Targeted insertion of cysteine by decoding UGA codons with mammalian selenocysteine machinery.
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 codons 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.
Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Xue-Ming
XM
Xu
Anton A
AA
Turanov
Bradley A
BA
Carlson
Min-Hyuk
MH
Yoo
Robert A
RA
Everley
Renu
R
Nandakumar
Irina
I
Sorokina
Steven P
SP
Gygi
Vadim N
VN
Gladyshev
Dolph L
DL
Hatfield
D000818:Animals; D018388:Codon, Terminator; D003545:Cysteine / Q000096:biosynthesis* / Q000235:genetics*; D004032:Diet; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D041681:NIH 3T3 Cells; D010710:Phosphates / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism; D012643:Selenium / Q000008:administration & dosage / Q000378:metabolism; D017279:Selenocysteine / Q000235:genetics / Q000378:metabolism*; D054481:Thioredoxin Reductase 1 / Q000235:genetics / Q000378:metabolism; D014166:Transferases / Q000235:genetics / Q000378:metabolism
D018388:Codon, Terminator; D007527:Isoenzymes; D010710:Phosphates; D012346:RNA, Transfer, Amino Acyl; D017279:Selenocysteine; D054481:Thioredoxin Reductase 1; D014166:Transferases; C060600:selenium transferase; D012643:Selenium; D003545:Cysteine; C035638:thiophosphoric acid