ABSTRACT
Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3' untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level.
Subject(s)
3' Untranslated Regions , Gene Expression Regulation , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Selenoproteins/genetics , Animals , Cell Line, Tumor , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/isolation & purification , RNA, Messenger/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/isolation & purification , Rats , Selenoproteins/metabolism , NucleolinABSTRACT
The synthesis of selenoproteins requires the translational recoding of the UGA stop codon as selenocysteine. During selenium deficiency, there is a hierarchy of selenoprotein expression, with certain selenoproteins synthesized at the expense of others. The mechanism by which the limiting selenocysteine incorporation machinery is preferentially utilized to maintain the expression of essential selenoproteins has not been elucidated. Here we demonstrate that eukaryotic initiation factor 4a3 (eIF4a3) is involved in the translational control of a subset of selenoproteins. The interaction of eIF4a3 with the selenoprotein mRNA prevents the binding of SECIS binding protein 2, which is required for selenocysteine insertion, thereby inhibiting the synthesis of the selenoprotein. Furthermore, the expression of eIF4a3 is regulated in response to selenium. Based on knockdown and overexpression studies, eIF4a3 is necessary and sufficient to mediate selective translational repression in cells. Our results support a model in which eIF4a3 links selenium status with differential selenoprotein expression.
