Ribonuclease Activity of Rat Liver Perchloric Acid-soluble Protein, a Potent Inhibitor of Protein Synthesis

Abstract

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 μm led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [α-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3′-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5′- or 3′-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule. Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 μm led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [α-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3′-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5′- or 3′-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule. Rat liver perchloric acid-soluble protein (L-PSP) 1The abbreviations L-PSPrat liver perchloric acid-soluble proteinPSPperchloric acid-soluble protein is a 136-amino acid protein that inhibits protein synthesis (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Oka et al. (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) demonstrated that L-PSP, when added to a rabbit reticulocyte cell-free system, causes inhibition of a biphasic kinetic nature and also leads to the disaggregation of polysomes. This would be similar to the mode of inhibition of translation by the heme-regulated eukaryotic initiation factor 2α kinase (2Chen J.J. Crosby J.S. London I.M. Biochimie (Paris). 1994; 76: 761-769Crossref PubMed Scopus (23) Google Scholar) (however, see our results below). Based on these data, it was suggested that the protein inhibits the initiation step rather than the elongation step (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). rat liver perchloric acid-soluble protein perchloric acid-soluble protein A 14-kDa translational inhibitor protein remarkably similar to L-PSP has been characterized in human monocytes and mouse liver (3Schmiedeknecht G. Kerkhoff C. Orso E. Stohr J. Aslanidis C. Nagy G.M. Knuechel R. Schmitz G. Eur. J. Biochem. 1996; 242: 339-351Crossref PubMed Scopus (85) Google Scholar, 4Samuel S.J. Tzung S.-P. Cohen S.A. Hepatology. 1997; 25: 1213-1222Crossref PubMed Scopus (48) Google Scholar, 5Ceciliani F. Faotto L. Negri A. Colombo I. Berra B. Bartorelli A. Ronchi S. FEBS Lett. 1996; 393: 147-150Crossref PubMed Scopus (60) Google Scholar). A homology search revealed that these proteins belong to a new group of small proteins named the YER057c/YJGF family (3Schmiedeknecht G. Kerkhoff C. Orso E. Stohr J. Aslanidis C. Nagy G.M. Knuechel R. Schmitz G. Eur. J. Biochem. 1996; 242: 339-351Crossref PubMed Scopus (85) Google Scholar), which is of unknown physiological function. The protein sequences of these family members are highly conserved in prokaryotes (including cyanobacteria), fungi, and eukaryotes, suggesting that the proteins may be involved in a basic cellular process. Indeed, mRNA of the translational inhibitor p14.5, the human homologue of L-PSP, becomes significantly up-regulated with the induction of differentiation to macrophages (3Schmiedeknecht G. Kerkhoff C. Orso E. Stohr J. Aslanidis C. Nagy G.M. Knuechel R. Schmitz G. Eur. J. Biochem. 1996; 242: 339-351Crossref PubMed Scopus (85) Google Scholar), and the synthesis of PSP from rat kidney increases from the 17th fetal day to the fourth postnatal week and then enters a steady-state level (6Asagi A. Oka T. Arao K. Suzuki I. Thakur M.K. Izumi K. Natori Y. Nephron. 1998; 79: 80-90Crossref PubMed Scopus (29) Google Scholar). In contrast, the expression of PSP from rat kidney in renal tumor cells was down-regulated (6Asagi A. Oka T. Arao K. Suzuki I. Thakur M.K. Izumi K. Natori Y. Nephron. 1998; 79: 80-90Crossref PubMed Scopus (29) Google Scholar). Recently, Schmiedeknecht et al. (7Schmiedeknecht G. Buchler C. Schmitz G. Biochem. Biophys. Res. Commun. 1997; 241: 59-67Crossref PubMed Scopus (20) Google Scholar) have identified the functional promoter of the human p14.5 translational inhibitor. They reported a head-to-head orientation of p14.5 with the gene for the protein subunit hPOP1 of RNase P and with RNase MRP ribonucleoproteins; the promoter region between p14.5 and hPOP1 acts as a bidirectional promoter. Because bidirectional transcription units commonly encode proteins that are different in structure but have similar biological function (8Wright K.L. White L.C. Kelly A. Beck S. Trowsdale J. Ting J.P.