HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 17, 15142-15146, April 26, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/17/15142    most recent M108115200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saxena, S. K. Articles by Youle, R. J. Search for Related Content PubMed PubMed Citation Articles by Saxena, S. K. Articles by Youle, R. J. Social Bookmarking                      What's this?

Entry into Cells and Selective Degradation of tRNAs by a Cytotoxic Member of the RNase A Family^*

Shailendra K. Saxena^§, Ravi Sirdeshmukh^¶, Wojciech Ardelt^§, Stanislaw M. Mikulski^§, Kuslima Shogen^§, and Richard J. Youle

From the  Biochemistry Section, Surgical Neurology Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892-1414, ^§ Molecular Biology Laboratory, Alfacell Corporation, Bloomfield, New Jersey 07003-3500, and ^¶ Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

Received for publication, August 22, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA, which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Onconase is a ribonuclease found in eggs and early embryos of the leopard frog, Rana pipiens (1). When incubated with mammalian cells in culture, the protein has cytotoxic and cytostatic activity (2) unlike homologous mammalian members of the RNase A family. The protein expresses RNase activity that appears essential for its biological activities and is insensitive to inhibition by the ribonuclease inhibitor (1, 3, 4). Resistance to ribonuclease inhibitor is thought to explain the greater toxicity of onconase compared with mammalian members of the same RNase family (4-6). Onconase consists of 104 amino acids with a molecular mass of 11.8 (K_d) and an isoelectric point of 9.7 (1). In contrast to mammalian RNase A family members including bovine pancreatic RNase, eosinophil-derived neurotoxin, and angiogenin (7, 8), the N-terminal pyroglutamyl residue, Pyr-1, is part of the active site, folding back against the N-terminal -helix and forming a hydrogen bond with Val-96 in the C-terminal -sheet (9).

Onconase displays antitumor activity in vivo (10, 11) and is presently in phase 3 clinical trials. Onconase at concentrations nontoxic to H9 cells has been found to specifically inhibit HIV-1 replication in HIV-1-infected human H9 leukemia cells without necessarily killing the virus-infected cell (12, 13). An examination of the mechanism of this inhibition reveals that onconase enters the infected cells and degrades HIV-1 RNA without degrading ribosomal RNA or the three different cellular messenger RNAs analyzed.

The primary molecular substrates and molecular mechanism of RNA selectivity for onconase anticancer and antiviral activity remain unclear. Here we describe the mechanism of a very specific elimination of tRNA and activation of tRNA synthesis by onconase in cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Onconase was prepared at Alfacell Corporation from R. pipiens oocytes by a modification of the previously published method (1). The enzyme was first isolated from the cell extract on SP-Sepharose and then further purified by re-chromatography on the same exchanger followed by the size-exclusion on Sephacryl S-100 in 75 mM ammonium bicarbonate and lyophilized. The final preparation was homogenous on SDS-PAGE and isoelectric focusing. Rabbit reticulocyte lysate (nuclease-treated) was from Promega. All-trans-retinoic acid was purchased from Calbiochem. CD4-positive H9 lymphocyte cells uninfected and persistently infected with HIV-1 IIIB strain were obtained from Dr. Robert Gallo (NCI, National Institutes of Health, Bethesda, MD). Purified Escherichia coli 23 S and 16 S ribosomal RNA and 5 S ribosomal RNA were purchased from Roche Molecular Biochemicals. Purified tRNA^Phe and 0.1-1.0-kb RNA marker were purchased from Sigma.

DNA Probes for Detecting tRNA Species-- Three DNA oligonucleotide probes for detecting the tRNA, tRNA (14), and tRNA^Phe (15) were synthesized. Each probe was 18-nucleotides long and complementary to the last 18 nucleotides at the 3' end of their respective tRNA. The probe oligonucleotide sequences were 5'-TGGCGCCCGAACAGGGAC-3' (for tRNA), 5'- TGGCGCCCAACGTGGGGC-3' (for tRNA), and 5'-TGGTGCCGAAACCCGGGA-3' (for tRNA^Phe).

