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J. Biol. Chem., Vol. 277, Issue 44, 41428-41437, November 1, 2002

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Pokeweed Antiviral Protein Regulates the Stability of Its Own mRNA by a Mechanism That Requires Depurination but Can Be Separated from Depurination of the -Sarcin/Ricin Loop of rRNA^*

Bijal A. Parikh^§, Chris Coetzer, and Nilgun E. Tumer^§¶

From the  Biotechnology Center for Agriculture and the Environment and the Department of Plant Biology and Pathology Cook College, Rutgers University and the ^§ Graduate Program in Microbiology and Molecular Genetics, Rutgers University, New Brunswick, New Jersey 08901-8520

Received for publication, June 3, 2002, and in revised form, August 2, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, -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 yeast Saccharomyces 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 the N-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, -sarcin/ricin (S/R) loop in the large rRNA (1-3). 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 (4, 5). 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 (6-10). Because of its cytotoxicity to dividing cells, PAP is currently under clinical trials as a potent anticancer agent (11). 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 (12). 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 (13, 14). 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 (15). 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 (3). 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 (3). 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 (16). PAP depurinates the RNA downstream of the cap at specific sites (16). 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 (16).

In our previous studies with transgenic plants, we observed that mRNA corresponding to wild type PAP was not detected by Northern blot analysis (13) even though PAP protein was detected (12). In contrast, PAP mRNA was detected in transgenic lines expressing the inactive form, PAPx (or PAP_E176V), which contains the point mutation, E176V, at its active site (13). In the present study, we examined the effect of PAP on the stability of its own mRNA and cellular mRNAs in the yeast, Saccharomyces cerevisiae where PAP expression can be tightly controlled. We have previously shown that PAP expression in yeast duplicates the effects of PAP in plant cells (12, 17). 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 rRNA in vivo. We discuss the significance of these observations for the biological function of RIPs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Media and Growth Conditions-- 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 (17, 18). 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 A₆₀₀ of 0.6. At zero time the medium was replaced with 300 ml of selective medium (SDLeu) containing 2% galactose to a starting A₆₀₀ 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 (A₆₀₀) at various times post-induction. The medium was diluted periodically to maintain the cells in the logarithmic phase (A₆₀₀ 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 SDLeu, 2% raffinose to an initial A₆₀₀ of 0.6. Cells were then pelleted, washed once, and resuspended in 13 ml of SDLeu, 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.

Plasmids-- PAP expression plasmids used in this study were described previously (3, 17). Expression of PAP in NT188 and the nontoxic PAP variants PAP_E176V and PAP_L71R 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 (18) downstream of the GAL1 promoter in YEp351.

RNase Protection Assays-- Total RNA from cells expressing PAP, PAP_E176V, and PAP_L71R was analyzed with the RNase protection assay according to Tumer et al. (18). 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 (18). A 90-nt U3-specific probe was generated from a T3 RNA polymerase run-off transcript of SspI-digested pJD161 (19). 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 the XRN1 gene from pXRN1 cloned into the BamHI/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-nt PGK1-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).

Analysis of Protein Expression-- Protein from frozen yeast cells expressing PAP, PAP_E176V, and PAP_L71R harvested during the time course of induction, was extracted as described by Hudak et al. (15). 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.

rRNA Depurination Assay-- Ribosomal RNA depurination was assayed by primer extension analysis as described previously (20). 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 [-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. (3). Extension products were separated on a 7 M urea, 5% polyacrylamide denaturing gel and visualized and quantified on a Phosphor-Imager (Amersham Biosciences). 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, PAP_E176V, or PAP_L71R, 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 (20). Ribosomes were then either incubated in buffer alone or depurinated with 250 ng of purified PAP to completion as described previously (20). To quantify the extent of depurination, the target RNA was hybridized initially in the presence of excess amounts (700 pmol) of the two [-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 7 M 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.