-Y. J. Exp. Med. 1995; 181: 1459-1472Crossref PubMed Scopus (205) Google Scholar, 9Chen S. Nagy P.L. Zalkin H. Nucleic Acids Res. 1997; 25: 1809-1816Crossref PubMed Scopus (23) Google Scholar), the authors suggested that the p14.5-hPOP1 cluster may encode functionally related proteins as well. As the first step in our endeavor to understand the physiological role of the PSP proteins, we studied the mechanism of action of the translational inhibitor L-PSP. The following procedures were either described or cited previously (10Hase M. Endo Y. Natori Y. Biochim. Biophys. Acta. 1982; 698: 102-104Crossref PubMed Scopus (7) Google Scholar, 11Endo Y. Wool I.G. J. Biol. Chem. 1982; 257: 9054-9060Abstract Full Text PDF PubMed Google Scholar, 12Endo Y. Huber P.W. Wool I.G. J. Biol. Chem. 1983; 258: 2662-2667Abstract Full Text PDF PubMed Google Scholar): preparation of rabbit reticulocyte lysate, sucrose density gradient analysis of polysomes, preparation of ribosomes, extraction of RNA with phenol and SDS, preparation of plasmid pBR322, analysis of the nucleic acids by polyacrylamide gel electrophoresis, separation of nucleotides by thin-layer chromatography, the methods used for the sequencing of 5 S rRNA, and the source of materials such asl-[U-14C]leucine, human placental ribonuclease inhibitor (133 units/ml), [α-32P]UTP, and SP6 RNA polymerase. Both forms of L-PSP were purified as described previously (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 13Oka T. Nishimoto Y. Sasagawa T. Kanouchi H. Kawasaki Y. Natori Y. Cell. Mol. Life Sci. 1999; 55: 131-134Crossref PubMed Scopus (8) Google Scholar). Briefly, the 5% perchloric acid-soluble fraction of a rat liver lysate was loaded onto CM-Sephadex C-25, and pure L-PSP was recovered in the flow-through fraction. Recombinant PSP was expressed inEscherichia coli as a fusion protein with glutathioneS-transferase using plasmid pGEX-PSP. The fusion protein was purified from the extract with glutathione-Sepharose 4B and then incubated with thrombin, and recombinant L-PSP was isolated using the same affinity column as described above. The recombinant protein was confirmed to be L-PSP by its mobility on SDS-polyacrylamide gels and by direct sequencing of the eight N-terminal amino acids. Furthermore, the recombinant protein and authentic L-PSP were immunologically identical. The standard cell-free protein synthesis system derived from rabbit reticulocyte lysate (10Hase M. Endo Y. Natori Y. Biochim. Biophys. Acta. 1982; 698: 102-104Crossref PubMed Scopus (7) Google Scholar) contained the following in a final volume of 30 μl: 15 μl of micrococcal nuclease-treated lysate (containing 25 μmhemin), 25 mm Hepes (pH 7.6), 2 mmdithiothreitol, 1 mm ATP, 20 μm GTP, 8 mm creatine phosphate, 1.2 μg of creatinine phosphokinase, 25 μm of each of the 20 amino acids, 0.1 μCi of l-[U-14C]leucine (13.3 mCi/mmol), 90 mm potassium acetate, 1 mm magnesium acetate, 0.6 mm spermidine, and 2 μg of mRNA. Endogenous globin synthesis was done under the same conditions as described above using lysate that was not treated with micrococcal nuclease. mRNA having the cap structure was synthesized with [α-32P]UTP and the four nucleotide triphosphates using phage SP6 RNA polymerase and linearized plasmid pSP65 containing the gene coding for E. coli form 1 dihydrofolate reductase as template (14Endo Y. Otsuzuki S. Ito K. Miura K. J. Biotechnol. 1992; 25: 221-230Crossref PubMed Scopus (64) Google Scholar). The transcript is 1079 nucleotides long and consists of a coding region of 477 nucleotides close to the 5′ end and a 3′ noncoding region of 565 nucleotides. L-PSP inhibits protein synthesis at the elongation step rather than the initiation step. We started with experiments to confirm that L-PSP inhibits protein synthesis with a similar mode of action as heme-regulated eukaryotic initiation factor 2α kinase, as reported previously (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Incubation of micrococcal nuclease-treated rabbit reticulocyte lysate containing exogenous capped mRNA encoding dihydrofolate reductase and all other components necessary for protein synthesis at 30 °C led to the incorporation of [14C]leucine into newly synthesized proteins. In agreement with the previous report (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), the addition of L-PSP to the system resulted in inhibition of protein synthesis in a concentration-dependent manner (Fig.1 A). The estimated IC50 (51 nm) was in the same range as the previously reported value (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). A L-PSP concentration of 1 μm almost completely abolished the capacity of the cell-free system to support protein synthesis. However, when similar experiments were done using endogenous globin mRNA, inhibition by L-PSP appeared to be about 10 times less efficient (IC50, 0.68 μm) (Fig. 1 B). The observed difference in the IC50 can be ascribed to the character of the two translation systems: in the latter system, a large population of ribosomes is already engaged with globin mRNA (as polysomes) at the start of incubation, whereas practically no mRNA is associated with ribosomes in the former system. Thus, the results suggested that the lysate containing a higher proportion of polysomes was less susceptible to L-PSP. An interesting and probably important observation here is that the kinetics of the inhibition is monophasic in both systems, rather than biphasic, as reported previously (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In fact, careful examination of the earlier data does not reveal an inhibition lag, and the discrepancy may stem from an incorrect explanation of the kinetic data. Inhibition of translation initiation by proteins such as heme-regulated eukaryotic initiation factor 2α kinase shows biphasic kinetics (2Chen J.J. Crosby J.S. London I.M. Biochimie (Paris). 