RNA-binding proteins that specifically recognize the selenocysteine insertion sequence of human cellular glutathione peroxidase mRNA.
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 SDS-polyacrylamide 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.
Shen
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Department of Pediatrics, University of Massachusetts Medical School, Worcester 01655, USA.
McQuilkin
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Newburger
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PE
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D005979:Glutathione Peroxidase / Q000096:biosynthesis*; D006678:HIV / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017279:Selenocysteine / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D017931:DNA Primers; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017279:Selenocysteine; D005979:Glutathione Peroxidase