Targeted insertion of cysteine by decoding UGA codons with mammalian selenocysteine machinery

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 107; no. 50; pp. 21430 - 21434
• Main Authors: Xu, Xue-Ming, Turanov, Anton A., Carlson, Bradley A., Yoo, Min-Hyuk, Everley, Robert A., Nandakumar, Renu, Sorokina, Irina, Gygi, Steven P., Gladyshev, Vadim N., Hatfield, Dolph L., Valentine, Joan Selverstone
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 14.12.2010; National Acad Sciences
• Subjects: Adenosine triphosphatase; Amino acids; Animals; Biological Sciences; Biosynthesis; Cells; Codon, Terminator; Codons; Cysteine - biosynthesis; Cysteine - genetics; Diet; Isoenzymes - genetics; Isoenzymes - metabolism; Liver; Liver - enzymology; Mammals; Mice; Mice, Inbred C57BL; Mutagenesis, Insertional; NIH 3T3 Cells; Phosphates - metabolism; Proteins; RNA, Transfer, Amino Acyl - genetics; RNA, Transfer, Amino Acyl - metabolism; Rodents; Selenium; Selenium - administration & dosage; Selenium - metabolism; Selenocysteine - genetics; Selenocysteine - metabolism; Selenoproteins; Thioredoxin Reductase 1 - genetics; Thioredoxin Reductase 1 - metabolism; Transfer RNA; Transferases - genetics; Transferases - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1009947107

Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA [Ser]Sec and its insertion was dependent on the Sec insertion sequence element in the 3' UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA [Ser]Sec to generate Cys-tRNA [Ser]Sec . Cys was inserted in vivo at UGA condons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinery-based mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.