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.
Highlights
The best studied function for selenium in the mammalian diet is its incorporation as selenocysteine (Sec)1 into a distinct set of proteins that carry out a diverse array of cellular functions
Development of a Sec Incorporation Reporter System—In order to quantitatively examine the efficiency of Sec incorporation in vitro, we developed a reporter system utilizing a luciferase coding region containing a Sec codon followed by a functional SECIS element
In addition we have found that of the known Sec incorporation factors, SBP2 is the only one limiting in rabbit reticulocyte lysates (RRL)
Summary
Selenocysteine; SECIS, Sec insertion sequence; UTR, untranslated region; PBS, phosphate-buffered saline; DAPI, 4Ј,6-diamidino-2-phenylindole; RRL, rabbit reticulocyte lysates; eEFSec, a Sec-specific translation elongation factor. Several reports have attempted to investigate efficiency in transfected cells [6, 15,16,17], but a quantitative study of the efficiency of Sec incorporation as measured by the ratio of translation termination products to Sec incorporation both in vitro and in transfected cells has not been performed. This question is vitally important to our understanding of Sec incorporation because the regulation of efficiency may be the primary determinant of variable selenoprotein expression. We have analyzed the contribution of SBP2 expression and subcellular localization, eEFSec, and SectRNASec to the efficiency of Sec incorporation
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