocysteine synthesis from serine and decodes UGA is demonstrated for this species as well. The lack of any Sec tRNA species including either the 1-methyladenosine or mcm5Um modifications, as well as the conformational response to selenium status described above, suggests that the study of selenium metabolism in Drosophila will yield novel insights into the evolution of the translation of selenium-containing proteins. In addition, the relatively low abundance of Sec tRNA[Ser]Sec in Drosophila (Fig. 1) as compared with higher vertebrates (2Lee B.J. Park S.I. Park J.M. Chittum H.J. Hatfield D.L. Mol. Cell. 1996; 6: 509-520Google Scholar, 3Hatfield D.L. Gladyshev V.N. Park J.M. Park S.I. Chittum H.S. Huh J.R. Carlson B.A. Kim M. Moustafa M.E. Lee B.J. Kelly J.W. Comprehensive Natural Products Chemistry. 4. Elsevier Science Ltd., Oxford, England1999: 353-380Google Scholar) suggests that because the intracellular levels of tRNAs reflect their requirements for protein synthesis (see Refs. 2Lee B.J. Park S.I. Park J.M. Chittum H.J. Hatfield D.L. Mol. Cell. 1996; 6: 509-520Google Scholar and 3Hatfield D.L. Gladyshev V.N. Park J.M. Park S.I. Chittum H.S. Huh J.R. Carlson B.A. Kim M. Moustafa M.E. Lee B.J. Kelly J.W. Comprehensive Natural Products Chemistry. 4. Elsevier Science Ltd., Oxford, England1999: 353-380Google Scholar and references therein), a lower amount of selenoprotein biosynthesis and perhaps a variety of selenoproteins synthesized occurs in flies. We express sincere appreciation to Dr. Carl Wu for the generous gift of D. melanogaster embryos used throughout this study and for the D. melanogaster adult flies used to raise our own fly colony and to Drs. Carl Wu and P. Georgel for the Drosophila embryo extract used as a source of seryl-tRNA synthetase. We also appreciate the advice and assistance of Drs. Siddhartha Roy and Dan L. Sackett with theK m studies.