1994; 76: 761-769Crossref PubMed Scopus (23) Google Scholar). However, this typical biphasic shape, in which protein synthesis proceeds at the initial rate for several minutes before an abrupt decline occurs, can only be expected when the globin-synthesizing system is used because only this system has an initial rate (run-off of polysomes). Obviously, inhibitors of initiation show monophasic kinetics of translation inhibition in the exogenous mRNA programmed system, which has no initial rate because there is no initial protein synthesis. The inhibition of protein synthesis by L-PSP was not prevented by the ribonuclease inhibitor from human placenta that tightly binds and inhibits the activity of the ribonuclease A family (data not shown) (15Blackburn P. Wilson G. Moore S. J. Biol. Chem. 1977; 252: 5904-5910Abstract Full Text PDF PubMed Google Scholar). In experiments that used higher amounts of mRNA than the standard reaction, the inhibitory effect on protein synthesis tended to be less. For instance, when mRNA was 20 μg instead of 2 μg in a 30 μl system, 20 nm L-PSP did not show significant inhibition (data not shown). To gain more insight into the mode of action of this small protein, we examined the effect of preincubation of mRNA with L-PSP. For these experiments, mRNA was first incubated with a low amount of L-PSP for a prolonged time, and then the activity of the mRNA of a small portion of the preincubation reaction mixture was tested in the standard translation system. This strategy minimizes the effect of L-PSP on the translation system because the concentration of L-PSP in the translation reaction mixture is very low. As shown in Fig.1 C, preincubation of mRNA with L-PSP results in a significant decrease of protein synthesis. Recombinant L-PSP affected the template quality of mRNA in a similar manner, making it unlikely that the observed inactivation of mRNA was due to contaminants in the L-PSP preparation. However, the activity on mRNA of authentic L-PSP differed significantly from that of recombinant PSP, as had been found previously (13Oka T. Nishimoto Y. Sasagawa T. Kanouchi H. Kawasaki Y. Natori Y. Cell. Mol. Life Sci. 1999; 55: 131-134Crossref PubMed Scopus (8) Google Scholar). Although we cannot be certain, the difference is likely to be due to a loss of activity because of the expression of recombinant PSP in a prokaryotic system. A similar but even larger discrepancy has been reported for the human homologue of PSP, p14.5: the authentic form is more active than the recombinant form by 3 orders of magnitude (3Schmiedeknecht G. Kerkhoff C. Orso E. Stohr J. Aslanidis C. Nagy G.M. Knuechel R. Schmitz G. Eur. J. Biochem. 1996; 242: 339-351Crossref PubMed Scopus (85) Google Scholar). Nevertheless, the results indicate that the main target of the protein is mRNA rather than the translation system. There are at least two alternative mechanisms for mRNA inactivation: 1) L-PSP might specifically modify mRNA at its 5′-untranslated region, including the cap structure (m7GpppG) that is nearly essential for the initiation reaction, or 2) the protein might degrade mRNA nucleolytically. To test the alternative possibilities, we performed experiments using sucrose density gradient centrifugation to investigate polysome profiles in the presence of cycloheximide. Incubation of the globin synthesizing lysate system with L-PSP resulted in a disaggregation of polysomes into 80 S ribosomes (Fig.2 A), supporting the earlier result (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The addition of the ribonuclease inhibitor at the start of the incubation did not prevent polysome disaggregation. An important feature of this experiment was that both the incubation and the analysis were performed in the presence of a low concentration of cycloheximide at which the antibiotic freezes the elongation reaction but not initiation. Polysome disaggregation in the presence of L-PSP demonstrates that the protein does not affect the initiation process. Rather, the results strongly suggested that L-PSP disintegrates polysomes through fragmentation of polysomal mRNA because of ribonuclease activity. To obtain direct evidence, micrococcal nuclease-treated rabbit reticulocyte lysate was incubated as described above with 32P-labeled dihydrofolate reductase mRNA in the presence of both the ribonuclease inhibitor and L-PSP. After incubation for various periods of time (Fig. 2 B), RNA was extracted and separated on polyacrylamide gels. Consistent with our hypothesis, the radioautograph shows intensive digestion of mRNA into small fragments in the presence of L-PSP, whereas the mRNA was fairly stable