Squires, Jeffrey E.2011-07-212011-07-2120089780549787778http://hdl.handle.net/10125/20460Thesis (Ph.D.)--University of Hawaii at Manoa, 2008.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 cotranslationally 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.Selenophosphate synthetase 1 (SPS1) and selenophosphate synthetase 2 (SPS2) have been implicated as essential components in selenoprotein biosynthesis. In vitro studies have demonstrated that SPS2, but not SPS1, generates the active selenium donor for Sec synthesis, monoselenophosphate. To establish the functions of these two proteins in vivo, siRNAs were used to knockdown SPS1 and SPS2 expression, and DNA constructs were designed to overexpress the two proteins in HEK-293 and MSTO-211H cells. Knockdown of SPS1 and SPS2 decreased the protein levels of several selenoproteins whereas the overexpression of SPS1 and SPS2 increased selenoprotein levels. These results reconfirm the role of SPS2 and establish SPS1 as a factor that regulates selenoprotein biosynthesis.Includes bibliographical references (leaves xxx-xxx).Also available by subscription via World Wide Web83 leaves, bound 29 cmSelenium 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 cotranslationally 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. Selenophosphate synthetase 1 (SPS1) and selenophosphate synthetase 2 (SPS2) have been implicated as essential components in selenoprotein biosynthesis. In vitro studies have demonstrated that SPS2, but not SPSI, generates the active selenium donor for Sec synthesis, monoselenophosphate. To establish the functions of these two proteins in vivo, siRNAs were used to knockdown SPS1 and SPS2 expression, and DNA constructs were designed to overexpress the two proteins in HEK-293 and MSTO-211H cells. Knockdown of SPS1 and SPS2 decreased the protein levels of several selenoproteins whereas the overexpression of SPS1 and SPS2 increased selenoprotein levels. These results reconfirm the role of SPS2 and establish SPS1 as a factor that regulates selenoprotein biosynthesis.en-USAll UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.Thesis