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• W2051276497 abstract "The highly conserved protein eIF5A found in Archaea and all eukaryotes uniquely contains the posttranslationally formed amino acid hypusine. Despite being essential the functions of this protein and its modification remain unclear. To gain more insight into these functions temperature-sensitive mutants of the human EIF5A1 were characterized in the yeast Saccharomyces cerevisiae. Expression of the point mutated form V81G in a ΔeIF5A strain of yeast led to a strongly temperature-sensitive phenotype and to a significantly reduced protein level at restrictive temperature. The mutant showed accumulation of a subset of mRNAs that was also observed in nonsense-mediated decay (NMD)-deficient yeast strains. After short incubation at restrictive temperature the mutant exhibited increased half-lives of the intron containing CYH2 pre-mRNA and mature transcripts of NMD-dependent genes. Reduced telomere silencing and shortening was detected in the V81G mutant further supporting similarities to NMD-deficient strains. Our data suggest that eIF5A mediates important cellular processes like cell viability and senescence through its effects on the stability of certain mRNAs. The highly conserved protein eIF5A found in Archaea and all eukaryotes uniquely contains the posttranslationally formed amino acid hypusine. Despite being essential the functions of this protein and its modification remain unclear. To gain more insight into these functions temperature-sensitive mutants of the human EIF5A1 were characterized in the yeast Saccharomyces cerevisiae. Expression of the point mutated form V81G in a ΔeIF5A strain of yeast led to a strongly temperature-sensitive phenotype and to a significantly reduced protein level at restrictive temperature. The mutant showed accumulation of a subset of mRNAs that was also observed in nonsense-mediated decay (NMD)-deficient yeast strains. After short incubation at restrictive temperature the mutant exhibited increased half-lives of the intron containing CYH2 pre-mRNA and mature transcripts of NMD-dependent genes. Reduced telomere silencing and shortening was detected in the V81G mutant further supporting similarities to NMD-deficient strains. Our data suggest that eIF5A mediates important cellular processes like cell viability and senescence through its effects on the stability of certain mRNAs. The cellular physiology of mRNA processing, transport, localization, and turnover is central to the process of gene expression at the posttranscriptional level. Increasing evidence has been found for a close connection between mRNA degradation processes and the steps of translation. The highly conserved hypusine-containing protein eIF5A has been implicated in both of these aspects of RNA metabolism; however, its precise cellular function is not yet fully understood.Hypusine formation is a two-step enzymatic reaction catalyzed by deoxyhypusine synthase and then deoxyhypusine hydroxylase (1Park M.H. J. Biochem. 2006; 139: 161-169Crossref PubMed Scopus (250) Google Scholar). The disruption of genes encoding either eIF5A or deoxyhypusine synthase in yeast leads to a lethal phenotype (2Sasaki K. Abid M.R. Miyazaki M. FEBS Lett. 1996; 384: 151-154Crossref PubMed Scopus (110) Google Scholar) demonstrating that the deoxyhypusine residue is essential for the function of eIF5A and thus for proliferation and cell survival. The genome of Saccharomyces cerevisiae contains two HYP genes (HYP1, alias TIF51B or ANB1, and HYP2, alias TIF51A) coding for eIF5A. These genes are differentially expressed under aerobic and anaerobic conditions (3Wöhl T. Klier H. Ammer H. Lottspeich F. Magdolen V. Mol. Gen. Genet. 1993; 241: 305-311Crossref PubMed Scopus (61) Google Scholar) and share an identity of 90%. HYP genes of other higher eukaryotes, e.g. one of the two human HYP genes (encoding EIF5A1), can functionally replace these yeast-specific genes (4Magdolen V. Klier H. Wöhl T. Klink F. Hirt H. Hauber J. Lottspeich F. Mol. Gen. Genet. 