In Vivo [³⁵S]Methionine Incorporation-- Yeast cells were grown to an A₆₀₀ of 0.6 in SDLeu, Met, 2% raffinose. Cells were then resuspended at an A₆₀₀ 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-Schmid et al. (21) 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 A₆₀₀ reading. Rates of translation were determined from these results and tabulated as CPM/A₆₀₀/min. To obtain translation inhibition in PAP_E176V 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PAP Inhibits the Growth of S. cerevisiae-- 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 PAP_E176V, which contains a point mutation (E176V) at its active site and produces an inactive protein, and PAP_L71R, 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 PAP_E176V or the vector alone (YEp351) were able to grow on SDLeu plates containing galactose (17). However, yeast cells harboring the wild type PAP plasmid (NT188) failed to grow on plates containing galactose (17). As shown in Fig. 2, the growth of cells expressing the wild type PAP but not PAP_E176V was inhibited in liquid SDLeu medium containing galactose compared with cells harboring the same vector (YEp351) with luciferase cDNA (VC). The doubling time of cells expressing PAP_E176V 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 PAP_L71R 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 PAP_E176V resulted 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.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Diagram of wild type PAP and the PAP variants. The mature PAP protein is 262 amino acids in length. Twenty-two amino acids are cleaved from the N terminus and 29 amino acids (29 a.a.) from the C terminus during processing of the PAP precursor to the mature protein. The amino acid changes and their positions are shown for each nontoxic variant, NT224 and NT538.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Growth 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 SDLeu medium containing 2% galactose. An A600 reading was taken for growth measurement at the indicated times after induction.

Correlation between Inhibition of Growth and Inhibition of Translation-- To determine whether reduction of growth is correlated with inhibition of translation, we examined total translation in cells expressing PAP, PAP_E176V, and PAP_L71R 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 (21). As shown in Fig. 3A, cells grown in galactose for 4 h to induce expression of PAP_E176V were not significantly inhibited in translation as compared with vector control cells. The rate of translation in cells expressing PAP_E176V, judged from the slope of the curve in Fig. 3A, 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 PAP_E176V.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Analysis of total translation in cells expressing PAP, PAPE176V, and PAPL71R. Yeast harboring PAP, PAPL71R, or PAPE176V or vector control were grown in SDLeu and Met and 2% galactose for 4 (A) or 10 h (B) to induce protein expression. At time zero, [35S]methionine was added to cells growing on galactose that express PAP (), PAPE176V (), PAPL71R (), or vector control (), and incorporation was determined at the indicated times (in minutes). Each point was assayed in duplicate, and the translation rates were determined from three separate experiments.

The growth inhibition observed in the presence of anisomycin in Fig. 2 suggested that inhibition of growth might be due to inhibition of translation. Under these conditions, we would expect cells expressing PAP_L71R to be inhibited in translation. As shown in Fig. 3A, total translation was significantly inhibited in cells expressing PAP_L71R at 4 h post-induction. The rate of translation in cells expressing PAP_L71R 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 PAP_L71R but not in PAP_E176V (Fig. 3B). These results provide evidence that the reduction in growth observed in cells expressing wild type PAP and PAP_L71R correlates with the inhibition of translation observed in these cells.

PAP Has a Specific Effect on the Stability of Its Own mRNA in Yeast-- Because our previous results had indicated that PAP can inhibit translation by depurinating capped RNAs (3), 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. 4A). 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, PAP_E176V, and PAP_L71R 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 (19). Samples were separated electrophoretically, and the intensities of the protected bands were quantified using a Phosphor- Imager. The ratios for signals of the PAP, PAP_E176V, or PAP_L71R mRNAs to the U3 snoRNA were used as relative measures of the steady state abundance of the PAP, PAP_E176V, or PAP_L71R 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. 4A). As expression of wild type PAP was induced, the level of PAP mRNA decreased dramatically relative to the U3 snoRNA (Fig. 4A). By 10 h post-induction, PAP mRNA levels decreased to about 10% of the levels observed at 4 h post-induction (Fig. 4B). In contrast, PAP_E176V mRNA levels increased up to 10 h post-induction and reached steady state levels after 10 h. Although the overall increase in PAP_E176V 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 PAP_E176V was between 2.5 and 8 times greater than that at 4 h. The error bars in Fig. 4B represent 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, PAP_E176V, is expressed in yeast, indicating that mRNA destabilization is due to the N-glycosidase activity of PAP. RNase protection analysis beyond 10 h post-induction indicated that PAP mRNA is not detectable after 12 h, whereas PAP_E176V mRNA remains at steady state levels (data not shown).

