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• W2039290476 abstract "Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, α-sarcin/ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeastSaccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to theN-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs. Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, α-sarcin/ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeastSaccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to theN-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs. pokeweed antiviral protein ribosome-inactivating protein sarcin/ricin synthetic dropout medium nucleotide(s) small nucleolar RNA glucose-6-phosphate dehydrogenase Pokeweed antiviral protein (PAP),1 a single chain ribosome-inactivating protein (RIP) isolated from the leaves of pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, α-sarcin/ricin (S/R) loop in the large rRNA (1Endo Y. K. T. J. Biol. Chem. 1988; 263: 8735-8739Abstract Full Text PDF PubMed Google Scholar, 2Hartley M.R. Legname G. Osborn R. Chen Z. Lord M.J. FEBS Lett. 1991; 290: 65-68Crossref PubMed Scopus (125) Google Scholar, 3Hudak K.A. Wang P.G. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar). The enzymatic removal of specific purines from the S/R loop has been reported to interfere with the binding of eEF-2 (elongation factor 2) and inhibit protein synthesis at the translocation step (4Montanaro L. Sperti S. Mattioli A. Testoni G. Stirpe F. Biochem. J. 1975; 146: 127-131Crossref PubMed Scopus (122) Google Scholar, 5Osborn R.W. Hartley M.R. Eur. J. Biochem. 1990; 193: 401-417Crossref PubMed Scopus (51) Google Scholar). RIPs are protein toxins produced by organisms ranging from bacteria to plants. Because of their selective toxicity, they have been used as biological weapons, to protect plants against pathogens, and as therapies against cancer. Their biological function in the organisms that produce them is unknown. PAP is thought to be a defense protein because it is a potent inhibitor of animal and plant viral pathogens, including human immunodeficiency virus, poliovirus, herpes simplex virus, influenza, potato virus X, and brome mosaic virus (6Aron G. Irvin J.D. Antimicrob. Agents Chemother. 1980; 17: 1032-1033Crossref PubMed Scopus (72) Google Scholar, 7Lodge J.K. Kaniewski W.K. Tumer N.E. Proc. Natl. Acad. Sci. 1993; 90: 7089-7093Crossref PubMed Scopus (231) Google Scholar, 8Tomlinson J.A. Walker V.M. Flewett T.H. Barclay G.R. J. Gen. Virol. 1974; 22: 225-232Crossref PubMed Scopus (88) Google Scholar, 9Ussery M.A. Irvin J.D. Hardesty B. Ann. N. Y. Acad. Sci. 1977; 284: 431-440Crossref PubMed Scopus (68) Google Scholar, 10Zarling J.M. Moran P.A. Haffar O. Sias J. Richman D.D. Spina C.A. Myers D.E. Kuebelbeck V. Ledbetter J.A. Uckun F.M. Nature. 1990; 347: 92-95Crossref PubMed Scopus (195) Google Scholar). Because of its cytotoxicity to dividing cells, PAP is currently under clinical trials as a potent anticancer agent (11Waurzyniak B. Schneider E.A. Tumer N. Yanishevski Y. Gunther R. Chelstrom L.M. Wendorf H. Myers D.E. Irvin J.D. Messinger Y., Ek, O. Zeren T. Langlie M.C. Evans W.E. Uckun F.M. Clin. Cancer Res. 1997; 3: 881-890PubMed Google Scholar). The mechanism by which PAP inhibits cell growth or viral infection is not well understood. Translation inhibition by PAP and the resulting host cell death have been hypothesized to be responsible for the antiviral activity of PAP. However, a nontoxic C-terminal deletion mutant of PAP inhibited viral infection without depurinating host ribosomes, indicating that antiviral activity could be separated from rRNA depurination (12Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar). Furthermore, expression of nontoxic forms of PAP in transgenic plants induced a stress-associated signal transduction pathway and provided resistance to viral and fungal infection (13Zoubenko O. Uckun F. Hur Y. Chet I. Tumer N. Nat. Biotechnol. 1997; 15: 992-996Crossref PubMed Scopus (75) Google Scholar, 14Zoubenko O. Hudak K. Tumer N.E. Plant Mol. Biol. 2000; 44: 219-229Crossref PubMed Scopus (56) Google Scholar). PAP is very active against both animal and plant ribosomes. It accesses the S/R loop by binding to ribosomal protein L3 (RPL3), a highly conserved protein associated with the peptidyltransferase center of ribosomes (15Hudak K.A. Dinman J.D. Tumer N.E. J. Biol. Chem. 1999; 274: 3859-3864Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Our recent results indicate that in a cell-free system, PAP can inhibit translation of mRNAs and viral RNAs that are capped by recognizing the cap structure and depurinating the capped RNAs (3Hudak K.A. Wang P.G. