Selenium deficiency, which ultimately leads to the reduction of selenoprotein expression, has been associated with many diseases, including but not limited to viral infection, reproductive dysfunction, cardiac and skeletal muscle disorders, AIDS progression, and increased incidence of several types of cancer. While much progress has been made in deciphering the mechanism of selenoprotein synthesis, less is known about its regulation. For example, when selenium are limiting, expression of phospholipid hydroperoxide glutathione peroxidase (PhGPx) in the rat liver is preserved in the expense of glutathione peroxidase 1 (GPx1) and other selenoproteins, suggesting a hierarchy of selenoprotein expression. Although it is unclear what dictates the hierarchy, we believe it is maintained by the interplay of several regulatory proteins, which is not likely to be explained by a single mechanism. We recently discovered that eukaryotic initiation factor 4a3 (elF4a3), a putative RNA helicase, is a likely candidate for the hypothesized regulatory protein. Our preliminary studies show that elF4a3 can distinguish between selenoprotein mRNAs by binding to the selenocysteine insertion sequence (SECIS), an element in the 3'-UTR required for selenoprotein expression. It preferentially binds to the SECIS from GPx1, which ranks low in the hierarchy, but not to the PhGPx, which ranks high in the hierarchy. It also reduces selenoprotein translation directed by GPx1 SECIS in vitro, suggesting that it may be involved in regulating the hierarchy of selenoprotein expression. In order to test this hypothesis, three aims will be proposed. First, we will identify the functional interactions between elF4a3 and the GPx1 SECIS using footprinting and mutational analysis. Results from this aim will help us to establish motifs within elF4a3 and SECIS elements that are important for interaction. Second, we will determine the function of elF4a3 in regulating selenoprotein expression. We will use cell based assays to assess the impact of both reduction and overexpression of elF4a3 protein on selenoprotein expression. Third, we will investigate the mechanism by which elF4a3 regulates selenoprotein translation. In this aim we will focus on answering basic questions regarding the mechanism of action of elF4a3 in reducing selenoprotein translation and also the impact of selenium deficiency on the regulation of elF4a3 expression.
We believe that the successful pursuit of these aims will result in a better understanding of the regulatory pathways that control selenoprotein expression in mammalian cells, which may ultimately lead to new therapeutic strategies for regulating selenoprotein gene expression at the translational level in vivo. ? ? ?
|Budiman, Michael E; Bubenik, Jodi L; Driscoll, Donna M (2011) Identification of a signature motif for the eIF4a3-SECIS interaction. Nucleic Acids Res 39:7730-9|
|Miniard, Angela C; Middleton, Lisa M; Budiman, Michael E et al. (2010) Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression. Nucleic Acids Res 38:4807-20|
|Budiman, Michael E; Bubenik, Jodi L; Miniard, Angela C et al. (2009) Eukaryotic initiation factor 4a3 is a selenium-regulated RNA-binding protein that selectively inhibits selenocysteine incorporation. Mol Cell 35:479-89|