A phosphodiesterase characterized by a generally higher activity on 2'-5' than on 3'-5' phosphodiester bonds was isolated from mouse L cells treated with interferon. A similar enzyme was purified from mouse reticulocytes. The phosphodiesterase 2'-PDi splits the 2'-phosphate bond of pppA2'p5'A2'p5'A, the oligonucleotide activator of ribonuclease F. The level of phosphodiesterase 2'-PDi is increased by interferon treatment of L cells. The phosphodiesterase was also shown to degrade the C-C-A terminus of tRNA and to reduce the amino acid acceptance of tRNA in cell-free extracts, thereby causing a tRNA-reversible inhibition of mRNA translation.

FEBS LettersVolume 95, Issue 2 p. 257-264 Full-length articleFree Access Interferon action: Isolation of Nuclease F, A translation inhibitor activated by interferon-induced (2′–5′) oligo-isoadenylate A. Schmidt, A. Schmidt Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorA. Zilberstein, A. Zilberstein Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorL. Shulman, L. Shulman Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorP. Federman, P. Federman Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorH. Berissi, H. Berissi Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorM. Revel, M. Revel Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this author A. Schmidt, A. Schmidt Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorA. Zilberstein, A. Zilberstein Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorL. Shulman, L. Shulman Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorP. Federman, P. Federman Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorH. Berissi, H. Berissi Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorM. Revel, M. Revel Department of Virology, Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this author First published: November 15, 1978 https://doi.org/10.1016/0014-5793(78)81006-0Citations: 136AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 D.H. Metz, Cell, 6, (1975), 429– 439. 2 E. Yakobson, C. Prives, J.R. Hartman, E. Winocour, M. Revel, Cell, 12, (1977), 73– 81. 3 M. Revel, E. Gilboa, A. Kimchi, A. Schmidt, L. Shulman, E. Yakobson, A. Zilberstein, B.F.C. Clark Proc. 11th FEBS Meet. Copenhagen 43, (1978), Pergamon Press Oxford 47– 58. 4 A. Zilberstein, P. Federman, L. Shulman, M. Revel, FEBS Lett., 68, (1976), 119– 124. 5 B. Lebleu, G.C. Sen, S. Shaila, B. Cabrer, P. Lengyel, Proc. Natl. Acad. Sci. USA, 73, (1976), 3107– 3111. 6 J. Cooper, P.J. Farrell, Biochem. Biophys. Res. Commun., 77, (1977), 124– 131. 7 W.K. Roberts, A. Hovanessian, R.E. Brown, M.J. Clemens, I.M. Kerr, Nature, 264, (1976), 477– 480. 8 I.M. Kerr, R.E. Brown, Proc. Natl. Acad. Sci. USA, 75, (1978), 256– 260. 9 A. Zilberstein, A. Kimchi, A. Schmidt, M. Revel, Proc. Natl. Acad. Sci. USA, (1978), in press 10 J. Content, B. Lebleu, U. Nudel, A. Zilberstein, H. Berissi, M. Revel, Eur. J. Biochem., 54, (1975), 1– 10. 11 G.C. Sen, S.L. Gupta, G.E. Brown, B. Lebleu, M.A. Rebello, P. Lengyel, J. Virol., 17, (1976), 191– 203. 12 R. Falcoff, E. Falcoff, J. Sanceau, J.A. Lewis, Virology, 86, (1978), 507– 515. 13 G.C. Sen, B. Lebleu, G.E. Brown, M. Kawakita, E. Slattery, P. Lengyel, Nature, 264, (1976), 370– 373. 14 C.E. Samuel, D.A. Farris, D.A. Eppstein, Virology, 83, (1977), 56– 71. 15 L. Ratner, R.C. Wiegand, P.J. Farrell, G.C. Sen, B. Cabrer, P. Lengyel, Biochem. Biophys. Res. Commun., 81, (1978), 947– 954. 16 M.J. Clemens, B.R.G. Williams, Cell, 13, (1978), 565– 572. 17 C. Baglioni, M.A. Minks, P.A. Maroney, Nature, 273, (1978), 684– 687. 18 G.M. Tener, Methods Enzymol., 12, (1967), 398– 404. 19 H. Soreq, U. Nudel, R. Salomon, M. Revel, U.Z. Littauer, J. Mol. Biol., 88, (1974), 233– 245. 20 W. Prensky, D.M. Steffensen, W.L. Hughes, Proc. Natl. Acad. Sci. USA, 70, (1973), 1860– 1864. 21 M. Revel, A. Kimchi, A. Schmidt, L. Shulman, A. Zilberstein, Proc. 12th FEBS Meet. Dresden (1978), Pergamon Press Oxford in press 22 A.G. Hovanessian, R.E. Brown, I.M. Kerr, Nature, 268, (1977), 537– 540. 23 M.D. White, S. Bauer, Y. Lapidot, Nuc. Acid. Res., 4, (1977), 3029– 3038. 24 M.J. Hewlett, S. Rozenblatt, V. Ambros, D. Baltimore, Biochemistry, 16, (1977), 2763– 2767. Citing Literature Volume95, Issue2November 15, 1978Pages 257-264 ReferencesRelatedInformation