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar). Incubation of brome mosaic virus RNAs or capped luciferase RNA with PAP resulted in depurination of either RNA. In contrast, uncapped luciferase RNA was not depurinated after incubation with identical concentrations of PAP (3Hudak K.A. Wang P.G. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar). Analysis of the interaction between the cap structure and PAP indicated that PAP binds to the m7GpppG cap structure but does not remove the cap (16Hudak K.A. Bauman J.D. Tumer N.E. RNA. 2002; 8: 1148-1159Crossref PubMed Scopus (56) Google Scholar). PAP depurinates the RNA downstream of the cap at specific sites (16Hudak K.A. Bauman J.D. Tumer N.E. RNA. 2002; 8: 1148-1159Crossref PubMed Scopus (56) Google Scholar). The relative affinity of PAP for capped RNA is similar to its affinity for the S/R loop of rRNA, suggesting that rRNA might not be the only target of PAP (16Hudak K.A. Bauman J.D. Tumer N.E. RNA. 2002; 8: 1148-1159Crossref PubMed Scopus (56) Google Scholar). In our previous studies with transgenic plants, we observed that mRNA corresponding to wild type PAP was not detected by Northern blot analysis (13Zoubenko O. Uckun F. Hur Y. Chet I. Tumer N. Nat. Biotechnol. 1997; 15: 992-996Crossref PubMed Scopus (75) Google Scholar) even though PAP protein was detected (12Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar). In contrast, PAP mRNA was detected in transgenic lines expressing the inactive form, PAPx (or PAPE176V), which contains the point mutation, E176V, at its active site (13Zoubenko O. Uckun F. Hur Y. Chet I. Tumer N. Nat. Biotechnol. 1997; 15: 992-996Crossref PubMed Scopus (75) Google Scholar). In the present study, we examined the effect of PAP on the stability of its own mRNA and cellular mRNAs in the yeast, Saccharomyces cerevisiaewhere PAP expression can be tightly controlled. We have previously shown that PAP expression in yeast duplicates the effects of PAP in plant cells (12Tumer N.E. Hwang D.J. Bonness M. Proc. Natl. Acad. Sci. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar, 17Hur Y. Hwang D.J. Zoubenko O. Coetzer C. Uckun F.M. Tumer N.E. Proc. Natl. Acad. Sci. 1995; 92: 8448-8452Crossref PubMed Scopus (56) Google Scholar). Our results demonstrate that PAP overexpression leads to a pronounced decrease of its mRNA levels. By comparison, PAP mRNA abundance is not affected in cells expressing an active site mutant, indicating that an intact active site is necessary for down-regulation of PAP expression. By examining the relationship between rRNA depurination and mRNA decay, we establish that PAP regulates the stability of its mRNA by a mechanism that can be separated from rRNA depurination and inhibition of translation. To our knowledge, this is the first report demonstrating that a ribosome inactivating protein targets its own mRNA, in addition to rRNAin vivo. We discuss the significance of these observations for the biological function of RIPs. S. cerevisiae strain W303 (MATa ade2-1 trp1-1 ura3-1 leu2-3, 112 his3-11, 15 can1-100) was used for all of the assays. Yeast cells were grown at 30 °C in YPD rich medium (1% yeast extract, 2% peptone, and 2% glucose) or synthetic dropout (SD) medium (0.67% Bacto-yeast nitrogen base) supplemented with the appropriate amino acids (17Hur Y. Hwang D.J. Zoubenko O. Coetzer C. Uckun F.M. Tumer N.E. Proc. Natl. Acad. Sci. 1995; 92: 8448-8452Crossref PubMed Scopus (56) Google Scholar, 18Tumer N.E. Parikh B.A., Li, P. Dinman J.D. J. Virol. 