Identification and molecular cloning of a human selenocysteine insertion sequence-binding protein. A bifunctional role for DNA-binding protein B.
Prokaryotic and eukaryotic cells incorporate the unusual amino acid selenocysteine at a UGA codon, which conventionally serves as a termination signal. Translation of eukaryotic selenoprotein mRNA requires a nucleotide selenocysteine insertion sequence in the 3'-untranslated region. We report the molecular cloning of the binding protein that recognizes the selenocysteine insertion sequence element in human cellular glutathione peroxidase gene (GPX1) transcripts and its identification as DNA-binding protein B, a member of the EFIA/dbpB/YB-1 family. The predicted amino acid sequence contains four arginine-rich RNA-binding motifs, and one segment shows strong homology to the human immunodeficiency virus Tat domain. Recombinant DNA-binding protein B binds the selenocysteine insertion sequence elements from the GPX1 and type I iodothyronine 5'-deiodinase genes in RNA electrophoretic mobility shift assays and competes with endogenous GPX1 selenocysteine insertion sequence binding activity in COS-1 cytosol extracts. Addition of antibody to DNA-binding protein B to COS-1 electromobility shift assays produces a slowly migrating "supershift" band. The molecular cloning and identification of DNA-binding protein B as the first eukaryotic selenocysteine insertion sequence-binding protein opens the way to the elucidation of the entire complex necessary for the alternative reading of the genetic code that permits translation of selenoproteins.
Shen
Q
Q
Department of Pediatrics, University of Medical Center, Worcester, Massachusetts 01605, USA.
Wu
R
R
Leonard
J L
JL
Newburger
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry*; D015696:Gene Products, tat / Q000737:chemistry; D005979:Glutathione Peroxidase / Q000096:biosynthesis*; D006801:Humans; D007453:Iodide Peroxidase / Q000235:genetics; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D014176:Protein Biosynthesis / Q000235:genetics; D011506:Proteins; D016601:RNA-Binding Proteins / Q000032:analysis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000235:genetics; D017279:Selenocysteine / Q000378:metabolism*; D051140:Selenoproteins; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D051140:Selenoproteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D017279:Selenocysteine; D007453:Iodide Peroxidase; D005979:Glutathione Peroxidase