Use of RNA affinity chromatography to investigate factors involved in selenoprotein synthesis and nonsense decay

Abstract

Selenocysteine, the 21st amino acid, is the defining component of selenoproteins, a family whose members function in cellular oxidative defenses, male fertility, and thyroid function. Selenium deficiency has been linked to the pathogenesis of cancers, diabetes, cardiovascular disease, neurodegenerative diseases and other diseases in which cumulative oxidative damage is implicated. Selenoprotein mRNAs recode UGA, which typically signals termination, to instead specify insertion of selenocysteine. This recoding process involves assembly of complexes at specific secondary structures in the 3' untranslated regions of these mRNAs termed SECIS elements. When selenium is limiting, UGA is recognized as a termination codon and can trigger nonsense‐mediated decay (NMD) of the corresponding mRNAs. Different selenoprotein mRNAs exhibit differential sensitivity to NMD, and the mechanistic basis for this is not known. We are using glutathione‐RNA (GRNA) chromatography to isolate RNA‐binding proteins that bind selenoprotein mRNAs, to further elucidate the mechanism of selenoprotein synthesis and the factors involved in conferring sensitivity or resistance to NMD. Using GRNA with w.t. and mutant SECIS elements, we have identified three new SECIS‐binding proteins and are characterizing the physiological functions of these proteins in selenoprotein synthesis and NMD.Supported by NIH grant DK‐47320 to MJB.