Ribonuclease Activity of Onconase in H9 Cells-- H9 lymphocyte cells uninfected or persistently infected with HIV-1 were grown in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum and 50 µg/ml gentamycin (12). 25-ml cultures of uninfected H9 cells (2 × 10⁵ cells/ml) in 75-cm² flasks were treated with onconase at concentrations ranging from 1 × 10⁹ to 1 × 10⁵ M. After 20-h incubation, cells were processed to isolate total cellular RNA. Persistently infected H9 cells were washed extensively to reduce levels of free virus and resuspended at 2 × 10⁵ cells/ml for further use. Onconase at 5 × 10⁸ M was added to washed cells, and cultures were sampled daily over a 4-day period. At the end of each incubation, cells were harvested by centrifuging the culture at 400 × g for 10 min. Cell pellets were processed to isolate total cellular RNA. Total RNA was analyzed on 1.4% agarose gel and 10% polyacrylamide gel containing 7.5 M urea.

Ribonuclease Activity of Onconase in 9L Cells in the Presence of Retinoic Acid-- 9L rat glioma cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 10 µg/ml gentamycin (4). Retinoic acid stock solution (15 mM in Me₂SO) was diluted into leucine-free RPMI 1640 medium without fetal calf serum to 10 µM. The same dilution of Me₂SO was added in the control solutions. After removing the Dulbecco's modified Eagle's medium containing 10% fetal calf serum, cells were incubated in the above leucine-free RPMI 1640 medium containing increasing concentrations of onconase with or without retinoic acid for 20 h followed by isolation of total cellular RNA. Total RNA was analyzed on 1.4% agarose gel and 10% polyacrylamide gel containing 7.5 M urea.

Ribonuclease Activity of Onconase in Rabbit Reticulocyte Lysate-- RNA specificity of onconase was also tested in rabbit reticulocyte lysate using phosphate-buffered saline as a reaction buffer. The reaction mixture (50 µl) containing rabbit reticulocyte lysate and onconase at 1 × 10⁹ to 1 × 10⁷ M concentrations were incubated for 15 min at 30 °C. At the end of the incubation, the total RNA was isolated from the reaction mixture using RNAzol^TM, and the RNA pellet was dissolved in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA. Approximately 1.5 µg of total RNA was analyzed in either 10% polyacrylamide gel containing 7.5 M urea or 1.4% agarose gel. The RNA was visualized after staining the gel with ethidium bromide. In the experiment shown in Fig. 3, purified E. coli rRNA was added to the lysate, and the onconase action was studied.

Ribonuclease Activity of Onconase toward Purified RNA from Rabbit Reticulocyte Lysate-- The reaction mixture (50 µl) containing purified RNA from rabbit reticulocyte lysate and onconase at 1 × 10⁹ to 1 × 10⁷ M concentrations were incubated for 15 min at 30 °C in phosphate-buffered saline as reaction buffer. At the end of the incubation, the total RNA from the reaction mixture was isolated using RNAzol. The RNA pellet was dissolved in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA. Approximately 1.5 µg of total RNA was analyzed on 10% polyacrylamide gel containing 7.5 M urea.

RNA Isolation-- Total RNA from H9 and 9L cells and rabbit reticulocyte lysate was extracted using RNAzol, according to the protocol supplied by Tel-Test Inc. (Friendswood, TX). Cells were homogenized in RNAzol (2 ml RNAzol/1 × 10⁷ cells). RNA was then extracted with 0.1 volume of chloroform, precipitated with 1 volume of isopropyl alcohol, and finally washed with 75% ethanol. The RNA pellet was dissolved in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA.