View larger version (67K): [in this window] [in a new window]   Fig. 4.   RNase protection of PAP, PAPE176V, and PAPL71R expression in yeast. A, expression of wild type PAP, PAPE176V, and PAPL71R was induced by growing cells in SDLeu containing 2% galactose. Total RNA (15 µg) extracted from yeast cells harvested at 0, 2, 4, 6, 8, and 10 h post-induction was analyzed by RNase protection analysis. The positions of the PAP and U3 probes alone are indicated in lanes marked "PAP " and "U3." The "tRNA " lane represents RNase protection using tRNA hybridized with both PAP and U3 probes. B, the mRNAs corresponding to PAP and U3 were quantified, and the ratios of PAP mRNA to U3 snoRNA were plotted at various times after induction of expression.

RNase protection analysis of yeast expressing PAP_L71R indicated that as observed with wild type PAP, mRNA levels increased up to 4 h post-induction. However, PAP_L71R mRNA remained at elevated steady state levels and was not destabilized after 4 h of induction (Fig. 4). These results indicated that PAP_L71R behaves similarly to wild type PAP during the early stages of induction. Although both growth and translation were inhibited in cells expressing PAP_L71R, mRNA was not destabilized, indicating that the L71R mutation did not affect the ability of PAP to inhibit translation but did affect its function in mRNA destabilization.

PAP Did Not Affect the Stability of the Cellular mRNAs Examined-- Because PAP is cytotoxic to yeast (17, 18), its expression could lead to a general reduction in the steady state levels of all mRNAs in the cell. To test this hypothesis we analyzed the steady state levels of four yeast genes that are constitutively expressed: XRN1, LEU2, PGK1, and RPL3 (22-27). These genes encode the major 5'-to-3' exoribonuclease (XRN1); isopropylmalate dehydrogenase (LEU2) involved in leucine biosynthesis; 3-phosphoglycerate kinase (PGK1) involved in glucose metabolism; and ribosomal protein L3 (RPL3). As shown in Fig. 5, the levels of mRNA corresponding to these four genes were unaffected in yeast expressing either PAP or PAP_E176V. In both PAP and PAP_E176V expressing cells, the levels of XRN1, LEU2, and RPL3 transcripts remained relatively unchanged; however, the level of PGK1 mRNA decreased. Because PGK1 mRNA levels decreased in the presence of both PAP and PAP_E176V, this effect may not be due to the activity of PAP (Fig. 5). These results indicate that PAP has a specific effect on the accumulation of its own mRNA.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   RNase protection analysis of yeast cellular mRNAs. RNase protection analysis was carried out with probes specific for XRN1, LEU2, RPL3, and PGK1. Total RNA (10 µg) extracted from yeast cells expressing PAP or PAPE176V at different times after induction was analyzed by RNase protection assay as described under "Experimental Procedures." The mRNAs corresponding to each gene were quantified, and the ratios of XRN1, LEU2, RPL3, and PGK1 to CYH2 mRNA were plotted at various times after induction of expression.

