Qichang Shen

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.

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. J. Cell. Biochem. 77:507–516, 2000.

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. J. Cell. Physiol.

Selenium-regulated translation control of heterologous gene expression: Normal function of selenocysteine-substituted gene products

In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a “selenium translation element” in the 3′ untranslated region of mRNA. The selenium translation element is required to re‐interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium‐dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in‐frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3′ untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor β1. UGA (opal) mutant‐STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium‐dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild‐type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium‐dependent translational control to the expression of a wide variety of target genes, allows facile 75Se radioisotopic labeling of the heterologous proteins, and permits site‐specific heavy atom substitution.

Phosphorylation of p53 serine 18 upregulates apoptosis to suppress Myc-induced tumorigenesis

ATM and p53 are critical regulators of the cellular DNA damage response and function as potent tumor suppressors. In cells undergoing ionizing radiation, ATM is activated by double-strand DNA breaks and phosphorylates the NH2 terminus of p53 at serine residue 18. We have previously generated mice bearing an amino acid substitution at this position (p53S18A) and documented a role for p53 phosphorylation in DNA damage–induced apoptosis. In this present study, we have crossed Eμmyc transgenic mice with our p53S18A mice to explore a role for ATM-p53 signaling in response to oncogene-induced tumorigenesis. Similar to DNA damage induced by ionizing radiation, expression of c-Myc in pre–B cells induces p53 serine18 phosphorylation and Puma expression to promote apoptosis. Eμmyc transgenic mice develop B-cell lymphoma more rapidly when heterozygous or homozygous for p53S18A alleles. However, Eμmyc-induced tumorigenesis in p53S18A mice is slower than that observed in Eμmyc mice deficient for either p53 or ATM, indicating that both p53-induced apoptosis and p53-induced growth arrest contribute to the suppression of B-cell lymphoma formation in Eμmyc mice. These findings further reveal that oncogene expression and DNA damage activate the same ATM-p53 signaling cascade in vivo to regulate apoptosis and tumorigenesis. Mol Cancer Res; 8(2); 216–22

Nuclease Sensitive Element Binding Protein 1 Gene Disruption Results in Early Embryonic Lethality

Nuclease sensitive element binding protein 1 (NSEP1) is a member of the EFIA/NSEP1/YB‐1 family of DNA‐binding proteins whose members share a cold shock domain; it has also been termed DNA‐binding protein B and Y box binding protein‐1 because of its recognition of transcriptional regulatory elements. In addition, NSEP1 functions in the translational regulation of renin, ferritin, and interleukin 2 transcripts, and our laboratory has reported that it plays a role in the biosynthesis of selenium‐containing proteins. To test the functional importance of NSEP1 in murine embryonic development, we have utilized a clone of ES cells in which the NSEP1 gene had been disrupted by integration of a plasmid gene‐trapping vector into the seventh exon. Injection of these cells into C57BL/6 blastocysts resulted in 11 high percentage chimeric mice; crosses to wild type C57BL/6 mice generated 82 F1 agouti mice, indicating germ line transmission of the ES cell clone, but genotyping showed no evidence of the disrupted allele in any of these agouti offspring even though spermatozoa from four of five tested mice contained the targeted allele. Embryos harvested after timed matings of chimeric male mice demonstrated only the wildtype allele in 27 embryos tested at E7.5, E12.5, and E18.5. These results suggest that gene targeting of NSEP1 induces a lethal phenotype in early embryos, due to either haploinsufficiency of NSEP1 or formation of a dominant negative form of the protein. In either case, these data indicate the functional importance of the NSEP1 gene in murine early embryonic development. J. Cell. Biochem.