Making Sense out of Nonsense: Mechanistic Insight into Selenoprotein Syntiiesis Factors a Dissertation Submitted to the Graduate Dmsion of the Unnersity

Selenium is an essential micronutrient linked to various aspects of health. Selenium exerts its biological activity through incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationaIly at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Selenoprotein mRNAs are potential targets for degradation via nonsense-mediated decay, due to the presence of in-frame UGA codons. When UGA recoding is inefficient, as occurs when selenium is limiting, termination occurs at these positions. Based on predicted exon-intron structure, fourteen of the 25 human selenoprotein mRNAs are predicted to be sensitive to nonsense-mediated decay. Among these, sensitivity varies, resulting in a hierarchy of selenoprotein synthesis. Potential factors dictating this hierarchy are the SECIS binding proteins, SBP2 and nucleolin. To investigate the role of these proteins in the hierarchy of selenoprotein synthesis, we carried out knockdowns of SBP2 expression and assessed the effects on selenoprotein mRNA levels. We also investigated in vivo binding of selenoprotein mRNAs by SBP2 and nucleolin, via immunoprecipitation and quantitation of bound mRNAs. We report that SBP2 exhibits preferential binding to some selenoprotein mRNAs over others, whereas nucleolin exhibits minimal binding differences. Thus SBP2 is a determinant in dictating the hierarchy of selenoprotein synthesis via differential selenoprotein mRNA translation and sensitivity to nonsense-mediated decay.