in the control sample. A similar digestion pattern of the mRNA was observed when the incubation was carried out in the absence of the ribonuclease inhibitor (data not shown). The amount of the ribonuclease inhibitor (266 units) used in Fig. 2, A andB, inhibits the activity of 1.33 μg of ribonuclease A by 50%, whereas the amount of L-PSP was 0.89 μg in a 30-μl reaction mixture. The result excludes the possibility that the inhibition by L-PSP is due to a contamination with ribonuclease A or other A-like ribonuleases, RNases known to be abundant in both animal tissues andE. coli. All of these results show that L-PSP is a ribonuclease that hydrolyzes phosphodiester bonds of RNA in an endonucleolytic fashion. We next determined the substrate specificity of the enzyme, and the results are shown in Fig. 3 A. The [32P]mRNA was incubated with l-psp,and then the sample was separated by thin-layer chromatography. Subsequent autoradiography showed the production of the four 3′ nucleotide monophosphates as identified by with standard nucleotides from ribonuclease The results are L-PSP the phosphodiester bonds of all four and In addition to the four are also in A that may small under the digestion the same digestion pattern of the RNA was using recombinant L-PSP (data not shown). the of the we determined the activity of L-PSP for the various indicate the of The mechanism of probably Because there are that both RNA and Biochem. PubMed Scopus Google Scholar), we determined L-PSP has activity. We as the substrate for the test because a in the of the plasmid a in that its mobility in gels. incubation of the plasmid with L-PSP μm) under conditions similar to those used for the RNA substrate did not result in significant effect on the of the plasmid (data not shown). Thus, we that the protein does not have activity. We next determined L-PSP is for or regions of We rat liver 5 S rRNA as substrate for the test because 1) the structure is and 2) the RNA has a highly structure J. A. R. S. Nucleic Acids Res. PubMed Scopus Google Scholar). 5 S rRNA was at its 3′ end with or at its 5′ end with and the sample was treated under conditions with either authentic PSP from rat liver or recombinant An and were with the of the protein on a polyacrylamide sequencing gel (Fig. Both L-PSP cleaved only in single-stranded regions of the molecule. recombinant L-PSP was about four times less active than authentic L-PSP. The addition of the ribonuclease inhibitor to the reaction did not the digestion pattern (data not shown). The results that L-PSP is a ribonuclease with unique liver with unique are the and related proteins Y. Huber P.W. Wool I.G. J. Biol. Chem. 1983; 258: 2662-2667Abstract Full Text PDF PubMed Google Scholar), and related T. J. M. Sci. S. A. PubMed Scopus Google Scholar), and which is important for Sci. S. A. PubMed Scopus Google Scholar). is a basic protein by the The of the action of the protein is an inhibition of protein synthesis by the inactivation of Although there are nucleotides in ribosomes, hydrolyzes only a phosphodiester between and of S This the and is responsible for the of the protein Y. Wool I.G. J. Biol. Chem. 1982; 257: 9054-9060Abstract Full Text PDF PubMed Google Scholar, 12Endo Y. Huber P.W. Wool I.G. J. Biol. Chem. 1983; 258: 2662-2667Abstract Full Text PDF PubMed Google Scholar, I.G. A. Endo Y. Biochem. Sci. 1992; Full Text PDF PubMed Scopus Google Scholar). that L-PSP acts on mRNA, we examined the protein other RNA A micrococcal cell-free system was incubated with L-PSP as described for Fig. 2 and RNA was were separated on and polyacrylamide and RNA were with (data not shown). a careful examination of the we did not significant of rRNA at μm of the authentic protein has an IC50 of 0.68 However, when similar experiments were done with higher μm) of the protein, significant fragmentation of rRNA be which the that L-PSP is a These results the possibility that L-PSP inhibits protein synthesis by rRNA or We that L-PSP inhibits cell-free protein synthesis by mRNA. These results may have an important on the physiological role of L-PSP and its related proteins. it may be to here that the of significant homology with other L-PSP also (1Oka T. Tsuji H. Noda C. Sakai K. Hong Y. Suzuki I. Munoz S. Natori Y. J. Biol. Chem. 1995; 270: 30060-30067Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). is known to be the acid in the activity of other R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, I. R. L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Recently, ribonuclease that has been reported T. K. T. K. T. H. 1999; PubMed Scopus Google Scholar): the of the inhibits protein synthesis in by several at a the 3′ of the nucleotide in the The the same even is the substrate, in which 5′ of the nucleotide that is at the The mechanism of a but the mechanism is A homology search between L-PSP and the revealed a by both acids in L-PSP and of the acid in but the functional of these to be