1994; 244: 646-652Crossref PubMed Scopus (38) Google Scholar, 5Schwelberger H.G. Kang H.A. Hershey J.W. J. Biol. Chem. 1993; 268: 14018-14025Abstract Full Text PDF PubMed Google Scholar).The eIF5A protein was first isolated from rabbit reticulocyte lysate ribosomes and classified as a translation initiation factor because in vitro the protein enhanced the building reaction of the first peptide bond measured as the yield of methioninepuromycin (6Kemper W. Berry K. Merrick W. J. Biol. Chem. 1976; 251: 5551-5557Abstract Full Text PDF PubMed Google Scholar). However, cell fractionation revealed that only a small fraction of the protein associated with ribosomes (7Thomas A. Goumans H. Amesz H. Benne R. Voorma H.O. Eur. J. Biochem. 1979; 98: 329-337Crossref PubMed Scopus (42) Google Scholar). Moreover depletion or inactivation of the protein in S. cerevisiae reduced the global protein synthesis rate by only 30% (8Kang H.A. Hershey J.W.B. J. Biol. Chem. 1994; 269: 3934-3940Abstract Full Text PDF PubMed Google Scholar, 9Zuk D. Jacobson A. EMBO J. 1998; 17: 2914-2925Crossref PubMed Scopus (162) Google Scholar). Thus, the role of eIF5A as a translation initiation factor remains to be confirmed.eIF5A is an RNA-binding protein (10Liu Y.P. Nemeroff M. Yan Y.P. Chen K.Y. Biol. Signals. 1997; 6: 166-174Crossref PubMed Scopus (32) Google Scholar), and it was hypothesized to regulate the translation of a subset of mRNAs that are needed for G1/S cell cycle progression because agents blocking deoxyhypusine hydroxylase (11Hanauske-Abel H.M. Slowinska B. Zagulska S. Wilson R.C. Staiano-Coico L. Hanauske A.R. McCaffrey T. Szabo P. FEBS Lett. 1995; 366: 92-98Crossref PubMed Scopus (71) Google Scholar) or deoxyhypusine synthase (12Jin B.F. He K. Hu M.R. Yu M. Shen B.F. Zhang X.M. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2003; 11: 325-328PubMed Google Scholar, 13Shi X.P. Yin K.C. Ahern J. Davis L.J. Stern A.M. Waxman L. Biochim. Biophys. Acta. 1996; 1310: 119-126Crossref PubMed Scopus (64) Google Scholar) induced a cell cycle arrest at the G1/S boundary in several mammalian cell types. These results were supported by the G1 arrest at 37 °C of yeast cells expressing a temperature-sensitive point mutated form of Tif51A (14Chatterjee I. Gross S.R. Kinzy T.G. Chen K.Y. Mol. Gen. Genet. 2006; 275: 264-276Crossref PubMed Scopus (57) Google Scholar).The finding that eIF5A is a cellular cofactor of human immunodeficiency virus type 1 Rev and human T-cell lymphotrophic virus type 1 Rex transactivator proteins in mRNA export and the interaction of eIF5A with the general exportin Crm1p for nuclear export signal-containing proteins in higher eukaryotes suggested it played a role in nucleoplasmatic shuttling of mRNA (15Bevec D. Jaksche H. Oft M. Wöhl T. Himmelspach M. Pacher A. Schebesta M. Koettnitz K. Dobrovnik M. Csonga R. Lottspeich F. Hauber J. Science. 1996; 271: 1858-1860Crossref PubMed Scopus (180) Google Scholar, 16Rosorius O. Reichart B. Kratzer F. Heger P. Dabauvalle M. Hauber J. J. Cell Sci. 1999; 112: 2369-2380Crossref PubMed Google Scholar, 17Ruhl M. Himmelspach M. Bahr G.M. Hammerschmid F. Jaksche H. Wolff B. Aschauer H. Farrington G.K. Probst H. Bevec D. J. Cell Biol. 1993; 123: 1309-1320Crossref PubMed Scopus (251) Google Scholar). However, although the protein was found to be located in the nucleus and the cytosol of COS-7 cells (18Jao D.L.-E. Chen K.Y. J. Cell. Biochem. 2002; 86: 590-600Crossref PubMed Scopus (39) Google Scholar), active shuttling between both compartments could not be confirmed. Also a direct interaction between Rev and eIF5A has not been shown (19Lipowski G. Bischoff F.R. Schwarzmaier P. Kraft R. Kostka. S. Hartmann E. Kutay U. Göhrlich D. EMBO J. 2000; 16: 4362-4371Crossref Scopus (159) Google Scholar). In addition yeast strains expressing temperature-sensitive mutants of Crm1p did not show a mislocalization of eIF5A to the nucleus (20Valentini S.R. Casolari J.M. Oliveira C.C. Silver P.A. McBride A.E. Genetics. 