PAP Protein Accumulates during Galactose Induction-- To verify that the effects of PAP on growth and mRNA abundance were due to PAP expression, immunoblot analysis was performed on aliquots harvested from the same cells, grown on galactose medium in Fig. 4. In Fig. 6, protein levels corresponding to mature PAP increased during the 10 h time course of induction in cells expressing the wild type PAP despite the translation inhibition and the reduction in PAP mRNA abundance. PAP is synthesized as a precursor and processed at both the N- and C-terminal ends to form the mature protein (7). The higher molecular weight form observed previously co-migrates with the precursor form of PAP, which is incompletely processed in yeast (15). Both forms accumulated in cells expressing PAP_E176V, since translation was not inhibited in these cells (Fig. 6). In cells expressing PAP_L71R, both forms accumulated by 4-6 h post-induction and remained at constant levels after 6 h, possibly due to inhibition of translation (Fig. 6). The immunoblots were stripped and reprobed with antibodies against G6PD to show that equal amounts of protein were loaded on the gels.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Immunoblot analysis of PAP and PAPE176V expression in yeast. Total protein (7.5 µg) from each time point in Fig. 4 was separated on 15% SDS-PAGE. Proteins were transferred to nitrocellulose and probed with polyclonal PAP serum (1:5000). Purified PAP (~10 ng) from pokeweed leaves was used as a standard. The immunoreactive species corresponding to mature PAP is indicated by an arrow. The blots were subsequently stripped and reprobed with anti-G6PD (1:5000) antibodies as loading controls.

Correlation between Inhibition of Translation and mRNA Accumulation-- RNase protection analysis (in Fig. 4) indicated that PAP mRNA levels peak at 4 h post-induction in cells expressing wild type PAP and PAP_L71R. This increase is not observed in cells expressing PAP_E176V even though the expression of all three genes is driven by the same promoter. PAP_E176V mRNA levels increase gradually up to 10 h, as reported with other genes driven by the GAL1 promoter, such as -galactosidase, which can require 6 h to reach maximal levels (28). These results suggested that the increase in PAP mRNA levels at 4 h post-induction could be due to inhibition of translation, because translation is inhibited in cells expressing both the wild type PAP and PAP_L71R but not PAP _E176V at 4 h post-induction (Fig. 3).

To determine whether inhibition of translation is responsible for the increase in mRNA levels, we added the translational inhibitor anisomycin to PAP_E176V cells at 2 h post-induction. Titration experiments indicated that the addition of 15 A₂₈₀ units (~5 µg) of anisomycin/ml to PAP_E176V-expressing cells resulted in a level of translation inhibition similar to that observed in PAP-expressing cells (Table I). As shown in Fig. 7A, RNase protection analysis indicated that PAP_E176V mRNA levels increased to higher levels after the addition of anisomycin compared with mRNA levels in the absence of anisomycin. PAP_E176V mRNA levels at 4 h after induction were 8-fold higher in the presence of anisomycin than the levels at 4 h in the absence of anisomycin (Fig. 7B). The PAP_E176V mRNA remained at high levels after 4 h in the presence of anisomycin. These results strongly suggest that the inhibition of translation elongation is responsible for the initial increase observed in mRNA levels in cells expressing PAP or PAP_L71R.

View larger version (71K): [in this window] [in a new window]   Fig. 7.   RNase protection of PAPE176V and PAPE176V + anisomycin expression in yeast. A, expression of PAPE176V was induced by growing cells in SDLeu containing 2% galactose. At 2 h post-induction, 15 A280 units of anisomycin (~5 µg/ml)/ml were added to one of two PAPE176V cultures. Total RNA (15 µg) extracted from yeast cells harvested at 0, 2, 4, 6, 8, and 10 h post-induction was analyzed by RNase protection analysis as described in the legend for Fig. 4B. The mRNAs corresponding to PAP and U3 were quantified, and the ratios of PAP mRNA to U3 snoRNA were plotted at the indicated times after induction of expression.

Immunoblot analysis indicated that PAP_E176V protein accumulated during the induction in the absence of anisomycin (Fig. 8). After the addition of anisomycin to PAP_E176V-expressing cells at 2 h post-induction, a slight increase in protein levels was observed between 2 and 4 h (Fig. 8); this could be due to the slow uptake of anisomycin into the yeast cells. After 4 h, however, both forms of PAP_E176V protein remained at constant levels and did not increase to the levels observed without anisomycin, confirming that translation was inhibited. The blot was stripped and reprobed with G6PD to demonstrate equal loading of protein on the gels.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Immunoblot analysis of PAPE176V and PAPE176V + anisomycin expression in yeast. Total protein (7.5 µg) loaded from each time point in Fig. 7 was separated on 15% SDS-PAGE. The amount of PAP protein expressed was determined as described in the legend for Fig. 6.

