Targeted insertion of cysteine by decoding UGA codons with mammalian selenocysteine machinery - PubMed (original) (raw)

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

Xue-Ming Xu et al. Proc Natl Acad Sci U S A. 2010.

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 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

In vitro Cys synthesis on tRNA[Ser]Sec by SecS. All reactions were carried out under anaerobic conditions in the presence of mSecS (unless otherwise indicated) and _O_-phospho-3H-seryl-tRNA[Ser]Sec. Cys synthesis was monitored by adding (A) SPO3, (B) mSPS2, Na2S, and ATP, (C) mSPS2 and Na2S, (D) cSPS2, Na2S, and ATP, (E) SelD, Na2S, and ATP, or (F) a control protein, Trx, that included mSPS2, Na2S, and ATP showed no activity in synthesizing Cys-tRNA[Ser]Sec. Migration of amino acid standards is shown below each panel. Experimental details are given in Materials and Methods.

Fig. 2.

Fig. 2.

Sulfide-dependent ATP hydrolysis by selenophosphate synthetases (SPSs). ATP hydrolysis reactions were carried out using [α-32P]ATP in the presence or absence (designated NC, negative control) of either 5.0 mM sodium sulfide or 0.1 mM selenide. Three SPSs, mSPS2, cSPS2, and SelD, were examined. At the end of the incubation period, compounds were separated on PEI TLC plates and visualized by exposing to a PhosphorImager screen. Experimental details are given in Materials and Methods.

Fig. 3.

Fig. 3.

Cys insertion into TR1 in NIH 3T3 cells in the presence of SPO3. (A) NIH 3T3 cells were cultured without (lanes 1, 2, duplicates) or with (lanes 3, 4, duplicates) 1 mM SPO3, labeled with 75Se, and analyzed by SDS-PAGE for selenoprotein expression. (Upper) 75Se labeling is shown, (Center) Western blot analyses of TR1, GPx4, and GPx1 are shown, and (Lower) Coomassie blue staining of proteins, used as a loading control, is shown. Experimental details are given in Materials and Methods. (B) NIH 3T3 cells were transfected with pGFP (the control vector encoding pGFP), pGFP-TR1-His-SECIS (encoding pGFP, TR1 with a His-tag at the C terminus and the SECIS element), or pGFP-TR1-His (encoding pGFP, TR1 with a His-tag at the C terminus but minus a SECIS element), grown either without (lanes 1–3) or with (lanes 4–6) 1 mM SPO3, labeled with 75Se, and analyzed by SDS-PAGE. (Upper) 75Se labeling is shown, (Center) Western blot analysis with either anti-TR1 or anti-His antibody as designated is shown, and (Lower) Coomassie blue staining of proteins, used as a loading control, is shown. Experimental details are given in Materials and Methods.

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References

    1. Arner ES. Selenoproteins—What unique properties can arise with selenocysteine in place of cysteine? Exp Cell Res. 2010;316:1296–1303. - PubMed
    1. Stadtman TC. Selenocysteine. Annu Rev Biochem. 1996;65:83–100. - PubMed
    1. Hatfield DL, Gladyshev VN. How selenium has altered our understanding of the genetic code. Mol Cell Biol. 2002;22:3565–3576. - PMC - PubMed
    1. Lu J, Holmgren A. Selenoproteins. J Biol Chem. 2009;284:723–727. - PubMed
    1. Low SC, Berry MJ. Knowing when not to stop: Selenocysteine incorporation in eukaryotes. Trends Biochem Sci. 1996;21:203–208. - PubMed

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