2002; 160: 393-405Crossref PubMed Google Scholar). Therefore the nuclear export hypothesis has been questioned.The implication of eIF5A in mRNA degradation initially came from studies of a temperature-sensitive HYP2 mutant (ts1159) that showed an accumulation and a strongly prolonged half-life of unspliced CYH2 mRNA (9Zuk D. Jacobson A. EMBO J. 1998; 17: 2914-2925Crossref PubMed Scopus (162) Google Scholar). The genetic background of this strain, however, reveals a functional HYP1 introducing the possibility that it might affect the observed phenotypic effects.Two studies revealed a connection between eIF5A and senescence processes showing a transcriptional up-regulation of deoxyhypusine synthase and eIF5A in aging tomato plants (21Wang T.-W. Lu L. Wang D. Thompson J.E. J. Biol. Chem. 2001; 276: 17541-17549Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 22Wang T.W. Lu L.G. Zhang C. Taylor C. Thompson J.E. Plant Mol. Biol. 2003; 52: 1223-1235Crossref PubMed Scopus (56) Google Scholar). However, in mammalian IMR-90 cells senescence led to a distinct attenuation of the hypusine formation (23Chen Z.P. Chen K.Y. J. Cell Physiol. 1997; 170: 248-254Crossref PubMed Scopus (14) Google Scholar). So far the cellular mechanism by which eIF5A is involved in cell aging is not known.Using yeast as a model system in which both native HYP genes were disrupted we describe the effects of expressing the point mutation V81G in the human hypusine-containing protein eIF5A1. We observed strong temperature sensitivities in these yeast strains. Expression of the mutant protein resulted in the accumulation of nonsense-containing RNAs, which are known to be specifically degraded by the polyadenylation-independent 5′-3′ mRNA decay pathway (nonsense-mediated decay (NMD) 2The abbreviations used are: NMD, nonsense-mediated decay; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDOA, high density oligonucleotide array; LTR, long terminal repeat; MTT, 3-(4,5-dimethylthizole-2yl)-2,5-diphenyltetrazolium bromide; ORF, open reading frame; ts, temperature-sensitive; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; Ty, transposon yeast; fmk, fluoromethyl ketone; PD, population doubling.2The abbreviations used are: NMD, nonsense-mediated decay; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDOA, high density oligonucleotide array; LTR, long terminal repeat; MTT, 3-(4,5-dimethylthizole-2yl)-2,5-diphenyltetrazolium bromide; ORF, open reading frame; ts, temperature-sensitive; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; Ty, transposon yeast; fmk, fluoromethyl ketone; PD, population doubling.). Additionally elongated half-lives of selected NMD transcripts and shortened telomeres were observed. These results suggest a functional connection of eIF5A with the NMD machinery that is coupled to translation initiation and reinforce the notion that the protein might influence essential cellular functions via its role in RNA processing.EXPERIMENTAL PROCEDURESYeast Strains, Plasmids, and Growth Conditions—Yeast cells were grown either on semisynthetic medium with 2% galactose or 2% glucose or on yeast-peptone-dextrose medium and yeast-peptone-galactose medium (24Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). The plasmid shuffle with and without 5-fluoroorotic acid as selective agent was performed as described previously (25Boeke J.D. Trueheart J. Natsoulis G. Fink G.R. Methods Enzymol. 1987; 154: 164-175Crossref PubMed Scopus (1067) Google Scholar). S. cerevisiae strains used in this study are listed in Table 1. The disruption of the HYP1 gene was performed with the haploid strain WDH#6-9[YEpHYP2] (3Wöhl T. Klier H. Ammer H. Lottspeich F. Magdolen V. Mol. Gen. Genet. 1993; 241: 305-311Crossref PubMed Scopus (61) Google Scholar) bearing a disruption of HYP2 by a 2.22-kb LEU2 fragment. The HYP1 gene (cloned as a 1.94-kb EcoRI-BamHI fragment in a plasmid derived from pBluescript; Stratagene) was disrupted by insertion of a 1.6-kb SmaI-AatII fragment carrying the TRP1 marker gene into the single SalI site of the HYP1 coding region. From the resulting vector a linear 3.6-kb EcoRI-BamHI fragment was used for homologous recombination according to Rothstein (26Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2015) Google Scholar). Correct integration was verified by Southern analysis. Isolation of high