Ribosome Depurination in Yeast Expressing PAP-- Previous results showed that inhibition of translation did not correlate with the decrease in mRNA accumulation in cells expressing PAP_L71R, suggesting that the translation inhibition is not due to the reduction in mRNA levels. Because PAP has been reported to inhibit translation by depurinating the S/R loop of the rRNA (4, 5), we examined rRNA depurination in cells expressing PAP. Ribosomes isolated from yeast expressing PAP, but not PAP_E176V, were previously shown to be depurinated (20). PAP may depurinate ribosomes in cis only during its translation. Alternatively, PAP may depurinate ribosomes in trans after it is synthesized in a manner that is independent of translation. To determine whether PAP depurinates yeast ribosomes in trans, we induced PAP expression by growing yeast cells for 1 h in galactose-containing medium followed by inhibition of PAP gene transcription by shifting cells to medium containing 2% glucose. As shown in Fig. 9A, PAP was not detected after 1 h on galactose but accumulated when transcription but not translation was repressed with glucose. To examine depurination of the rRNA, we used the primer extension assay described previously (3). Ribosomes were not depurinated after 1 h on galactose (Fig. 9B). However, depurination was observed 1 h after repression of transcription (Fig. 9B). To determine whether translation is required for depurination of rRNA, following a 1-h induction on galactose, cells were resuspended in medium containing glucose and cycloheximide to inhibit transcription and translation, respectively. PAP did not accumulate when transcription and translation were both inhibited (Fig. 9C). As shown in Fig. 9D, ribosomes were depurinated at 1 h after repression of transcription and translation equally as well as when only transcription was repressed (Fig. 9B). These results demonstrate that rRNA depurination does not require ongoing translation and can occur in trans in a way that is independent of translation.

View larger version (26K): [in this window] [in a new window]   Fig. 9.   Analysis of rRNA depurination during PAP induction. Yeast cells expressing wild type PAP were resuspended in SDLeu + galactose to induce PAP expression. Immediately prior to galactose addition, an aliquot was pelleted to serve as a raffinose control (Raf). After 1 h of galactose induction, a second aliquot of cells was collected (Gal), and either glucose alone (+ Glucose) or glucose plus cycloheximide (100 µg/ml final concentration; + Glucose + CHX) was added. At the indicated times (in hours) aliquots were removed for depurination (B and D) and immunoblot analysis (A and C).

Correlation between mRNA Destabilization and rRNA Depurination-- To examine the relationship between rRNA depurination and mRNA destabilization, a novel dual-oligo primer extension assay was developed to quantify the extent of rRNA depurination at different times after induction of PAP expression. Equimolar amounts of the two oligonucleotides were end-labeled and hybridized to total RNA. One primer hybridized downstream of the depurination site and was used to examine the extent of depurination, whereas the other primer hybridized upstream of the depurination site close to the 5' end of the 25 S rRNA and was used to quantify the total amount of rRNA (Fig. 10A). The ratio of the depurination fragment compared with the control fragment allowed for accurate quantification of the extent of depurination. This ratio was compared with the level of depurination obtained when ribosomes were treated with purified PAP in vitro (PAP in Fig. 10B). The primer extension products of total RNA from the same cells expressing PAP and PAP_L71R shown in Fig. 4 were then resolved on a denaturing polyacrylamide gel. As shown in Fig. 10B, primer extension analysis indicated that rRNA depurination was detected 2 h after induction in cells expressing wild type PAP. Maximal depurination of rRNA occurred at 4 h after induction and then decreased rapidly (Fig. 10C). Depurination of rRNA was also detected in cells expressing PAP_L71R at 2 h post-induction. Depurination increased up to 4 h post-induction, and in contrast to cells expressing wild type PAP, it remained at constant levels after 4 h (Fig. 10C). These studies indicated that not all of the ribosomes are depurinated in yeast expressing wild type PAP or PAP_L71R. Maximal depurination in vivo corresponded to 47% of the depurination observed in vitro (Fig. 10C). Furthermore, as shown in Fig. 10D, the temporal pattern of rRNA depurination was similar to the temporal pattern of mRNA accumulation in cells expressing wild type PAP and PAP_L71R, indicating that there is a direct relationship between rRNA depurination and mRNA abundance. These results suggest that PAP may depurinate the rRNA and destabilize its own mRNA by a common mechanism.