1998; 72: 1036-1042Crossref PubMed Google Scholar). To induce expression of PAP and PAP variants, transformed yeast were grown initially at 30 °C in 150 ml of selective medium containing 2% raffinose to a starting A 600 of 0.6. At zero time the medium was replaced with 300 ml of selective medium (SD−Leu) containing 2% galactose to a startingA 600 of 0.3. Subsequently, 5 ml of culture was taken for protein isolation, 25 ml for RNA isolation, and 1 ml for a growth reading (A 600) at various times post-induction. The medium was diluted periodically to maintain the cells in the logarithmic phase (A 600 between 0.3 and 0.6). Doubling times were calculated based on exponential growth between 4 and 10 h post-induction. For measuring the effect of translation and transcription on rRNA depurination, yeast were grown at 30 °C in 150 ml of SD−Leu, 2% raffinose to an initialA 600 of 0.6. Cells were then pelleted, washed once, and resuspended in 13 ml of SD−Leu, 2% galactose to induce PAP expression. At 1 h post-induction, 2% glucose with or without cycloheximide to a final concentration of 100 μg/ml was added to the medium, and 2 ml of pellets were collected for RNA and protein isolation at the indicated times. PAP expression plasmids used in this study were described previously (3Hudak K.A. Wang P.G. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar, 17Hur Y. Hwang D.J. Zoubenko O. Coetzer C. Uckun F.M. Tumer N.E. Proc. Natl. Acad. Sci. 1995; 92: 8448-8452Crossref PubMed Scopus (56) Google Scholar). Expression of PAP in NT188 and the nontoxic PAP variants PAPE176V and PAPL71R in NT224 and NT538, respectively, is under control of the galactose-inducible GAL1 promoter in the YEp351-based high-copy plasmid. The NT616 contained the firefly luciferase cDNA from pLUC0 (18Tumer N.E. Parikh B.A., Li, P. Dinman J.D. J. Virol. 1998; 72: 1036-1042Crossref PubMed Google Scholar) downstream of the GAL1 promoter in YEp351. Total RNA from cells expressing PAP, PAPE176V, and PAPL71R was analyzed with the RNase protection assay according to Tumer et al. (18Tumer N.E. Parikh B.A., Li, P. Dinman J.D. J. Virol. 1998; 72: 1036-1042Crossref PubMed Google Scholar). RNase protection assay was performed using several gene-specific antisense RNA probes to measure the steady state levels of mRNA. A 281-nt CYH2-specific probe was generated from an SP6 RNA polymerase run-off transcript of HincII-digested p3433 (18Tumer N.E. Parikh B.A., Li, P. Dinman J.D. J. Virol. 1998; 72: 1036-1042Crossref PubMed Google Scholar). A 90-nt U3-specific probe was generated from a T3 RNA polymerase run-off transcript of SspI-digested pJD161 (19Hagan K.W. Ruizechevarria M.J. Quan Y. Peltz S.W. Mol. Cell. Biol. 1995; 15: 809-823Crossref PubMed Google Scholar). The U3 small nucleolar RNA (snoRNA), constitutively expressed from an RNA polymerase III promoter, was used as loading control. A 252-nt RNA probe that hybridizes to the 3′ end of PAP mRNA was generated from a SP6 RNA polymerase transcript of XhoI-digested pMON8588. A 200-nt XRN1-specific probe was generated from a T3 RNA polymerase run-off transcript of DdeI-digested pNT404. The pNT404 has a 2-kb BglII/HincII fragment of theXRN1 gene from pXRN1 cloned into theBamHI/HincII sites of pBluescript KS+ (Stratagene). A 260-nt LEU2-specific probe was generated from a T3 RNA polymerase transcript of EcoRI-digested pNT403. pNT403 was constructed by inserting a 745-bp HincII/ClaI fragment of the LEU2 gene from YEp351 into pGEM3Zf(+) digested with the same restriction enzymes. A 250-nt RPL3-specific probe was generated from a T7 RNA polymerase transcript of XbaI-digested pRPL3, which carries the ribosomal protein gene RPL3. A 180-ntPGK1-specific probe was generated from a T7 RNA polymerase run-off transcript of SalI-digested pRS314-PGK1. Protected fragments were separated on a 7 m urea 5% acrylamide denaturing gel, visualized with radiographic film (Kodak), and quantified on a PhosphorImager (Amersham Biosciences). Protein from frozen yeast cells expressing PAP, PAPE176V, and PAPL71Rharvested during the time course of induction, was extracted as described by Hudak et al. (15Hudak K.A. Dinman J.D. Tumer N.E. J. Biol. Chem. 1999; 274: 3859-3864Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Total protein (7.5 μg) from each time point was separated on 15% SDS-PAGE, transferred to nitrocellulose, and probed with affinity-purified anti-PAP polyclonal antibody (1:5000). PAP was visualized by chemiluminescence using the Renaissance kit (PerkinElmer Life Sciences). The blots were then stripped for 30 min with 8 m guanidine hydrochloride and reprobed with antibody to glucose-6-phosphate dehydrogenase (G6PD) (1:5000) as an internal loading control. Ribosomal RNA depurination was assayed by primer extension analysis as described previously (20Hudak K.A. Hammell A.B. Yasenchak J. Tumer N.E. Dinman J.D. Virology. 