Translational incorporation of the unusual amino acid selenocysteine in eukaryotes requires a coding region UGA codon (which otherwise serves as a termination signal), a selenocysteine insertion sequence (SECIS) in the 3-untranslated region of the mRNA, and selenocysteyl-tRNA. The mechanisms involved in SECIS recognition by the eukaryotic translational machinery remain unknown. We report the detection of RNA-binding proteins that specifically recognize the SECIS from human cellular glutathione peroxidase (GPX1) transcripts. RNA gel shift assays showed three retarded bands after incubation with COS-1 whole cell lysate or S-100 cytosol fraction or with extracts from hepatoma cell lines HepG2 and Hep3B. The specificity of the binding was demonstrated by competition by cold unlabeled SECIS RNA and by lack of competition by other RNA species with similar stem-loop secondary structures, such as the human immunodeficiency virus (HIV) transactivation-response region of HIV mRNA element, and mutated SECIS constructs. UV cross-linking and SDSpolyacrylamide gel electrophoresis revealed at least two proteins, with estimated molecular masses of 55,000 and 65,000 Da, that bind to the SECIS. Examination of a series of insertion and deletion SECIS mutants indicated recognition of the SECIS primarily through the basal stem region, although the upper stem, loop, and two of three short conserved sequences also appear to contribute to the affinity of the binding.A wide variety of sequence elements have recently been identified in the 5Ј-and 3Ј-untranslated regions (UTR) 1 of eukaryotic mRNAs. The resultant RNA secondary structures and the RNA-binding proteins that recognize them play important roles in the regulation of mRNA stability and translation (1-6).One such sequence is the selenocysteine insertion sequence (SECIS; also termed the selenium translation element) in the 3Ј-UTR of eukaryotic gene transcripts encoding selenoproteins (7-9). Members of this unique group of proteins contain one or more selenocysteine residues, often at their active sites, and several catalyze important oxidation/reduction reactions (10). The gene transcripts for these selenoproteins encode the atypical amino acid by a UGA codon, which normally functions as a termination signal (11,12). In eukaryotes, incorporation of selenocysteine into the polypeptide chain at this UGA codon requires the presence of at least one SECIS, located in the 3Ј-UTR as much as 1200 nucleotides downstream (8, 11). Comparison of the sequences of SECIS in rat and human iodothyronine deiodinase, cellular glutathione peroxidase, and selenoprotein P mRNAs (7, 8, 13) has revealed only three very short conserved sequences, but all share a common computer-predicted secondary structure featuring a long stem, several bulges, and an apical loop with three short conserved sequences. These characteristic features are illustrated in the diagram of the SECIS from the human cellular glutathione peroxidase (GPX1) gene transcript in Fig. 1A.We have shown that this SECIS is necessary for trans...