Electrophoresis-- Electrophoresis of RNA samples was carried out in 1.4% agarose gel using a horizontal gel electrophoresis apparatus model H5 (Invitrogen) or in 10% polyacrylamide gel (19:1, acrylamide:bis-acrylamide) containing 7.5 M urea using a vertical gel electrophoresis apparatus model V16 (Invitrogen). In both cases, 1× TBE (89 mM Tris, 89 mM borate, 2 mM EDTA, pH 8.3) was used as a running buffer (16).

Northern Blot Analysis-- Northern blot analysis was carried out according to the protocols described previously (16). Approximately, 1.5 µg of the total RNA from each set was denatured by heating at 70 °C for 15 min in 1× formamide gel-loading buffer (10% formamide, 2 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue) followed by a quick chill in icy water. The gel was pre-run for 30 min at 25 watts before samples were loaded. These samples were electrophoresed on 10% polyacrylamide gel containing 7.5 M urea at 20 watts for 2 h using 1× TBE (89 mM Tris, 89 mM borate, and 2 mM EDTA, pH 8.3) as a running buffer. After electrophoresis, the gel was fixed in 7% acetic acid for 30 min. The gel was photographed after staining with ethidium bromide in the 1× TBE for 10 min. The gel was electroblotted at 200 mA for 16 h onto Nytran Plus membranes (Schleicher & Schuell) using the 1× TBE as a running buffer. RNA was fixed onto filters by UV cross-linking (UV Stratalinker, Stratagene, La Jolla, CA). Pre-hybridization was carried out at 42 °C for 6 h in the hybridization buffer (0.2% polyvinyl-pyrrolidone (M_r = 40,000), 0.2% Ficoll (M_r = 400,000), 0.2% bovine serum albumin, 0.05 M Tris-HCl, pH 7.5, 1 M sodium chloride, 0.1% sodium pyrophosphate, 1% SDS, 10% dextran sulfate (M_r = 500,000), and 0.1 mg/ml denatured salmon sperm DNA). Following pre-hybridization, hybridization was carried out for 20 h at 42 °C using the ³²P-5'-end-labeled ([-³²P]ATP, catalog number NEG-002Z, PerkinElmer Life Sciences) DNA probes for tRNA, tRNA (14), and tRNA^Phe (15). The blot was washed twice in the 2× SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0)/1% SDS at room temperature for 15 min each and once with the 0.2× SSC/1% SDS at 65 °C for 30 min. Autoradiography was carried out with intensifying screens at 70 °C and developed by X-Omat (Eastman Kodak Company).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Onconase Degrades tRNA Species in Living Cells-- Onconase is known to be cytotoxic to mammalian cells by virtue of its RNase activity (3). In H9 cells, onconase is cytotoxic above 1 × 10⁷ M (12), whereas at 5 × 10⁸ M concentrations, it slightly inhibits cell growth but does not degrade either cellular 28 S and 18 S rRNA or certain mRNA species (13). We examined the effect of extracellular onconase on cultured H9 cells at concentrations ranging from noncytotoxic (1 × 10⁹ M) to cytotoxic (1 × 10⁵ M) and examined the degradation of different species of cellular RNA in intact cells (Fig. 1). The gel analysis of total RNA obtained from H9 cells showed that onconase from 10⁷ to 10⁵ M concentrations decreased tRNA levels (Fig. 1A), whereas even at the cytotoxic concentration of 1 × 10⁵ M, onconase did not degrade 28 S and 18 S rRNA (Fig. 1B). This tRNA specificity is also observed when reticulocyte lysate is treated with onconase (Fig. 2A, lanes 2-4). Specific tRNA degradation products (30-40 residues long) are visible even at 1 × 10⁹ M onconase concentration (Fig. 2A, lane 2). It appears that at least 10-fold higher concentrations of onconase are required outside cells to see tRNase activity inside the cells compared with that in the reticulocyte lysate.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Onconase cleaves tRNA in cells. H9 cells were grown in RPMI 1640 medium containing 10% fetal calf serum and 50 µg/ml gentamycin. 25-ml cultures of H9 cells (2 × 105 cells/ml) in 75-cm2 flasks were treated with various concentrations of onconase (from 1 × 109 to 1 × 105 M). After 20-h incubation, cells were processed for total RNA isolation using the RNAzol as described under "Experimental Procedures." The RNA pellet was dissolved in 100 µl of 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA. Approximately 1.5 µg of RNA was analyzed on 10% polyacrylamide gel containing 7.5 M urea (A) and over 1.4% agarose gel (B) as described under "Experimental Procedures." RNA was visualized by ethidium bromide staining. In the case of urea-PAGE analysis, the gels were pre-run for 30 min at 25 watts before loading the RNA samples.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   tRNA specificity of onconase in reticulocyte lysate. Rabbit reticulocyte lysate (35 µl) or equivalent amount of purified rabbit reticulocyte lysate RNA was incubated with the specified concentration of onconase (from 1 × 109 to 1 × 107 M) in 50 µl of reaction mixture containing phosphate-buffered saline. After incubation at 30 °C for 15 min, the total RNA was isolated using RNAzol, and the RNA pellet was dissolved in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA. A, approximately 1.5 µg of total RNA was analyzed on 10% polyacrylamide gel. Positions of tRNA bands indicated in A are based on the mobility of purified rabbit reticulocyte lysate RNA (lanes 2 and 5) along with an 0.1-1.0-kb RNA ladder (lanes 1 and 6), purified tRNAPhe (lane 3), and 5 S RNA (lane 4) on a 10% polyacrylamide gel containing 7.5 M urea (B). XC, the migration of xylene cyanol dye on the gel.