View larger version (39K): [in this window] [in a new window]   Fig. 10.   Analysis of rRNA depurination during induction of PAP and PAPL71R. A, schematic representation of dual-oligo primer extension assay. Two different end-labeled primers (Depurination primer and 25S Control primer) were annealed to rRNA and reverse-transcribed. The resulting fragments represent extension products that have stopped prematurely at the depurination site (indicated with an asterisk) and extension products that have stopped at the 5' end of the 25 S rRNA. B, the primer extension products for PAP and PAPL71R representing the extent of depurination and the amount of total 25 S rRNA present at the indicated times (in hours) from the same samples assayed in Fig. 4 were resolved on a denaturing polyacrylamide gel. Ribosomes treated in vitro with purified PAP or buffer alone are designated as PAP and No PAP, respectively. Primers were also extended separately as marked in the first two lanes. C, the extent of depurination shown in B was quantified by calculating the ratio of the depurination fragment to the 25 S rRNA fragment; this ratio was expressed as a percent of the depurination observed in vitro. The quantification was repeated twice more with similar results. D, comparison of the temporal pattern of rRNA depurination (in C) and PAP mRNA abundance (Fig. 4B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results presented here demonstrate that in addition to depurinating the rRNA, PAP regulates its own expression by reducing the abundance of its own mRNA. This effect is dependent on the N-glycosidase activity of PAP, as an active site mutant fails to alter its mRNA levels. We present evidence that inhibition of growth observed in PAP-expressing cells is due to inhibition of translation. Depurination of the rRNA and inhibition of translation by PAP leads to up-regulation of the steady state levels of PAP mRNA, which occurs prior to mRNA destabilization. Using a PAP variant that depurinates rRNA, inhibits translation, but does not destabilize its own mRNA, we show that destabilization of PAP mRNA can be separated from rRNA depurination and translation inhibition. We examine the relationship between the rRNA depurination and destabilization of PAP mRNA and present evidence that they may be mechanistically related.

Total translation was reduced by 65-75% in cells expressing wild type PAP and PAP_L71R at 4 h post-induction and remained at that level throughout the time course. In contrast, total translation was not significantly inhibited in cells expressing PAP_E176V. These results indicate that protein synthesis is inhibited but not completely abolished in cells expressing PAP. Many RIPs have been shown to depurinate DNA, RNA, and poly(A) RNA (29). We have previously shown that PAP can inhibit translation in a cell-free system by depurinating capped RNAs. To determine whether PAP targets capped RNAs in vivo, we examined the stability of PAP mRNA and cellular mRNAs in yeast and showed that induction of PAP expression led to a dramatic decrease in PAP mRNA abundance. Steady state levels of four different cellular mRNAs were not affected by PAP, demonstrating that PAP mRNA is not destabilized simply as a consequence of host cell death. These results indicate that PAP expression does not destabilize every RNA and therefore exhibits specificity. DNA microarray analysis of yeast expressing PAP confirmed these results and indicated that PAP expression affects the abundance of specific mRNAs in yeast (data not shown). We observed no effect on U3 snoRNA, which is capped with a trimethyl guanosine. These results are consistent with our prior observations, which indicate that PAP destabilizes mRNAs containing a 7-methyl guanosine cap and suggest that PAP may not recognize the trimethyl guanosine cap present on the U3 RNA.

