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Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay. Intriguingly, both EFsec and SBP2 localization differed depending on the cell line but significant colocalization of the two proteins was observed in cells where SBP2 levels were detectable. We identify functional nuclear localization and export signals in both proteins, demonstrate that SBP2 undergoes nucleocytoplasmic shuttling, and provide evidence that SBP2 levels and localization may influence EFsec localization. Our results suggest a mechanism for the nuclear assembly of the selenocysteine incorporation machinery that could allow selenoprotein mRNAs to circumvent nonsense-mediated decay, thus providing new insights into the mechanism of selenoprotein translation.Selenocysteine, the 21st amino acid, is the defining component of selenoproteins, a family whose members exhibit a wide range of functions, including roles in cellular oxidative status, male fertility, and thyroid function. Selenoprotein mRNAs are unique in that they recode UGA, which typically signals termination to the protein synthesis machinery, to instead specify cotranslational insertion of selenocysteine. In eukaryotes, this recoding process involves the assembly of complexes, termed Sec insertion sequence (SECIS) elements, at specific secondary structures in the 3Ј untranslated regions of these mRNAs (5). The complexes include a SECIS binding protein (SBP2) (13) and a dedicated elongation factor (EFsec) (19, 35) complexed with selenocysteyl-tRNA [Ser]Sec (for a review, see references 4 and 17). Disrupting the functions of the factors involved in selenocysteine incorporation or limiting selenium availability results in premature termination of translation at UGA codons (6,7,13,26,30,39). Under these circumstances, selenoprotein mRNAs are susceptible to degradation through a process termed nonsense-mediated decay (NMD) (for a review, see references 23 and 31), with different mRNAs exhibiting differential sensitivities to the NMD pathway (33, 38).NMD or mRNA surveillance targets mRNAs containing premature termination codons for degradation, ensuring that they do not produce prematurely terminated polypeptides. In higher eukaryotes, a nonsense codon is usually recognized as premature if it is located more than 50 to 55 nucleotides upstream of the last intron in the pre-mRNA. Sensitivity to NMD is conferred by the deposition of an exon-junction complex upstream of exon-exon boundaries during mRNA splicing and export (23). Immunity to NMD is thought to be acquired upon removal of these proteins during the first round of translation. However, if an mRNA escapes NM...

The type 2 monodeiodinase (D2) is an endoplasmic reticulum-resident membrane selenoprotein responsible for catalyzing the first step in thyroid hormone action, T(4) deiodination to T(3). Its short half-life is due to ubiquitination and proteolysis by proteasomes, a mechanism that is accelerated by D2 interaction with T(4). To identify proteins involved in D2 ubiquitination, a FLAG-tagged selenocystine133-to-Cys mutation of the human D2 (CysD2) was created and expressed in Saccharomyces cerevisiae using the GAL1 gene promoter. CysD2 activity was detected in the microsomes, indistinguishable from transiently expressed CysD2 in vertebrate cells. Treatment with 100 mg/ml cycloheximide or 30 micro M T(4) caused rapid loss of CysD2 (t(1/2) = approximately 30 min). Clasto-lactacystin beta-lactone not only increased galactose-inducible CysD2 but also stabilized CysD2 in the presence of cycloheximide or T(4). Immunoprecipitation with anti-FLAG antibody combined with Western analysis with antiubiquitin revealed that CysD2 is heavily ubiquitinated. Expression of CysD2 in yeast strains that lack the ubiquitin conjugases Ubc6p or Ubc7p stabilized CysD2 half-life by markedly reducing CysD2 ubiquitination, whereas no difference was detected in Ubc1p-deficient mutants. Similarly, expression of CysD2 in UBC6 and UBC7 mutants also impaired the substrate-induced loss of CysD2 activity and protein. In conclusion, Ubc6p and Ubc7p are required for normal and substrate-induced ubiquitination and proteolysis of D2.