Onconase Selectivity for tRNA Species-- When RNA from reticulocyte lysate is purified and incubated with onconase, no selectivity for tRNA is seen (Fig. 2A, lanes 6-8). In fact, perhaps because of its large size, rRNA is more susceptible to onconase than tRNA (Fig. 2A, lane 8). One possible explanation for the different RNA specificities of onconase observed in cells and in cell lysates from that of purified RNAs could be that cellular rRNA is bound to proteins and thus protected from onconase in cells and cell lysates, whereas the tRNA is more exposed. Purified rRNA, protein-depleted and more accessible perhaps because of its large size, could engage the ribonuclease at several susceptible sites and thus could be degraded faster than tRNA (Fig. 2A, lane 8), and this abundance of rRNA substrate may also compete for the degradation of purified tRNA. The protein-RNA complex could also be responsible, at least in part, for the apparent protection of several intracellular mRNAs against the ribonucleolytic activity of onconase, whereas the tRNA species being relatively protein-free remain accessible to onconase catalytic action. To examine whether the association with proteins limits the accessibility of rRNA to onconase, we carried out a spiking experiment in which purified E. coli ribosomal 23 S and 16 S RNA was added to rabbit reticulocyte lysate. The E. coli 23 S and 16 S ribosomal RNAs migrate at different positions than 28 S and 18 S rRNA, allowing protein bound and unbound rRNAs to be assayed in the same reaction milieu. The incubation of the rabbit reticulocyte lysate containing E. coli rRNAs with 1 × 10⁸ and 1 × 10⁷ M onconase resulted in clear degradation of the exogenously added E. coli 23 S and 16 S ribosomal RNA and no degradation of the 28 S and 18 S rRNA in ribosomes (Fig. 3). This finding is consistent with the model that the onconase specificity for tRNA seen in our experiments stems from the shielding of the substrate by associated proteins.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Onconase degrades purified E. coli 23 S and 16 S ribosomal RNA when added to reticulocyte lysates. Rabbit reticulocyte lysate (35 µl) alone or in the presence of purified E. coli 23 S and 16 S ribosomal RNA (3 µl and 4 µg/µl, respectively) was incubated with 1 × 108 and 1 × 107 M onconase in 50 µl of reaction mixture containing phosphate-buffered saline. After incubation at 30 °C for 15 min, the total RNA was isolated as described under "Experimental Procedures." The RNA pellet was dissolved in 35 µl of 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA. 2.5 µl of each RNA sample was analyzed on 1.4% agarose gel, and RNA bands were visualized after staining with ethidium bromide.