2001; 279: 292-301Crossref PubMed Scopus (20) Google Scholar). Briefly, 2 μg of total yeast RNA from cells expressing PAP was hybridized with 106 cpm of reverse primer (5′-AGCGGATGGTGCTTCGCGGCAATG-3′). This depurination primer was end-labeled by T4 kinase (Invitrogen) in the presence of [γ-32P]ATP, and it hybridized 73 nt 3′ of the depurination site. The presence or absence of depurination was noted by synthesis of a 73-nt extension product that terminated at the depurination site. Superscript II-reverse transcriptase (Invitrogen) was used in the primer extension assay following the protocol described in Hudak et al. (3Hudak K.A. Wang P.G. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar). Extension products were separated on a 7 m urea, 5% polyacrylamide denaturing gel and visualized and quantified on a Phosphor-Imager (AmershamBiosciences). Further studies requiring more accurate quantification of depurination employed the use of a second primer serving as an internal control. For these analyses, either 1.25 μg of total yeast RNA isolated from yeast expressing PAP, PAPE176V, or PAPL71R, as described above, or 1.0 μg of rRNA isolated from ribosomes was hybridized to two different reverse primers. The second primer hybridized upstream of the depurination site close to the 5′ end of the 25 S rRNA. For in vitro depurination assays, yeast ribosomes were isolated as described previously (20Hudak K.A. Hammell A.B. Yasenchak J. Tumer N.E. Dinman J.D. Virology. 2001; 279: 292-301Crossref PubMed Scopus (20) Google Scholar). Ribosomes were then either incubated in buffer alone or depurinated with 250 ng of purified PAP to completion as described previously (20Hudak K.A. Hammell A.B. Yasenchak J. Tumer N.E. Dinman J.D. Virology. 2001; 279: 292-301Crossref PubMed Scopus (20) Google Scholar). To quantify the extent of depurination, the target RNA was hybridized initially in the presence of excess amounts (700 pmol) of the two [γ-32P]ATP end-labeled negative strand primers. The depurination primer described above annealed 73 nt 3′ of the depurination site (A3137) on the 25 S rRNA. The 25 S control primer (5′-TTCACTCGCCGTTACTAAGG-3′) annealed 100 nt 3′ of the 25 S rRNA 5′ end. To allow for accurate quantification, the labeled 25 S control primer was diluted 1:4 with unlabeled 25 S control primer. Superscript II-reverse transcriptase was used in the primer extension assay as above. Extension products for the control and depurination fragments (100 and 73 nt, respectively) were separated on a 7m urea, 5% polyacrylamide denaturing gel and visualized and quantified on a PhosphorImager. The amount of total yeast RNA and rRNA used was determined previously to be in the linear range of detection. Yeast cells were grown to an A 600 of 0.6 in SD−Leu, −Met, 2% raffinose. Cells were then resuspended at anA 600 of 0.3 in 2% galactose for 4–10 h to induce either wild type PAP or PAP variant expression. At time zero, [35S]methionine was added to cells growing on galactose. At the times indicated, 800 μl of yeast cells were removed for growth measurements, and additional aliquots of 800 μl were assayed for methionine incorporation in duplicate as described by Carr-Schmidet al. (21Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar) with minor modifications. Briefly, the yeast were added to 200 μl of 100% trichloroacetic acid, and the mixture was incubated for 10 min on ice followed by 20 min at 70 °C. The precipitate was then filtered through 24-mm glass microfiber filters (VWR), washed with ice-cold 5% trichloroacetic acid followed by ice cold 95% ethanol. Filters were dried for several hours, and incorporation was quantified in a scintillation counter. The cpm was normalized to the A 600 reading. Rates of translation were determined from these results and tabulated as CPM/A 600/min. To obtain translation inhibition in PAPE176V cells comparable to wild type PAP inhibition at 4 h post-induction, ∼5 μg/ml final concentration of anisomycin was added to the medium at 2 h post-induction. This amount was titrated previously to achieve 75% translation inhibition (data not shown). To determine whether PAP affects the stability of its own mRNA in vivo,cDNAs encoding the wild type PAP or nontoxic variants were placed under the regulation of the GAL1 promoter and expressed in the yeast S. cerevisiae. The variants used in this study are shown in Fig. 1. They include the nontoxic PAPE176V, which contains a point mutation (E176V) at its active site and produces an inactive protein, and PAPL71R, which contains a point mutation (L71R) at the putative RNA binding domain and has reduced