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

Authors

Xue-Ming Xu, NCI-NIH, Bethesda, MDFollow
Anton A. Turanov, University of Nebraska - LincolnFollow
Bradley A. Carlson, NCI-NIH, Bethesda, MDFollow
Min-Hyuk Yoo, NCI-NIH, Bethesda, MDFollow
Robert A. Everley, Harvard Medical SchoolFollow
Renu Nandakumar, University of Nebraska-LincolnFollow
Irina Sorokina, Midwest Bio Services, Overland Park, KS
Steven P. Gygi, Harvard Medical SchoolFollow
Vadim N. Gladyshev, Harvard Medical SchoolFollow
Dolph L. Hatfield, National Institutes of HealthFollow

Date of this Version

12-2010

Citation

PNAS, December 14, 2010, vol. 107, no. 50, pp. 21430–21434; doi: 10.1073/pnas.1009947107

Comments

This article is a U.S. government work, and is not subject to copyright in the United States.

Abstract

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 machinerybased mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.