Potentiation of Onconase Cytotoxicity Does Not Alter Its RNA Specificity-- Inside the cell, ribonucleases appear to route into the cytosol through the Golgi apparatus. Retinoic acid appears to disrupt the Golgi apparatus (17) and thus facilitates the transport of onconase into the cytosol. Onconase alone was shown to have an IC₅₀ of 1 × 10⁶ M in 9L rat glioma cells, whereas in the presence of 10 µM retinoic acid, the cytotoxicity was increased 100-fold to an IC₅₀ of ~1 × 10⁸ M (4). An analysis of total RNA obtained from 9L cells treated with onconase at 1 × 10⁷ and 1 × 10⁵ M concentrations for 20 h with and without all-trans-retinoic acid demonstrated that onconase selectively degraded tRNA, and this activity was potentiated by the presence of retinoic acid (Fig. 4A). The loss of tRNA at 1 × 10⁷ M onconase with retinoic acid (Fig. 4A, lane 5) was the same or greater than the loss of tRNA at 1 × 10⁵ M onconase alone (Fig. 4A, lane 3). These results show that the cytotoxic effects of the enzyme correlate well with the degradation of cellular tRNA.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Retinoic acid potentiates onconase-mediated tRNA cleavage. 9L rat glioma cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 10 µg/ml gentamycin. 15-ml cultures of 9L cells (2 × 105 cells/ml) in 75-cm2 flasks were treated with 1 × 107 and 1 × 105 M onconase with or without 10 µM retinoic acid. After the 20-h incubation at 37 °C in leucine-free RPMI 1640 medium without fetal calf serum, cells were trypsinized, washed, and processed for total RNA isolation as described under "Experimental Procedures." Approximately 1.5 µg of total RNA was analyzed on 10% polyacrylamide gels containing 7.5 M urea (A) and 1.4% agarose gel (B). RNA bands were visualized after staining with ethidium bromide.

Degradation and Transcription Activation of Individual tRNAs-- Onconase has anti-HIV activity in tissue culture cells (12, 13). Although we previously observed degradation of viral RNA and inhibition of viral replication in HIV-1-infected H9 cells exposed to onconase at nontoxic doses, the primary molecular targets of onconase were not well understood. On the basis of the results presented above, the susceptibility of HIV mRNA to degradation by onconase described earlier may stem from relatively less protection by proteins. An initial step in the replication of the HIV-1 is the conversion of the HIV-1 RNA genome into DNA by reverse transcriptase (RT). RT requires a primer for initiation of DNA synthesis, which in the case of HIV-1 is a cellular tRNA species. This primer tRNA found in HIV-1 particles is selected from the host cell tRNA population during virus assembly. The 3'-terminal 18 nucleotides of the primer tRNA bind to a complementary region of the 5'-U5 region in the long terminal repeat termed as a primer binding site of the HIV-1 RNA genome.

Although we have found that onconase inhibits HIV-1 replication at nontoxic doses, its molecular mechanism of antiviral activity remains unknown. HIV-1 mRNA seems to be particularly sensitive to onconase and, based on the results shown here, may stem from a lack of protection by native cellular proteins. However, considering that onconase selectively degrades tRNA, the priming of RT may be inhibited by onconase. Therefore, we examined the tRNA levels in HIV-1-infected cells treated with onconase.