toxicity compared with wild type PAP. We have previously shown that yeast cells transformed with plasmids carrying PAPE176V or the vector alone (YEp351) were able to grow on SD−Leu plates containing galactose (17Hur Y. Hwang D.J. Zoubenko O. Coetzer C. Uckun F.M. Tumer N.E. Proc. Natl. Acad. Sci. 1995; 92: 8448-8452Crossref PubMed Scopus (56) Google Scholar). However, yeast cells harboring the wild type PAP plasmid (NT188) failed to grow on plates containing galactose (17Hur Y. Hwang D.J. Zoubenko O. Coetzer C. Uckun F.M. Tumer N.E. Proc. Natl. Acad. Sci. 1995; 92: 8448-8452Crossref PubMed Scopus (56) Google Scholar). As shown in Fig.2, the growth of cells expressing the wild type PAP but not PAPE176V was inhibited in liquid SD−Leu medium containing galactose compared with cells harboring the same vector (YEp351) with luciferase cDNA (VC). The doubling time of cells expressing PAPE176V was similar to the vector control, 3.9 ± 0.1 h, whereas the doubling time of cells expressing wild type PAP was 11.9 ± 1.7 h. Growth of cells expressing PAPL71R was also inhibited in liquid medium containing galactose but not to the same extent as wild type PAP. This was evident from its doubling time of 8.5 ± 0.8 h and ability to grow on plates containing galactose (data not shown). The addition of anisomycin to cells expressing PAPE176Vresulted in complete inhibition of growth (doubling time: 24.9 ± 2.0 h). These results suggested that the inhibition of growth observed in cells expressing wild type PAP might be due to inhibition of translation.Figure 2Growth of yeast expressing PAP and the PAP variants. Expression of PAP, PAPL71R, PAPE176V, PAPE176V + anisomycin, and vector control (VC) was induced by growing cells in SD−Leu medium containing 2% galactose. An A 600 reading was taken for growth measurement at the indicated times after induction.View Large Image Figure ViewerDownload (PPT) To determine whether reduction of growth is correlated with inhibition of translation, we examined total translation in cells expressing PAP, PAPE176V, and PAPL71R compared with control cells harboring the same vector with luciferase cDNA. Total translation was examined by [35S]methionine incorporation at 4 and 10 h post-induction (21Carr-Schmid A. Valente L. Loik V.I. Williams T. Starita L.M. Kinzy T.G. Mol. Cell. Biol. 1999; 19: 5257-5266Crossref PubMed Google Scholar). As shown in Fig.3 A, cells grown in galactose for 4 h to induce expression of PAPE176V were not significantly inhibited in translation as compared with vector control cells. The rate of translation in cells expressing PAPE176V, judged from the slope of the curve in Fig.3 A, was 88.7 ± 2.9% of the rate of translation in vector control cells (Table I). The rate of translation in yeast expressing active PAP was 27.4 ± 3.0% of the vector control as determined by averaging the results of five independent experiments (Table I). These results indicate that total translation is significantly inhibited in cells expressing PAP but not PAPE176V.Table ITranslation rates of PAP and PAP variantsPAP or PAP variantTranslation RateaTranslation rates were determined at 4 h post-induction by measuring the slope of the methionine incorporation curve shown in Fig. 3 A and expressed as a percent of the translation rate for the vector control. The rates were confirmed by at least two independent experiments.% of controlPAP27.4 ± 3.0%PAPE176V88.7 ± 2.9%PAPL71R33.8 ± 0.7%PAPE176V + anisomycinbApproximately 5 μg/ml anisomycin was added to cells expressing PAPE176V at 2 h post-induction, and translation was analyzed at 4 h as described in footnote a.35.0 ± 5.0%a Translation rates were determined at 4 h post-induction by measuring the slope of the methionine incorporation curve shown in Fig. 3 A and expressed as a percent of the translation rate for the vector control. The rates were confirmed by at least two independent experiments.b Approximately 5 μg/ml anisomycin was added to cells expressing PAPE176V at 2 h post-induction, and translation was analyzed at 4 h as described in footnote a. Open table in a new tab The growth inhibition observed in the presence of anisomycin in Fig. 2suggested that inhibition of growth might be due to inhibition of translation. Under these conditions, we would expect cells expressing PAPL71R to be inhibited in translation. As shown in Fig.3 A, total translation was significantly inhibited in cells expressing