molecular weight genomic yeast DNA, cloning, and in vitro mutagenesis of DNA were performed as described previously (24Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar, 31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). PCR amplification was used to clone the coding regions of different wild-type and point mutated HYP genes according to the properties of multiple cloning sides in the target vectors. The plasmids used and produced for this study are listed in Table 2. The constructs for HYP expression either harbored the coding sequence of the HYP2 gene (S. cerevisiae) or the cDNA of the human HYP1 gene EIF5A1. Transformation of yeast strains was performed using the lithium ion method (32Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1766) Google Scholar).TABLE 1Yeast strains used in this studyStrainGenotypeSource or Ref.W303-1MATaMATa, leu2-3, 112, ura3-1, 112, his3-11, 15, trp1-l, 15, ade2-l, can1-10026WDH#6-9[YEpHYP2]MATa, hyp2::LEU2, HYP2, YEp (URA3) else isogenic to W303-1MATa4W303Δh1h2MATa, hpy1::TRPI, hyp2::LEU2, HYP2, YEp (URA3) else isogenic to W303-1MATaThis studyWDHyp2-GalMATa, hyp1::TRPI, hyp2::LEU2, HYP2, pRSThis studyWDH(hum)GalMATa, hyp1::TRPI, hyp2::LEU2, EIF5A1, pRSThis studyWDHG81GalMATa, hyp1::TRPI, hyp2::LEU2, EIF5A1-V81G, pRSThis studyPLY118MATa upf1-Δ1::URA3 ura3-52 trp1-7 leu2-3, 112 2 his4-3827 Open table in a new tab TABLE 2Plasmids used in this studyPlasmidDescriptionRef.YEp3522μ, LacZ, amp, MCS, URA328YEpHYP2YEp352 containing yeast wild-type HYP2 including its genomic promoter (cloned as PstI fragment)This studypRS315pBluescript, LEU2, CEN6, ARSH4, AmpR, LacZ29pRSG313pRS313 containing the GAL1 promotor fragment cloned EcoRI BamHI3pRSG316pRS316 containing the GAL1 promotor fragment cloned NotI SalIThis studypRSG313-HYP2pRSG313 expressing the yeast wild-type Hyp2p3pRSG313-EIF5A1pRSG313 expressing human wild-type EIF5A1This studypRSG313-EIF5A1G81pRSG313 containing point mutated (V81G) human EIF5A1 fragment cloned BamHI/XbaIThis studypAA79LEU2, CEN6, ARSH4, UPF1 (genomic promotor)27pCM189URA3, ampr, lacZ, CEN6, tTA (tetR promotor moiety)30pCM189-EIF5A1pCM189 containing human EIF5A1 fragment cloned BamHI/PstIThis studypCM189-EIF5A1G81pCM189 containing point mutated (V81G) human EIF5A1 fragment cloned BamHI/PstIThis study Open table in a new tab Anti-eIF5A Western Blotting—To determine eIF5A levels in V81G mutant strains, cells were grown to an A600 of 1 at 25°C or shifted to 37 °C and incubated at this temperature for a further 6 h. SDS-PAGE and immunoblotting were performed as described previously (33Knop M. Siegers K. Pereira G. Zachariae W. Winsor B. Nasmyth K. Schiebel E. Yeast. 1999; 15: 963-972Crossref PubMed Scopus (802) Google Scholar) using a 1:10,000 dilution of polyclonal antiserum against human EIF5A1 and enhanced chemiluminescence detection (GE Healthcare).MTT Cell Viability Test—An MTT viability assay of mutant and wild-type yeast cells was performed as described previously (34Hodgson V. Walker G.M. Button D. FEMS Microbiol. Lett. 1994; 120: 201-206Crossref PubMed Scopus (34) Google Scholar).Terminal Deoxynucleotidyltransferase-mediated dUTP Nick End Labeling (TUNEL)—The TUNEL assay and appropriate cell preparations were performed as described previously (35Madeo F. Fröhlich E. Froehlich K.-U. J. Cell Biol. 1997; 139: 729-734Crossref PubMed Scopus (676) Google Scholar) using the In Situ Cell Death Detection kit (Roche Applied Science). Coverslips were mounted with a drop of 5% n-propyl gallate (Sigma) in glycerol (100%). Slides were analyzed by fluorescence microscopy.Measurement of Caspase Activation—In vivo staining of caspase activity by flow cytometric analysis was performed as described previously (36Madeo F. Herker E. Maldener C. Wissing S. Laechelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Froehlich K.