Recognition and binding of the human selenocysteine insertion sequence by nucleolin.
Prokaryotic and eukaryotic cells cotranslationally incorporate the unusual amino acid selenocysteine at a UGA codon, which conventionally serves as a termination signal. Translation of selenoprotein gene transcripts in eukaryotes depends upon a "selenocysteine insertion sequence" in the 3'-untranslated region. We have previously shown that DNA-binding protein B specifically binds this sequence element. We now report the identification of nucleolin as a partner in the selenoprotein translation complex. In RNA electromobility shift assays, nucleolin binds the selenocysteine insertion sequence from the human cellular glutathione peroxidase gene, competes with binding activity from COS cells, and shows diminished affinity for probes with mutations in functionally important, conserved sequence elements. Antibody to nucleolin interferes with the gel shift activity of COS cell extract. Antibody to DNA-binding protein B co-extracts nucleolin from HeLa cell cytosol, and the two proteins co-sediment in glycerol gradient fractions of ribosomal high salt extracts. Thus, nucleolin appears to join DNA-binding protein B and possibly other partners to form a large complex that links the selenocysteine insertion sequence in the 3'-untranslated region to other elements in the coding region and ribosome to translate the UGA "stop" codon as selenocysteine.
Wu
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Department of Pediatrics, University of Massachusetts Medical School, and the University of Massachusetts Cancer Center, Worcester, Massachusetts 01605, USA.
Shen
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Newburger
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PE
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins; D019556:COS Cells; D002499:Centrifugation, Density Gradient; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017279:Selenocysteine / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; D000097796:Nucleolin
D022762:CCAAT-Enhancer-Binding Proteins; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012313:RNA; D016601:RNA-Binding Proteins; D017279:Selenocysteine; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; D000097796:Nucleolin; C070856:YBX1 protein, human