As summarized in Table II, analysis of PAP mRNA accumulation in two different PAP mutants with reduced toxicity indicated that destabilization of PAP mRNA required an intact active site, because the active site mutant, PAP_E176V did not decrease the abundance of its mRNA. The rRNA was depurinated and translation and growth were inhibited in cells expressing PAP_L71R just like wild type PAP. However, mRNA was not destabilized, indicating that destabilization of PAP mRNA can be dissociated from depurination of the S/R loop and inhibition of translation (Table II). Unlike previously characterized pathways of mRNA degradation, which are interconnected with translation (30), PAP-mediated degradation of PAP mRNA occurs when translation is inhibited.

Inhibition of translation correlated well with the up-regulation of PAP mRNA levels in cells expressing wild type PAP and PAP_L71R at 4 h post-induction. This increase was not observed in cells expressing PAP_E176V, which did not inhibit translation. To determine whether inhibition of translation elongation would lead to stabilization of PAP mRNA levels, we added the elongation inhibitor anisomycin to cells expressing PAP_E176V. RNase protection analysis indicated that PAP_E176V mRNA levels increased dramatically after addition of anisomycin. These results suggest that the increase observed in PAP mRNA levels at 4 h post-induction in cells expressing wild type PAP and PAP_L71R is due to inhibition of translation elongation. They are consistent with previous reports in which elongation inhibitors have been shown to stabilize mRNAs in yeast (30).

We show that very small amounts of PAP synthesized during 1 h of induction on galactose depurinated ribosomes in trans, demonstrating that ribosome depurination occurs in a way that is independent of translation. Similar results are reported with ricin, where one molecule of ricin has been shown to inactivate 300 ribosomes in trans (31). A novel dual-oligo primer extension assay was devised to examine the relationship between rRNA depurination and mRNA decay. In cells expressing wild type PAP, depurination of rRNA was detected prior to destabilization of PAP mRNA, indicating that rRNA can be depurinated under conditions in which PAP mRNA is not degraded. Because rRNA depurination can be separated from mRNA destabilization, PAP may destabilize its own mRNA in trans when it is on the ribosome but depurinate the ribosomes whether or not they translate its mRNA. Our previous observations indicate that PAP mRNA degradation most likely occurs on the ribosomes. We demonstrated that PAP is associated with and binds to ribosomes in yeast through its ability to interact physically with RPL3 (15). A chromosomal mutant of yeast harboring the mak8-1 allele of RPL3 is resistant to PAP because PAP cannot interact with the mutant L3 in vivo. Although PAP protein accumulated in mak8-1 cells, PAP was not associated with ribosomes and ribosomes were not depurinated (15). RNase protection analysis showed that PAP transcripts were not destabilized in mak8-1 cells expressing PAP or PAP_E176V (15), suggesting that PAP mRNA destabilization occurs on the ribosome.

We present evidence that the temporal patterns of rRNA depurination in cells expressing wild type PAP and PAP_L71R are very similar to the patterns of mRNA accumulation (Fig. 10D), suggesting that they are mechanistically related. This is further supported by the observation that destabilization of PAP mRNA occurs independently of translation, suggesting that like rRNA, PAP destabilizes its own mRNA in trans. Analysis of PAP mRNA turnover in the presence of cycloheximide or anisomycin demonstrated that PAP mRNA is destabilized in trans in a manner that is independent of translation (data not shown).

Our previous results indicate that PAP depurinates capped but not uncapped RNAs in a cell-free system (3). We have recently characterized this activity further, showing that PAP binds to the cap structure (16). PAP does not remove the cap structure or depurinate the cap but depurinates the mRNA at specific adenine and guanines downstream of the cap (16). If a single site of PAP binds to both cap and purines, it implies that a single molecule of PAP cannot do so simultaneously. Incubation of ribosomes with PAP and increasing concentrations of the cap analog m⁷GpppG resulted in a decrease in the level of rRNA depurination, indicating that the cap structure competes with the rRNA for binding to PAP (16). We have shown that the affinity of PAP for capped message is only 4-fold lower than its affinity for rRNA (16), indicating that at increased levels such as those seen as a result of translation inhibition, capped RNA may become a substrate for PAP.