-U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar) using FITC-VAD-fmk (CaspACE, Promega) and a FACSCalibur system (BD Biosciences). Wild-type cells treated with 3 mm H2O2 served as a positive control.Generation of Gene Expression Datasets by Microarray Analysis—For high density oligonucleotide array (HDOA) analysis total RNA was isolated using the hot phenol method (37Sprague G.F. Jensen Jr., R. Herskowitz I. Cell. 1983; 32: 409-415Abstract Full Text PDF PubMed Scopus (55) Google Scholar) from cultures grown at 25 °C reaching early logarithmic phase. For each experiment RNA was independently isolated from four isogenic yeast strains of the wild type and the mutant (strains WDH(hum)Gal and WDHG81Gal), and in vitro transcriptions and hybridizations were performed. Thus, a total of eight chips were included in the analysis (4 replicates × 2 datasets for the wild type and the mutant V81G). RNA was further purified with RNeasy columns (Qiagen). In brief 30 μg of total RNA were subjected to a cDNA synthesis reaction to make the first strand using an oligo(dT) primer with a T7 promoter sequence added to the 5′-end. After synthesis of the second strand, double-stranded cDNA was purified by phenol/chloroform extraction, precipitated, and resuspended in nuclease-free water. Biotin-labeled cRNA was made by in vitro transcription using the High Yield Transcription kit (ENZO Diagnostics). The resulting cRNA was fragmented at 94 °C for 35 min in buffer A (40 mm Tris acetate, 100 mm KOAc, and 30 mm Mg(OAc)2). Affymetrix yeast S98 GeneChip ® arrays were hybridized, washed, stained, and scanned according to the manufacturer's specifications. Scanned raw data images were processed with Affymetrix GeneChip Version 3.2 software.Normalization and Statistical Analysis of Hybridization Data—Data from the arrays was normalized, and expression values based on an additive model were calculated according to the method of Irizarry et al. (38Irizarry R.A. Bolstad B.M. Collin F. Cope L.M. Hobbs B. Speed T.P. Nucleic Acids Res. 2003; 31: 1-8Crossref PubMed Scopus (3994) Google Scholar). Differentially expressed genes were identified by the permutation-based method of Tusher et al. (39Tusher V.G. Tibshirani R. Gilbert C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5115-5121Crossref Scopus (9704) Google Scholar). Briefly to control for multiple testing, a false discovery rate (40Benjamini Y. Hochberg Y. R. Stat. Soc. Ser. B. 1995; 57: 289-300Google Scholar) was calculated as the percentage of genes falsely detected as differentially expressed among all genes detected as differentially expressed. The q value is the lowest false discovery rate at which the gene is called significant. Significant genes were identified if they exhibited the lowest q value computed by SAM (“Significance Analysis of Microarrays”) software. Detailed information regarding the analysis performed on the microarray data can be found in the supplemental data section. Primary array datasets were published in the gene expression and hybridization array data repository of the National Center for Biotechnology Information (Gene Expression Omnibus (GEO), www.ncbi.nlm.nih.gov/geo/, accession number GSE5290). The transcriptomic profiles of V81G with NMD-deficient yeast were compared with the original .cel files published by He et al. (41He F. Spatrick P. Li X. Casillo R. Dong S. Jacobson A. Mol. Cell. 2003; 12: 1439-1452Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) and are available at www.ebi.ac.uk/miamexpress/under accession numbers E-MEXP-26 and E-MEXP-27. These datasets were analyzed in exactly the same way as the microarray data produced for this study.Northern Blotting and Determination of mRNA Half-lives—Yeast strains were cultured until they entered early log phase at either 25 or 37 °C. Total RNA was isolated as before (37Sprague G.F. Jensen Jr., R. Herskowitz I. Cell. 1983; 32: 409-415Abstract Full Text PDF PubMed Scopus (55) Google Scholar). Northern blotting was carried out as described previously (42Hatfield L. Beelman C.A. Stevens A. Parker R. Mol. Cell. Biol. 1996; 16: 5830-5838Crossref PubMed Scopus (145) Google Scholar). RNA was detected by hybridization to digoxigenin-labeled DNA probes of about 350 bp in length that were prepared by PCR using yeast genomic template DNA. After hybridization and stringent washing signal detection was performed using anti-digoxigenin antiserum (Roche Applied Science). Quantification of RNA bands was performed using densitometry. The stability of selected mRNAs was determined according to Parker et al. (43Parker R. Herrick D. Peltz S.W. Jacobson A. Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. 194. Academic Press, San Diego, CA1991: 415-423Google Scholar). Briefly 200 ml of mutant and wild-type yeast strains were grown to A600 = 0.7 and incubated for 1 h at either 25 or 37 °C. Next transcription was inhibited by the addition of thiolutin (20 μg/ml), and subsequent samples were taken at 0-, 4-, 8-, 16-, 32-, and 64-min intervals and frozen quickly in a dry ice-ethanol bath. Subsequently total RNA was isolated, and 10 μg of RNA/lane was analyzed by Northern blotting as described above.Quantitative Real Time (Reverse Transcriptional) PCR—Genespecific primers were designed with the PrimerExpress software (Applied Biosystems). The antisense primers were used for reverse transcription with avian myeloblastosis virus reverse transcriptase (Promega) according to the manufacturer's instructions and using 2 μg of total RNA. Real time PCR was performed using SYBR ® Green PCR Master Mix and a Gene-Amp5700 sequence detection system (Applied Biosystems) according to the protocols of the manufacturer. Relative expression levels were calculated, and PCR efficiencies were determined as described previously by Pfaffl (44Pfaffl M.W. Nucleic Acids Res. 2001; 29: 2002-2007Crossref Scopus (24904) Google Scholar) and Ramakers et al. (45Ramakers C. Ruijter J.M. Deprez R.H. Moorman A.F. Neurosci. Lett. 2003; 339: 62-66Crossref PubMed Scopus (2857) Google Scholar), respectively, using ACT1 expression for normalization. For each RNA preparation tested a minimum of three independent real time (reverse transcriptional) PCR experiments were conducted.Determination of Telomere Length by Southern Analysis—Telomere lengths were determined as described previously (46Lew J.E. Enomoto S. Berman J. Mol. Cell. Biol. 1998; 18: 6121-6130Crossref PubMed Scopus (65) Google Scholar). Briefly a probe containing a telomere repeat sequence was prepared by 5′-end labeling of the oligonucleotide Tel-Rep4 (CACCACACCCACACCCACACCACACCCACACACCCACAC) with digoxigenin. The sequence was identical to 2.5 repeats of the telomere template sequence from the chromosome 8 analogue to TLC1. Total genomic DNA was digested with PstI, and Southern blotting was performed as before. Signal development was achieved by using the DIG (digoxigenin) Luminescent Detection kit (Roche Applied Science) according to the manufacturer's instructions.RESULTSIsolation of the Temperature-sensitive Point Mutant V81G—To study the relationship between structure and function in the human EIF5A1 hypusine-containing protein point mutations were made throughout the entire length by exchanging phylogenetically conserved residues with related amino acids. A yeast strain was constructed (W303Δh1h2) in which both the genes HYP2 and HYP1 (ANB1) encoding for the hypusine-containing protein were disrupted (3Wöhl T. Klier H. Ammer H. Lottspeich F. Magdolen V. Mol. Gen. Genet. 1993; 241: 305-311Crossref PubMed Scopus (61) Google Scholar). The strain harbored a 2μ-URA3-plasmid-borne wild-type copy of the genomic HYP2, thus complementing the otherwise lethal phenotype. The constructs containing the point mutations to be tested were cloned into pRS313 containing the GAL promotor and transformed into the W303Δh1h2 cells. The exchange of the wild type for a mutated allele was performed by selective plasmid shuffling using fluoroorotic acid. Because hypusine is essential, cell death occurred if the mutant allele cloned in pRS313 was incapable of complementing the gene function of the wild type. This counterselection was used to screen all mutations in the human HYP homologue EIF5A1.The expression of certain mutated alleles resulted in temperature-sensitive yeast strains. The strongest temperature sensitivity was observed by the substitution of the valine residue at position 81 (Fig. 1). Proliferation of cells at the restrictive temperature was stopped after only one round of doubling when valine at position 81 