Selective inhibition of the polypeptide chain elongation in eukaryotic cells.

The effect of Cephalotaxus alkaloids--homoharringtonine and cephalotaxine--on translation in a cell-free system from rabbit reticulocytes and on phenylalanine polymerisation by human ribosomes was studied. The effect of the alkaloids on the nonenzymatic and the eEF-1-dependent Phe-tRNA(Phe) binding to poly(U)-programmed 80S ribosomes, diphenylalanine synthesis accompanying nonenzymatic Phe-tRNA(Phe) binding and acetylphenylalanyl-puromycin formation was examined. Homoharringtonine was shown to inhibit the formation of diphenylalanine and acetylphenylalanyl-puromycin catalysed by human and rat liver ribosomes, but was inactive as an inhibitor on the E. coli elongation system. Neither nonenzymatic nor enzymatic Phe-tRNA(Phe) binding was noticeably affected by the alkaloid. It has been proposed that the site of homoharringtonine binding to 80S ribosomes should overlap or coincide with the acceptor site of the ribosomal peptidyl transferase centre. The association constant of homoharringtonine for 80S human ribosomes was estimated to be (2.57 +/- 0.33).10(7) M-1 in the presence of puromycin. Cephalotaxine did not exert a significant influence on the polypeptide chain elongation.

Interplay between termination and translation machinery in eukaryotic selenoprotein synthesis.

Termination of translation in eukaryotes is catalyzed by eRF1, the stop codon recognition factor, and eRF3, an eRF1 and ribosome-dependent GTPase. In selenoprotein mRNAs, UGA codons, which typically specify termination, serve an alternate function as sense codons. Selenocysteine incorporation involves a unique tRNA with an anticodon complementary to UGA, a unique elongation factor specific for this tRNA, and cis-acting secondary structures in selenoprotein mRNAs, termed SECIS elements. To gain insight into the interplay between the selenocysteine insertion and termination machinery, we investigated the effects of overexpressing eRF1 and eRF3, and of altering UGA codon context, on the efficiency of selenoprotein synthesis in a transient transfection system. Overexpression of eRF1 does not increase termination at naturally occurring selenocysteine codons. Surprisingly, selenocysteine incorporation is enhanced. Overexpression of eRF3 did not affect incorporation efficiency. Coexpression of both factors reproduced the effects with eRF1 alone. Finally, we show that the nucleotide context immediately upstream and downstream of the UGA codon significantly affects termination to incorporation ratios and the response to eRF overexpression. Implications for the mechanisms of selenocysteine incorporation and termination are discussed.

Alkaloid homoharringtonine inhibits polypeptide chain elongation on human ribosomes on the step of peptide bond formation.

The aim of the present study was to investigate homoharringtonine alkaloid effect on: (i) the nonenzymatic and eEF-1-dependent Phe-tRNAPhe binding to poly(U)-programmed human placenta 80 S ribosomes; (ii) diphenylalanine synthesis accompanying nonenzymatic Phe-tRNAPhe binding; and (iii) acetylphenylalanyl-puromycin formation. Neither nonenzymatic nor eEF-1-dependent Phe-tRNAPhe binding were noticeably affected by the alkaloid, whereas diphenylalanine synthesis and puromycin reaction were strongly inhibited by homoharringtonine. It has been proposed that the site of homoharringtonine binding on 80 S ribosomes should overlap or coincide with the acceptor site of the ribosome.

Selenocysteine incorporation directed from the 3'UTR: characterization of eukaryotic EFsec and mechanistic implications.

The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid. The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA. Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.

Selenoprotein P expression, purification, and immunochemical characterization.

Most selenoproteins contain a single selenocysteine residue per polypeptide chain, encoded by an in-frame UGA codon. Selenoprotein P is unique in that its mRNA encodes 10-12 selenocysteine residues, depending on species. In addition to the high number of selenocysteines, the protein is cysteine- and histidine-rich. The function of selenoprotein P has remained elusive, in part due to the inability to express the recombinant protein. This has been attributed to presumed inefficient translation through the selenocysteine/stop codons. Herein, we report for the first time the expression of recombinant rat selenoprotein P in a transiently transfected human epithelial kidney cell line, as well as the endogenously expressed protein from HepG2 and Chinese hamster ovary cells. The majority of the expressed protein migrates with the predicted 57-kDa size of full-length glycosylated selenoprotein P. Based on the histidine-rich nature of selenoprotein P, we have purified the recombinant and endogenously expressed proteins using nickel-agarose affinity chromatography. We show that the recombinant rat and endogenous human proteins react in Western blotting and immunoprecipitation assays with commercial anti-histidine antibodies. The ability to express, purify, and immunochemically detect the recombinant protein provides a foundation for investigating the functions and efficiency of expression of this intriguing protein.

Expression and characterization of nonmammalian selenoprotein P in the zebrafish, Danio rerio.

BACKGROUND: Selenoprotein P is a protein of considerable intrigue, due to its unusual composition and requirements for its biosynthesis. Whereas most selenoproteins contain a single selenocysteine residue, the human, bovine and rodent selenoprotein P genes encode proteins containing 10-12 selenocysteines. Selenoprotein P genes have, to date, only been reported in mammals, and the function of the protein remains elusive. RESULTS: Herein, we report the identification and characterization of nonmammalian selenoprotein P in the zebrafish Danio rerio. Sequencing of the cDNA revealed the presence of 17 selenocysteine codons, the highest number reported in any protein. Two histidine-rich regions present in the mammalian selenoprotein P sequences are conserved in the zebrafish protein, and two SECIS elements are present in the 3' untranslated region. Whole-mount in situ hybridization of zebrafish embryos revealed high levels of expression of selenoprotein P mRNA in fertilized eggs and in the yolk sac of developing embryos. Transient transfection of the cDNA in mammalian cells resulted in efficient expression of the full-length secreted selenoprotein. A single N-glycosylation site is predicted, and shown to be utilized. CONCLUSIONS: Discovery of selenoprotein P in the zebrafish opens a previously unavailable avenue for genetic investigation of the functions of this unusual protein.

Decoding apparatus for eukaryotic selenocysteine insertion.

Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3' untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms. We have identified and characterized the first eukaryotic selenocysteyl-tRNA-specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor-SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl-tRNA-elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.