A model that takes these observations into account is one that separates early events (pre-destabilization) from late events (destabilization). Galactose induction causes PAP mRNA and protein to accumulate. Only a small amount of protein is required for S/R loop depurination to occur, and this depurination can proceed in trans. By 4 h post-induction, rRNA depurination reaches maximal levels, translation is inhibited, and there is a dramatic increase in PAP mRNA abundance, which is not observed in cells expressing PAP_E176V. Because translation is inhibited very early in cells expressing PAP, this inhibition most likely causes the accumulation. PAP mRNA may be sequestered on depurinated ribosomes by the interaction between PAP and the cap structure of the mRNA. Translation remains inhibited late in the time course, but PAP protein accumulates, indicating that its translation is somehow insensitive to PAP-mediated translation inhibition. Previous studies also showed that unlike ricin, PAP does not inhibit translation of its own mRNA in vitro in rabbit reticulocyte lysate (17). As the time course continues, enough PAP is translated to create a high concentration of protein on the ribosome. Although translation remains inhibited late in the time course, there is now enough active PAP present to depurinate the mRNA on the ribosomes. Under these conditions, the depurination of the PAP mRNA would occur independently of depurination of the rRNA and inhibition of translation. The depurinated RNA may be degraded as a result of cleavage by cellular lyases as observed for rRNA in wheat germ (32). RNase protection analysis indicates that PAP mRNA levels decrease and PAP mRNA is not detectable after 12 h. In some experiments, we have observed shorter fragments of PAP mRNA, indicating that it is not simply sequestered but is being degraded (data not shown).

Our model implies that the destabilization observed may not be specific only for PAP mRNA but may affect other mRNAs translated by depurinated ribosomes. We present evidence that PAP does not affect every capped mRNA in the cell. In vitro results also show that capped viral RNAs differ in their sensitivity to PAP, indicating that the presence of the cap structure is not the only determinant for depurination by PAP. This activity may be modulated in vivo by association with cellular proteins or through recognition of particular secondary structures in addition to cap, which would enable PAP to target particular transcripts.

Mature PAP is an extracellular protein stored in the apoplast of pokeweed plants where it can accumulate up to 0.5% of total soluble protein (33). This compartmentalization prevents access to pokeweed ribosomes (34). It has been reported that PAP preferentially enters virus-infected cells. It is estimated that 80% of the RNA in healthy cells is ribosomal, but during viral infection the level of viral RNA increases significantly beyond the normal level of capped messages and approaches the levels of rRNA (16). In this situation, PAP may target capped viral RNAs. Nontoxic variants of PAP exhibit antiviral activity in vivo, suggesting that they do not target every capped RNA in the cell but likely display specificity for viral RNAs or cellular messages that are involved in virus replication. RIPs are thought to be defense proteins; however, the mechanism by which they inhibit cell growth or pathogen infection is not well understood. The results reported here suggest that PAP may have a role in differentially regulating mRNA stability in vivo. Further studies will address the basis for the selectivity of this regulation and its role in the antiviral activity of PAP.

	ACKNOWLEDGEMENTS

We thank J. Dinman for plasmids used in this work and Drs. P. Day, J. Dinman, K. Hudak, E. Lam, S. Gunderson, and A. Shatkin for helpful discussions and reading the manuscript. We also thank Dr. T. Kinzy for comments on the manuscript and the members of T. Kinzy's laboratory for assistance with the yeast translation assays.

	FOOTNOTES

^* This work was supported by National Science Foundation Grant MCB 9982498 (to N. E. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Biotechnology Center, Cook College, Rutgers University, 59 Dudley Rd., New Brunswick, NJ 08901-8520. Tel.: 732-932-8165, ext. 215; Fax: 732-932-6535; E-mail: tumer@aesop.rutgers.edu.

Published, JBC Papers in Press, August 8, 2002, DOI 10.1074/jbc.M205463200

	ABBREVIATIONS

The abbreviations used are: PAP, pokeweed antiviral protein; RIP, ribosome-inactivating protein; S/R, sarcin/ricin; SD medium, synthetic dropout medium; nt, nucleotide(s); snoRNA, small nucleolar RNA; G6PD, glucose-6-phosphate dehydrogenase.

	REFERENCES

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