Efficiency of Mammalian Selenocysteine Incorporation

Abstract

Five components have thus far been identified that are necessary for the incorporation of selenocysteine (Sec) into ϳ25 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 ϳ40%, 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.

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

“…This assay, which is widely used in the field, has been validated to be specific for selenocysteine incorporation. 33,34 As shown in Figure 1B, maximal luciferase activity was obtained using 80 nM purified bacterially expressed SBP2-CT, which is comparable to reports in the literature that range from 40-300 nM. [35][36][37][38][39] In contrast, 0.4 nM in vitro translated SBP2-CT generates an equivalent signal to 40 nM of recombinant protein (compare Figs.…”

“…The luciferase reporter constructs luc/UGA 258 /PhGPx and luc/UGA 258 /Dio1 have been previously described. 26,33 The phospholipid hydroperoxide glutathione peroxidase (PHGPx), glutathione peroxidase 1 (GPx1), and deiodinase 2 (Dio2) SECIS probe sequences were also previously described. 26,53 DNA sequences The sequences used in the multiple alignment of SBP2-RBD were obtained primarily from GenBank and Ensembl databases.…”

The Selenocysteine-specific Elongation Factor Contains a Novel and Multi-functional Domain

“…The addition of recombinant His-tagged eEFSec to rabbit reticulocyte lysate, a system limiting for SBP2, did not increase Sec incorporation activity (13). Efforts to deplete eEFSec from reticulocyte lysate by immunoprecipitation have also been unsuccessful (data not shown).…”

“…1A). This method has been demonstrated to faithfully indicate Sec incorporation because the production of full-length and active luciferase is strictly SECIS-and SBP2-dependent (13).…”

“…In Vitro Translation and Sec Incorporation Assay-Sec incorporation activity in cell-free extracts was measured with a luciferase mRNA reporter containing a UGA-Sec codon at position 258 of the coding region and the rat GPX4 SECIS element at the 3Ј untranslated region (13). The luciferase reporter was also used to measure the translation activity by having an UGU-Cys codon instead of the UGA-Sec codon.…”