FEBS LettersVolume 68, Issue 1 p. 119-124 Full-length articleFree Access Specific phosphorylation in vitro of a protein associated with ribosomes of interferon-treated mouse L cells Asher Zilberstein, Asher Zilberstein Department of Virology, The Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorPerla Federman, Perla Federman Department of Virology, The Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorLester Shulman, Lester Shulman Department of Virology, The Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorMichel Revel, Michel Revel Department of Virology, The Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this author Asher Zilberstein, Asher Zilberstein Department of Virology, The Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorPerla Federman, Perla Federman Department of Virology, The Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorLester Shulman, Lester Shulman Department of Virology, The Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this authorMichel Revel, Michel Revel Department of Virology, The Weizmann Institute of Science, Rehovot, IsraelSearch for more papers by this author First published: September 15, 1976 https://doi.org/10.1016/0014-5793(76)80418-8Citations: 197AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume68, Issue1September 15, 1976Pages 119-124 ReferencesRelatedInformation

A phosphodiesterase characterized by a generally higher activity on 2'-5' than on 3'-5' phosphodiester bonds was isolated from mouse L cells treated with interferon. A similar enzyme was purified from mouse reticulocytes. The phosphodiesterase 2'-PDi splits the 2'-phosphate bond of pppA2'p5'A2'p5'A, the oligonucleotide activator of ribonuclease F. The level of phosphodiesterase 2'-PDi is increased by interferon treatment of L cells. The phosphodiesterase was also shown to degrade the C-C-A terminus of tRNA and to reduce the amino acid acceptance of tRNA in cell-free extracts, thereby causing a tRNA-reversible inhibition of mRNA translation. In extracts of interferon-treated cells, translation of mRNA into proteins is inhibited by several enzymatic pathways (1-3). One pathway is mediated by the double-stranded RNA (dsRNA)/ ATP-activated protein kinase PK-i, induced by interferon in the cells (1, 3), which phosphorylates eukaryotic initiation factor 2 and decreases Met-tRNAf et binding to 40S ribosomes (4). A second translational-inhibitory pathway is mediated through synthesis of (2'-5')pppApApA (5) by the dsRNA/ATP-dependent oligoisoadenylate synthetase E, another interferon-induced enzyme (1, 3, 6, 7). The oligonucleotide enhances mRNA degradation in cell extracts (8, 9), and its synthesis gives rise to the apparent dsRNA/ATP-dependent endonucleolytic activity of interferon-treated cell extracts (3, 10-12). This function of the oligonucleotide was demonstrated by the isolation of RNase F, whose nucleolytic activity is completely dependent on the continued presence of (2'-5')pppApApA, from both interferon-treated and untreated cells (13). The third pathway of translation inhibition does not require dsRNA, affects polypeptide chain elongation, and is reversed by the addition of tRNA (14-20). The mechanism that brings about this tRNA deficiency in interferon-treated cell extracts has not been elucidated. In the study of the oligoisoadenylate-dependent RNase F, we found (13) a phosphodiesterase that cleaves the (2'-5')pppApApA oligonucleotide into ATP and 5'-AMP, and inactivates RNase