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. 2004 Sep 3;279(36):37852-9.
doi: 10.1074/jbc.M404639200. Epub 2004 Jun 30.

Efficiency of mammalian selenocysteine incorporation

Anupama Mehta  1 , Cheryl M Rebsch, Scott A Kinzy, Julia E Fletcher, Paul R Copeland
Affiliations

Efficiency of mammalian selenocysteine incorporation

Anupama Mehta et al. J Biol Chem. .

Abstract

Five components have thus far been identified that are necessary for the incorporation of selenocysteine (Sec) into approximately 25 mammalian proteins. Two of these are cis sequences, a SECIS element in the 3'-untranslated region and a Sec codon (UGA) in the coding region. The three known trans-acting factors are a Sec-specific translation elongation factor (eEFSec), the Sec-tRNA(Sec), and a SECIS-binding protein, SBP2. Here we describe a system in which the efficiency of Sec incorporation was determined quantitatively both in vitro and in transfected cells, and in which the contribution of each of the known factors is examined. The efficiency of Sec incorporation into a luciferase reporter system in vitro is maximally 5-8%, which is 6-10 times higher than that in transfected rat hepatoma cells, McArdle 7777. In contrast, the efficiency of Sec incorporation into selenoprotein P in vitro is approximately 40%, suggesting that as yet unidentified cis-elements may regulate differential selenoprotein expression. In addition, we have found that SBP2 is the only limiting factor in rabbit reticulocyte lysate but not in transfected rat hepatoma cells where SBP2 is found to be mostly if not entirely cytoplasmic despite having a strong putative nuclear localization signal. The significance of these findings with regard to the function of known Sec incorporation factors is discussed.

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Figures

Fig. 1
Fig. 1. Design of luciferase-based Sec incorporation system
A, 10 ng of mRNA corresponding to Cys to Sec (U) mutants of luciferase mRNA were translated in a RRL Sec incorporation assay in the presence (black bars) or absence (white bars) of 200 ng of recombinant SBP2399–846. B, diagram of the constructs used in this study: L/Sec/p, luciferase coding region with UGASec at codon 258 and the wild-type PHGPx SECIS element in the 3′ UTR; L/Cys/p, same as L/Sec/P but with a Cys codon at position 258; L/Sec/mutP, same as L/Sec/P but with mutated PHPGx SECIS element lacking the conserved AUGA motif; L/UAA/p, same as L/Sec/P but with UAAterm at position 258. C, 200 ng of luciferase mRNA corresponding to three of the constructs diagrammed in B were translated in RRL in the presence of 160 ng of SBP2399–846.
Fig. 2
Fig. 2. Comparison of full-length and truncated SBP2
A, purified strep-tagged full-length (gray bars) or C-terminal SBP2399–846 (white bars) were added to a Sec incorporation assay with the L/Sec/P mRNA. Average luciferase activity ± S.E. is plotted against SBP2 concentration (n = 3). B, purified Strep-tagged SBP2399–846 was added in increasing amounts (2-fold serial dilution ranging from 930 to 7 nM) to a Sec incorporation assay including [35S]Met and luciferase mRNAs as indicated. Radioactive proteins were resolved by SDS-PAGE and detected by PhosphorImage analysis. The positions of full-length (FL) luciferase versus the pre-Sec peptide (pSp) are indicated on the right.
Fig. 3
Fig. 3. Calculation of the efficiency of Sec incorporation
A, 35S-labeled luciferase protein from a Sec incorporation reaction containing the indicated amount of SBP2399–846 was resolved by SDS-PAGE and detected by PhosphorImage analysis. B, percent efficiency of Sec incorporation. Black bars represent percent efficiency expressed as amount of full-length luciferase divided by total product (full-length plus pre-Sec peptide). Gray bars represent percent efficiency expressed as the amount of full-length Sec-containing luciferase protein divided by the amount of full-length Cys-containing luciferase protein as determined by PhosphorImage analysis. White bars represent percent efficiency expressed as the amount of luciferase activity derived from Sec-containing luciferase divided by the amount of activity derived from Cys-containing luciferase after correcting for differences in specific activity.
Fig. 4
Fig. 4. Efficiency of Sel P translation in vitro
A, all possible products of Sel P translation are listed with their predicted molecular masses. B, in vitro translation of Sel P mRNA in the presence (lane 1) or absence (lane 2) of recombinant SBP2399–846. Reactions lacking Sel P mRNA with and without added SBP2 are shown in lanes 3 and 4, respectively. Observed molecular masses are shown on the left.
Fig. 5
Fig. 5. SBP2 is the only limiting factor for Sec incorporation in RRL
Purified Sec-tRNASec was added in the amounts indicated to a standard Sec incorporation assay. Reactions contained either 1 pmol each of SBP2399–846 and eEFSec (black bars), 1 pmol of SBP2399–846 alone (white bars), or no added protein (gray bars). B, same reactions as described for A but in the presence of Phe-tRNAPhe as a control for nonspecific effects.
Fig. 6
Fig. 6. Efficiency of Sec incorporation in transfected cells
A, McArdle 7777 cells were transfected with varying amounts of L/Sec/P or L/Cys/P plasmids as indicated. Extracts were analyzed for luciferase activity (expressed as luminescence per mg of total protein). Activity for the L/Cys/P transfections was divided by a factor of 10 to allow visual comparison. B, percent efficiency of Sec incorporation was calculated by dividing activity derived from L/Sec/P and L/Cys/P at each of the DNA amounts used for transfection. The data was gathered from two luciferase assays for two independent experiments. C, Western blot analysis of 2% of the total protein (~85 μg) extracted from cells transfected with luciferase constructs as indicated. For L/Cys/P extracts, only 0.2% was loaded to enhance clarity. Full-length luciferase and the pre-Sec peptide are noted with arrows. D, Northern blot analysis of one half of the total RNA (~25 μg) extracted from cells transfected with 2, 0.5, 0.125, and 0.03 μg of L/Sec/P and L/Cys/P DNA (lanes 2–5 and 8–11) and 2 μg each of L/Sec/mutP and L/UAA/P DNA (lanes 6 and 12). RNA isolated from mock transfections was loaded in lanes 1 and 7. Bottom panel shows ethidium bromide-stained rRNA as a loading control.
Fig. 7
Fig. 7. Subcellular localization of SBP21–846
McArdle 7777 cells were transfected with SBP21–846 cDNA, and the V5/His-tagged SBP2 protein was detected with anti-V5 mouse monoclonal antibody followed by fluorescein-conjugated secondary antibody. Top panel shows SBP2 fluorescence. Bottom panel shows merged image of DAPI-stained nuclei and SBP2 fluorescence.
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References

    1. Driscoll DM, Copeland PR. Annu Rev Nutr. 2003;23:17–40. - PubMed
    1. Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R, Gladyshev VN. Science. 2003;300:1439–1443. - PubMed
    1. Sunde RA. In: Selenium: Its Molecular Biology and Role in Human Health. Hatfield DL, editor. Kluwer Academic Publishers; Boston: 2001. pp. 81–98.
    1. Moriarty PM, Reddy CC, Maquat LE. Mol Cell Biol. 1998;18:2932–2939. - PMC - PubMed
    1. Sun X, Li X, Moriarty PM, Henics T, LaDuca JP, Maquat LE. Mol Biol Cell. 2001;12:1009–1017. - PMC - PubMed

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