Mammalian Cell‐Free Protein Synthesis Directed by Viral Ribonucleic Acid

Abstract

1.A cell-free system from mouse Krebs I1 ascites cells is described which responds by increased amino acid incorporation into protein on addition of encephalomyocarditis virus (EMC-RNA). RNA from other sources does not produce this response.2 . The stimulation by EMC-RNA occurs over a narrow range of Mg2f concentrations and is maximal a t 5mM, which is optimal for the endogenous incorporation and lower than that required in the presence of poly(U).3. The EMC-RNA-directed product is distinguished from the endogenous products by its size and composition.4. After an initial lag of 4 min, during which the nascent chains are too short to be acidinsoluble, EMC-RNA-directed protein synthesis is active for more than 1 h.5. EMC-RNA-directed synthesis is particularly sensitive to the inhibitors cycloheximide and dextran sulphate. Using the latter, it has been shown that the initiation of protein synthesis is completed during the first 15 min of incubation.6. Only 20-3001, of the product of the cell-free system, whether endogenous or EMC-RNAdirected, is released from the ribosomes. Despite the size of the viral RNA, it did not appear to promote the formation of exceptionally large polysomes.7. It is concluded that the EMC-RNA is acting as a message for viral proteins in the ascites cell-free system. The demonstration that bacteriophage RNA can act as a messenger RNA in cell-free preparations from Escherichia coli [l] has greatly facilitated the study of several aspects of protein biospthesis in bacteria. We wished to extend this approach to mammalian systems using a homologous viral RNA. It is known that the RNA extracted from encephalomyocarditis virus (EMC) [2,3] and from several other mammalian viruses [4-61 is capable of stimulating amino acid incorporation when added to extracts of certain mammalian or avian cells and we have used the EMClKrebs I1 ascites cell system in this work. We report here the development of a highly sensitive cell-free preparation from mouse Krebs I1 ascites cells and describe some of the characteristics of the stimulation of protein synthesis elicited by encephalomyocarditis virus RNA (EMC-RNA). The fidelity of this system has recently been established by the demonstration that the product formed Unusual Abbreviations. EMC, encephalomyocarditis; Guo-5'-P2-CH,-P, 5'-guanylyl-methylenediphosphate; poly-(U), polyuridylic acid; S-30, the 30000 x g supernatant.Enzymes. Creatine phosphokinase (EC 2.7.3.2); pancreatic ribonuclease (EC 2.7.7.16).under the direction of EMC-RNA corresponds to material present in EMC-infected cells [7].

Double-Stranded RNA as an Inhibitor of Protein Synthesis and as a Substrate for a Nuclease in Extracts of Krebs II Ascites Cells

“…Procedures for handling Krebs II ascites cells and S-30 extracts for protein synthesis have been described (15,16 The effect of added dsRNA on the rate of EMC RNA translation by the ascites cell-free system is shown in Fig. la.…”

“…An extract was separated into ribosomal and S-150 fractions, as described in Methods, and activity against equal amounts of phage fi dsRNA or ssRNA of identical specific activity was measured. In the S-30 fraction, 72% of the dsRNA or 35% of the ssRNA was solubilized in 24 The dsRNA substrate, poly(G C), is described in Methods Ascites cells were washed by repeated sedimentation and suspension in medium RS (5.2 mM KCl -130 mM NaCl-7.4 mM MgC12) and were homogenized and fractionated as described (15 results suggest that a nuclease with preference for dsRNA is located in the cytoplasm of ascites tumor cells, perhaps in association with ribosomes.…”

“…Another cell-free extract that can translate various mammalian mRNAs faithfully is that prepared from Krebs II ascites cells - (14). This system has the advantage that it can translate efficiently exogenous RNA of both viral and cellular origin (15). We report here the effect of dsRNA on the translation of encephalomyocarditis (EMC) viral RNA and mouse globin mRNA in Krebs cell extracts.…”

Cell-free Synthesis of Adenovirus 2 Proteins Programmed by Fractionated Messenger RNA: A Comparison of Polypeptide Products and Messenger RNA Lengths

“…Postmitochondrial supernatant from these cells was prepared by the method of Mathews and Korner (11) (10) except that the incubation for polysome run-off was done directly with the postmitochondrial supernatant fraction (final A260 of 60 in the reaction mixture). The separated subunits were pooled (60 S to 40 S in an A260 ratio of 2.5:1), sedimented, resuspended at 100 A260 units/ml, and stored at -70°in 0.1-ml aliquots (without pH 5 enzyme).…”

Anti-threonyl-tRNA synthetase, a second myositis-related autoantibody.

“…An extract containing aminoacyl-tRNA synthetases was prepared using a modification of the method described previously (16). HeLa cells grown in suspension culture were harvested, washed twice in cold PBS, suspended in 2.5 vol of buffer E (10 mM Tris-HC1, pH 7.4, 1.5 mM MgCI~, 15 mM KCI, 0.5 mM DTT), and allowed to swell for 10 min on ice.…”