Polyacrylamide gel analysis of the total cellular RNA isolated from H9 cells uninfected and persistently infected with HIV-1 incubated with a noncytotoxic concentration of onconase (5 × 10⁸ M) showed degradation of tRNA only after 4 days (Fig. 5A, lane 6). It is hard to see any appreciable difference in total tRNA levels on the 1st, 2nd, and 3rd day of incubation. Examining certain individual tRNAs yielded a distinctly different result. Probing specifically for tRNA (Fig. 5B) by Northern blot analysis of the same gel with a tRNA specific ³²P-5'-end-labeled DNA probe revealed that levels of tRNA in the H9 cells uninfected and persistently infected with the HIV-1 increased within 1 day of treatment with onconase (Fig. 5B). The increased level of total tRNA continued through days 2 and 3 and fell back to control levels at day 4. Thus, as total tRNA levels stayed constant, there appeared to be a mixture of tRNA degradation and stimulated tRNA synthesis to maintain constant total levels. A tRNA precursor band is also seen running above the tRNA band at days 2 and 3. In addition, degraded forms of tRNA running at lower molecular weights can be seen as early as day 1 after onconase treatment, consistent with the model of onconase-induced degradation and accelerated resynthesis of tRNA.

View larger version (64K): [in this window] [in a new window]   Fig. 5.   Increased turnover of tRNA by onconase. A, uninfected and HIV-1 persistently infected H9 cells were grown in RPMI 1640 medium containing 10% fetal calf serum and 50 µg/ml gentamycin. 50 ml cultures of HIV-1, persistently infected H9 cells (2 × 105 cells/ml) in 75-cm2 flasks, were incubated with a noncytotoxic concentration of onconase (5 × 108 M) for 4 days. Each day, one flask was processed for isolation of total RNA using RNAzol as described under "Experimental Procedures." Total RNA was analyzed on 10% polyacrylamide gel containing 7.5 M urea after staining with ethidium bromide. Xylene cyanol dye front represents the migration position of ~50-55 nucleotides. B, Northern blot analysis of gel as in A showing the effect of onconase on tRNA species in HIV-1 persistently infected H9 cells. Urea-PAGE as shown in A was electroblotted onto Nytran Plus membranes. Following pre-hybridization, hybridization was carried out for 20 h at 42 °C using the tRNA-specific 32P-5'-end labeled DNA probe (18-mer). The blot was washed twice in 2× SSC (0.3 M sodium chloride, 0.03 M sodium citrate), 1% SDS at room temperature for 15 min each and once in 0.2× SSC, 1% SDS at 65 °C for 30 min followed by autoradiography.

The tRNAs range from 74 to 96 nucleotides in length and have a cloverleaf-like secondary structure. The tRNAs comprise ~15% total cellular RNA. Lysine-tRNA has three isoaccepting forms, and all three forms are 76 nucleotides long. The lysine isoacceptor tRNAs differ from the majority of the other tRNA species in containing 2'-O-methylribosylthymine in place of ribosylthymine in loop IV. The tRNA species differs from tRNA species by one base pair in the center of the anticodon stem. The anticodon sequence CUU is followed by N-threonyladenosine (18). The tRNA has the anticodon SUU and contains two highly modified thionucleosides where S is 2-thio-5-carboxymethyl-uridine methyl ester, and a further modified derivative of N-threonyladenosine (2-methylthio-N⁶-threonyladenosine) is on the 3' side of the anticodon. The tRNA differs in positions 14 and 16, respectively, from the other two isoacceptors (18). The human tRNA^Phe is also 76 nucleotides long and was tested and compared with tRNA and tRNA for sensitivity to onconase ribonuclease activity.

Probing the total RNA isolated from uninfected H9 cells treated with onconase for 24 h with tRNA, tRNA, and tRNA^Phe-specific DNA probes showed that onconase does degrade all three isoacceptor forms of tRNA^Lys as well as tRNA^Phe. The different degradation product patterns of the tRNA^Lys isoacceptors and the tRNA^Phe suggest that onconase cleaves these tRNA species at different sites (Fig. 6). Because all these species have the same number of nucleotides, onconase may recognize a target sequence located at a different position in the tRNA^Lys and the tRNA^Phe species, or onconase catalyzes cleavage of phosphodiester bond(s) at a different nucleotide sequence in these two species. However, the tRNAs may be cleaved at the same site, and differences in secondary structure may contribute to differences in gel mobility.