PAPL71R at 4 h post-induction. The rate of translation in cells expressing PAPL71R was 33.8 ± 0.7% of the vector control (Table I). Analysis of total translation at 10 h post-induction indicated that translation remained inhibited in cells expressing wild type PAP and PAPL71R but not in PAPE176V (Fig. 3 B). These results provide evidence that the reduction in growth observed in cells expressing wild type PAP and PAPL71R correlates with the inhibition of translation observed in these cells. Because our previous results had indicated that PAP can inhibit translation by depurinating capped RNAs (3Hudak K.A. Wang P.G. Tumer N.E. RNA. 2000; 6: 369-380Crossref PubMed Scopus (92) Google Scholar), to determine whether translation inhibition correlated with the activity of PAP on mRNAs in vivo, we examined the abundance of PAP mRNA and cellular mRNAs in yeast expressing the wild type and mutant forms of PAP. Cells were harvested at various times after induction on galactose, and the level of PAP mRNA was measured by RNase protection assay (Fig. 4 A). A 252-nt 32P-labeled antisense RNA probe corresponding to the 3′ end of PAP mRNA was transcribed and hybridized with total RNA extracted from cells harboring PAP, PAPE176V, and PAPL71R plasmids. A 90-nt 32P-labeled antisense probe specific for U3 snoRNA, which is constitutively expressed from an RNA polymerase III promoter, was used as a loading control (19Hagan K.W. Ruizechevarria M.J. Quan Y. Peltz S.W. Mol. Cell. Biol. 1995; 15: 809-823Crossref PubMed Google Scholar). Samples were separated electrophoretically, and the intensities of the protected bands were quantified using a Phosphor- Imager. The ratios for signals of the PAP, PAPE176V, or PAPL71RmRNAs to the U3 snoRNA were used as relative measures of the steady state abundance of the PAP, PAPE176V, or PAPL71R mRNAs. The cells harboring the vector alone (YEp351) grown for 8 h on raffinose or galactose did not show any detectable background (data not shown). Similarly, RNase protection analysis using tRNA did not show any protected fragments corresponding to either PAP or U3 (Fig. 4 A). As expression of wild type PAP was induced, the level of PAP mRNA decreased dramatically relative to the U3 snoRNA (Fig. 4 A). By 10 h post-induction, PAP mRNA levels decreased to about 10% of the levels observed at 4 h post-induction (Fig. 4 B). In contrast, PAPE176V mRNA levels increased up to 10 h post-induction and reached steady state levels after 10 h. Although the overall increase in PAPE176V mRNA was highly reproducible, the extent of the increase was subject to some variation. The amount of mRNA present at 10 h in cells expressing PAPE176V was between 2.5 and 8 times greater than that at 4 h. The error bars in Fig. 4 Brepresent averaging of three independent quantifications, and the RNase protection analysis was repeated at least three times with similar results. These results indicated that PAP mRNA is destabilized in cells expressing wild type PAP. This regulation is impaired when the active site mutant, PAPE176V, is expressed in yeast, indicating that mRNA destabilization is due to theN-glycosidase activity of PAP. RNase protection analysis beyond 10 h post-induction indicated that PAP mRNA is not detectable after 12 h, whereas PAPE176V mRNA remains at steady state levels (data not shown). RNase protection analysis of yeast expressing PAPL71Rindicated that as observed with wild type PAP, mRNA levels increased up to 4 h post-induction. However, PAPL71RmRNA remained at elevated steady state levels and was not destabilized after 4 h of induction (Fig. 4). These results indicated that PAPL71R behaves similarly to wild type PAP during the early stages of induction. Although both growth and translation were inhibited in cells expressing PAPL71R, mRNA was not destabilized, indicating that the L71R mutation did not affect the ability of PAP to i" @default.
• W2039290476 created "2016-06-24" @default.
• W2039290476 creator A5021019342 @default.
• W2039290476 creator A5037865763 @default.
• W2039290476 creator A5073028262 @default.
• W2039290476 date "2002-11-01" @default.
• W2039290476 modified "2023-09-27" @default.
• W2039290476 title "Pokeweed Antiviral Protein Regulates the Stability of Its Own mRNA by a Mechanism That Requires Depurination but Can Be Separated from Depurination of the α-Sarcin/Ricin Loop of rRNA" @default.
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