Nuclease sensitive element binding protein 1 associates with the selenocysteine insertion sequence and functions in mammalian selenoprotein translation.
Biosynthesis of selenium-containing proteins requires insertion of the unusual amino acid selenocysteine by alternative translation of a UGA codon, which ordinarily serves as a stop codon. In eukaryotes, selenoprotein translation depends upon one or more selenocysteine insertion sequence (SECIS) elements located in the 3'-untranslated region of the mRNA, as well as several SECIS-binding proteins. Our laboratory has previously identified nuclease sensitive element binding protein 1 (NSEP1) as another SECIS-binding protein, but evidence has been presented both for and against its role in SECIS binding in vivo and in selenoprotein translation. Our current studies sought to resolve this controversy, first by investigating whether NSEP1 interacts closely with SECIS elements within intact cells. After reversible in vivo cross-linking and ribonucleoprotein immunoprecipitation, mRNAs encoding two glutathione peroxidase family members co-precipitated with NSEP1 in both human and rat cell lines. Co-immunoprecipitation of an epitope-tagged GPX1 construct depended upon an intact SECIS element in its 3'-untranslated region. To test the functional importance of this interaction on selenoprotein translation, we used small inhibitory RNAs to reduce the NSEP1 content of tissue culture cells and then examined the effect of that reduction on the activity of a SECIS-dependent luciferase reporter gene for which expression depends upon readthrough of a UGA codon. Co-transfection of small inhibitory RNAs directed against NSEP1 decreased its expression by approximately 50% and significantly reduced luciferase activity. These studies demonstrate that NSEP1 is an authentic SECIS binding protein that is structurally associated with the selenoprotein translation complex and functionally involved in the translation of selenoproteins in mammalian cells.
Shen
Qichang
Q
Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Fan
Lin
L
Newburger
Peter E
PE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004251:DNA Transposable Elements / Q000235:genetics*; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017279:Selenocysteine / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D051840:Y-Box-Binding Protein 1 / Q000235:genetics / Q000378:metabolism*; D000094765:Glutathione Peroxidase GPX1
D004251:DNA Transposable Elements; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051840:Y-Box-Binding Protein 1; D017279:Selenocysteine; D005979:Glutathione Peroxidase; D000094765:Glutathione Peroxidase GPX1