was mutated to a glycine (V81G). In comparison with wild type the growth of the mutated strain at the permissive temperature was decelerated. Retransformation of the mutant strains with a single copy plasmid carrying wild-type human EIF5A1 completely restored the temperature sensitivity (Fig. 1C) indicating that it was not a recessive mutation. Several independent transformations and subsequent 5-fluoroorotic acid selections reproducibly showed the pronounced phenotype of the mutant V81G. These mutated strains were chosen for the subsequent phenotypic characterization experiments.eIF5A Protein Levels and Viability at 37 °C—Complete cell lysates were prepared from wild-type or mutant cells after incubation at permissive or restrictive temperatures (Fig. 2). The eIF5A protein levels in each case were determined by Western blotting. At 25 °C the mutated protein was expressed to nearly the same amount as the wild type. Within 60 min after heat shock induction the level of the eIF5A (V81G) protein declined more than 5-fold. A further 5-h incubation at 37 °C did not result in further changes in the protein level.FIGURE 2Protein expression of wild-type and V81G mutated human eIF5A in yeast. Cells were incubated at 25 or at 37 °C for the indicated times and were subsequently harvested to prepare complete cell lysates. Equal amounts of total protein from mutant and wild-type strains were separated by SDS-PAGE. Western blotting was performed with a rabbit polyclonal antiserum against the human EIF5A1 protein. Equal loading of all lanes was confirmed by densitometric scanning of the Coomassie-stained gel prior to blotting as well as quantification of the actin 1 protein on the Western blots that was equally expressed in wild-type and mutant strains. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The viability of V81G-expressing cells at both temperatures was checked with an MTT assay (Fig. 3). Independently isolated strains of the mutant V81G and the corresponding wild-type strains that were in early logarithmic growth phase were tested at both the restrictive and the permissive temperatures. Fig. 3 shows that the formation of blue formazan from the activity of dehydrogenases was similar for the wild-type cultures grown at either the restrictive or the permissive temperature. Slight differences were observed between wild-type HYP2 expression in yeast and heterologous (human) EIF5A1 expression. However, even at the permissive temperature, V81G, although capable of growth, displayed a 50% reduction in viability when compared with the wild type. After a 6-h incubation at 37 °C the viability was further decreased.FIGURE 3MTT viability assay of mutant and wild-type cultures incubated with MTT for 2 h at the permissive and restrictive temperatures. Two independently isolated clones of each strain (WDH(hum) TO, WDHyp2TO, and WDHG81TO) were tested. Cell density of all cultures was normalized to 5×108 cells/ml, and isopropanolic cell extracts were measured at 570 nm. This OD value was proportional to the amount of blue formazan that was formed during reduction time. Error bars indicate the standard deviation of triplicate measurements of each of the two isogenic strains. Student's t tests for independent samples performed on every pair of strains in all cases generated p values below 0.05, indicating significance of the results. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the mutant's loss of cell viability was due to apoptotic death a TUNEL assay was performed, and the results are shown in Fig. 4B. No fluorescence was detected in the control cells expressing wild-type eIF5A regardless of the growth conditions. However, addition of 3 mm H2O2 and incubation for 6 h at 37°C rendered the cells positive for DNA breaks, which served as a positive control. At the permissive temperature (25 °C) the V81G mutant did not show any fluorescent nuclei. In contrast, after 4 h at the restrictive temperature (37 °C) clear fluorescence in the nuclei was visible and increased with longer incubation times.FIGURE 4Test for apoptotic death in growth-arrested cells of V81G. A, i and" @default.