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Onconase induced tRNAs degradation in H9 cells. Uninfected H9 cells were grown in RPMI 1640 medium containing 10% fetal calf serum and 50 µg/ml gentamycin. 50-ml cultures of uninfected H9 cells (2 × 105 cells/ml) in 75-cm2 flasks were incubated with a noncytotoxic concentration of onconase (5 × 108 M) for 1 day followed by total RNA isolation using RNAzol. Total RNA was analyzed on 10% polyacrylamide gels containing 7.5 M urea followed by electroblotting onto Nytran Plus membranes. Following pre-hybridization, hybridization was carried out for 20 h at 42 °C using the specific 32P-5'-end-labeled DNA probes for tRNA, tRNA, and tRNAPhe. The blot was washed twice in 2× SSC, 1% SDS at room temperature for 15 min each and once in 0.2× SSC, 1% SDS at 65 °C for 30 min followed by autoradiography.

tRNA synthesis is increased upon onconase treatment of cells, and in some cases the level of individual tRNAs may decrease, increase, or stay the same. At high onconase levels, the overall level of tRNA drops, and cells eventually die.

	ACKNOWLEDGEMENTS

We thank Pat Johnson, Joan Barrick, Karen Sanders, and Dr. Motoshi Suzuki for technical assistance in this work.

	FOOTNOTES

^* 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. E-mail: youle@ helix.nih.gov.

Published, JBC Papers in Press, February 11, 2002, DOI 10.1074/jbc.M108115200

	ABBREVIATIONS

The abbreviation used is: TBE, Tris borate EDTA.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				D. M. Thompson, C. Lu, P. J. Green, and R. Parker tRNA cleavage is a conserved response to oxidative stress in eukaryotes RNA, October 1, 2008; 14(10): 2095 - 2103. [Abstract] [Full Text] [PDF]

				R. F. Gahl, M. Narayan, G. Xu, and H. A. Scheraga Dissimilarity in the oxidative folding of onconase and ribonuclease A, two structural homologues Protein Eng. Des. Sel., April 1, 2008; 21(4): 223 - 231. [Abstract] [Full Text] [PDF]

				R. Stein, M. J. Mattes, T. M. Cardillo, H. J. Hansen, C.-H. Chang, J. Burton, S. Govindan, and D. M. Goldenberg CD74: A New Candidate Target for the Immunotherapy of B-Cell Neoplasms Clin. Cancer Res., September 15, 2007; 13(18): 5556s - 5563s. [Abstract] [Full Text] [PDF]

				M. Rodriguez, G. Torrent, M. Bosch, F. Rayne, J.-F. Dubremetz, M. Ribo, A. Benito, M. Vilanova, and B. Beaumelle Intracellular pathway of Onconase that enables its delivery to the cytosol J. Cell Sci., April 15, 2007; 120(8): 1405 - 1411. [Abstract] [Full Text] [PDF]

				A. N. Suhasini and R. Sirdeshmukh Transfer RNA Cleavages by Onconase Reveal Unusual Cleavage Sites J. Biol. Chem., May 5, 2006; 281(18): 12201 - 12209. [Abstract] [Full Text] [PDF]

				C.-H. Chang, P. Sapra, S. S. Vanama, H. J. Hansen, I. D. Horak, and D. M. Goldenberg Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin Blood, December 15, 2005; 106(13): 4308 - 4314. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/17/15142    most recent M108115200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saxena, S. K. Articles by Youle, R. J. Search for Related Content PubMed PubMed Citation Articles by Saxena, S. K. Articles by Youle, R. J. Social Bookmarking                      What's this?