Selenocysteine is incorporated into selenoproteins during translation by an unconventional mechanism involving a dedicated Sec-tRNA that decodes UGA codons, which normally specify termination. This translational ?redefinition? of UGA is a highly regulated step that is of interest not only for our understanding of the mechanisms controlling selenoprotein synthesis but because it also provides an opportunity to examine more generally how the processes of translation elongation and decoding can be altered to regulate gene expression. Here we propose to examine the molecular mechanisms of UGA redefinition and its regulation by physiological conditions using a combination of new deep-sequencing approaches and established biochemical methods.
In Aim 1, we will test new hypotheses regarding the role of UGA-proximal ribosome pausing and its release when cells undergo endoplasmic reticulum (ER) stress. We propose this phenomenon to be a novel mechanism to rapidly induce selenoprotein expression in response to cellular stress. Further, we will address the biological role that these selenoproteins play in protection from ER stress and the progression to apoptosis.
In Aim 2, we examine the role of RNA structures in UGA-proximal ribosome pausing, response to ER stress, and sensing selenium. Finally, in Aim 3, we will utilize high throughput sequencing techniques, mass spectrometry, and RNA structural analysis of selenoprotein ribonucleoprotein complexes isolated directly from tissues of mice to examine the how selenium effects UGA redefinition and selenoprotein expression. These studies will provide new insights into how the control of translational elongation and the very process of decoding can be altered to regulate gene expression.
Selenium deficiency or excess has profound effects on the expression of selenoproteins and can lead to a variety of human health disorders. Despite significant advances in identification of the importance of selenium when it is incorporated into selenoproteins as the amino acid selenocysteine, there remain significant gaps in our knowledge of the molecular mechanisms controlling this process. The broad objectives of this proposal are to improve our understanding of how the synthesis of selenoproteins is affected by cellular and environmental conditions, how selenium is incorporated into selenoproteins, and ultimately how it affects human health and disease.