• W2051276497 created "2016-06-24" @default.
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• W2051276497 creator A5041718533 @default.
• W2051276497 creator A5057511722 @default.
• W2051276497 creator A5060671769 @default.
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• W2051276497 date "2006-11-01" @default.
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• W2051276497 title "Temperature-sensitive eIF5A Mutant Accumulates Transcripts Targeted to the Nonsense-mediated Decay Pathway" @default.
• W2051276497 cites W1484606707 @default.
• W2051276497 cites W1531987646 @default.
• W2051276497 cites W1537782690 @default.
• W2051276497 cites W1544068656 @default.
• W2051276497 cites W1559808711 @default.
• W2051276497 cites W1821435151 @default.
• W2051276497 cites W1849896953 @default.
• W2051276497 cites W1948628402 @default.
• W2051276497 cites W1964297184 @default.
• W2051276497 cites W1968185558 @default.
• W2051276497 cites W1978398494 @default.
• W2051276497 cites W1981482024 @default.
• W2051276497 cites W1985001103 @default.
• W2051276497 cites W1992478143 @default.
• W2051276497 cites W2014489014 @default.
• W2051276497 cites W2015982086 @default.
• W2051276497 cites W2016728080 @default.
• W2051276497 cites W2017641016 @default.
• W2051276497 cites W2025426978 @default.
• W2051276497 cites W2026774621 @default.
• W2051276497 cites W2030302003 @default.
• W2051276497 cites W2041097992 @default.
• W2051276497 cites W2049093430 @default.
• W2051276497 cites W2055204177 @default.
• W2051276497 cites W2058346978 @default.
• W2051276497 cites W2060337869 @default.
• W2051276497 cites W2061091174 @default.
• W2051276497 cites W2065626654 @default.
• W2051276497 cites W2067662127 @default.
• W2051276497 cites W2069271664 @default.
• W2051276497 cites W2069501735 @default.
• W2051276497 cites W2071417493 @default.
• W2051276497 cites W2072302116 @default.
• W2051276497 cites W2079242841 @default.
• W2051276497 cites W2083058646 @default.
• W2051276497 cites W2087576311 @default.
• W2051276497 cites W2088317037 @default.
• W2051276497 cites W2096650626 @default.
• W2051276497 cites W2100291823 @default.
• W2051276497 cites W2108244474 @default.
• W2051276497 cites W2109972881 @default.
• W2051276497 cites W2110425626 @default.
• W2051276497 cites W2115073295 @default.
• W2051276497 cites W2115162201 @default.
• W2051276497 cites W2118450214 @default.
• W2051276497 cites W2118718951 @default.
• W2051276497 cites W2121770428 @default.
• W2051276497 cites W2125699004 @default.
• W2051276497 cites W2136495792 @default.
• W2051276497 cites W2140951250 @default.
• W2051276497 cites W2149548143 @default.
• W2051276497 cites W2152231771 @default.
• W2051276497 cites W2160184214 @default.
• W2051276497 cites W2169117131 @default.
• W2051276497 cites W2169333620 @default.
• W2051276497 cites W2185323554 @default.
• W2051276497 cites W4236956086 @default.
• W2051276497 cites W4294107304 @default.
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