F. Degradation of (2'-5')pppApApA in crude cell extracts was also observed by others (21, 22). We report here the purification of this phosphodiesterase from interferontreated L cells and mouse reticulocytes. The enzyme is a 40,000 Mr protein with a generally higher affinity for 2'-5' dinucleotides than for their 3'-5' isomers. The phosphodiesterase, however, also degrades the C-C-A terminus of tRNA, thereby causing a tRNA-reversible inhibition of protein synthesis in the cell-free systems. We find a 4to 5-fold increase in this 2'phosphodiesterase activity in L cells after interferon treatment. This increased activity could explain the tRNA deficiency and elongation block observed in extracts of interferon-treated cells. In view of its unusual substrate specificity and enhancement by interferon, we call this phosphodiesterase 2'-PDi. MATERIALS AND METHODS Assay and Purification of 2'-PDi. Assays are described in the legend of Fig. 1. Routinely, degradation of (2'-5')ApA was measured in 15mM Hepes buffer, pH 7.5/90mM KC1/3.5mM MgCl2/0.7 mM dithiothreitol/7% (vol/vol) glycerol. After chromatography on polyethyleneimine (PEI)-cellulose, the nucleotides were eluted with 0.7 M MgCl2/20 mM Tris-HCI, pH 7.5, and absorbance at 260 nm was measured. Purification of 2'-PDi from mouse L cells treated 24 hr with 200 units of interferon per ml (1) is outlined in Table 1. Steps I-III were as for RNase F (13). At step IV, 2'-PDi eluted from hydroxylapatite at 50mM K phosphate, whereas RNase F eluted at higher phosphate (13). For mouse reticulocytes (23), the 100,000 X g supernatant (100 ml) was loaded on 300 ml of DEAE-cellulose in 20mM Hepes buffer, pH 7.5/25 mM KC1/5 mM MgCl2/1 mM dithiothreitol/10% (vol/vol) glycerol (buffer A). Proteins eluted by 150 mM KCl in buffer A (step IIa, 300 mg, 50 ml) were applied to a DEAE-cellulose column (1.6 X 15 cm) and eluted with a 200-ml 25-150 mM KCI gradient in buffer A. Fractions eluting at 60-80 mM KCl (step Ilb, 150 mg, 60 ml) were loaded on phosphocellulose (1.2 X 14 cm) in buffer A but at pH 6.7 and with no Mg2+, and were eluted with a 160-ml 25-800mM KCl gradient. Fractions around 280mM KCl (step III,60 mg, 40 ml) were applied to hydroxylapatite (1.6 X 10 cm) in 25 mM K phosphate buffer, pH 7.2/120 mM KCl/1 mM dithiothreitol. The 2'-PDi was eluted stepwise with 100 mM phosphate (step IV), while RNase F eluted in the 150 mM phosphate step. The 2'-PDi (step IV, 2.5 mg, 13 ml) was applied to a blue dextran-Sepharose (24) column (1 X 3.2 cm) and eluted by an 18-ml 120-700 mM KCI gradient in buffer A. Activity eluted around 200mM KCI (step V, 0.4 mg) and 0.24 mg in 0.4 ml was filtered through Bio-Gel P-100 (0.7 X 60 cm) in buffer A with 120 mM KCI (step VI, Fig. 2A). Effect of 2'-PDi on tRNA Activity. Aminoacyl-tRNA synthetases were prepared from rabbit reticulocytes by pH 5 precipitation and DEAE-cellulose chromatography (16, 25). Rat liver tRNA nucleotidyltransferase was partially purified as in ref. 26. Rabbit liver tRNA, 1.2 ,ug, was preincubated 10 min at 30'C, in 10 Al of 15 mM Hepes buffer, pH 7.5/3.5 mM MgCl2/90 mM KCI/0.7 mM dithiothreitol/7% (vol/vol) glycerol with the indicated amounts of 2'-PDi. After heating 10 min at 60°C, the reaction mixture was adjusted to 50,ul of 30mM Hepes buffer, pH 7.5/1 mM MgCl2/80mM KCI/2mM dithiothreitol/0.01 mM EDTA/3% (vol/vol) glycerol/i mM ATP/0.25 mM GTP/0.25 mM CTP (when indicated)/5 mM creatine phosphate/80,ug of creatine kinase per ml/60 AM each of 19 amino acids/4,M [3H]leucine (50 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels)/90,ug of aminoacyl-tRNA synthetase/0.01 unit of tRNA nucleotidyltransferase (when indicated). IncuAbbreviations: dsRNA, double-stranded RNA; 2'-PDi, interferoninduced 2'-phosphodiesterase; PEI-cellulose, polyethyleneimine-cellulose. 4788 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 76 (1979) 4789