Ribonucleotide sequences non-adjacent to poly(A) participate in the poly(A)-protein complex in 15S duck globin mRNP particles.

The study of the interaction between mRNA and proteins in the polyribosomal 15 S duck globin messenger ribonucleoprotein complex showed that proteins protect specific mRNA sequences against digestion by the nonspecific micrococcal nuclease (Nucleic Acids Research 6 (8) 2787, 1979). Here we report the isolation of the poly(A)-protein RNP complex from nuclease digested 15 S mRNP by two different methods: sucrose gradient sedimentation and oligo(dT)-cellulose chromatography. We show by fingerprint analysis, that aprt from the periodically fragmented poly(A) segment, mRNA sequences adjacent and non-adjacent to the poly(A) segment are protected by the poly(A) binding proteins against nuclease digestion. The duck globin poly(A)-protein RNP complex, with a sedimentation coefficient between 7 S and 10 S, shows a characteristic protein composition, with a major 73,000 MW polypeptide and some minor components. The results are discussed in view of a dynamic ribonucleoprotein structure.     

A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro

RNAs containing the polyadenylation sites for adenovirus L3 or E2a mRNA or for SV40 early or late mRNA are substrates for cleavage and poly(A) addition in an extract of HeLa cell nuclei. When polyadenylation reactions are probed with ribonuclease T1 and antibodies directed against either the Sm protein determinant or the trimethylguanosine cap structure at the 5' end of U RNAs in small nuclear ribonucleoproteins, RNA fragments containing the AAUAAA polyadenylation signal are immunoprecipitated. The RNA cleavage step that occurs prior to poly(A) addition is inhibited by micrococcal nuclease digestion of the nuclear extract. The immunoprecipitation of fragments containing the AAUAAA sequence can be altered, but not always abolished, by pretreatment with micrococcal nuclease. We discuss the involvement of small nuclear ribonucleoproteins in the cleavage and poly(A) addition reactions that form the 3' ends of most eukaryotic mRNAs.     

Polysome disassembly in cell extracts of cricket male accessory gland during sucrose gradient centrifugation

The stability of polysomes in cell extracts of cricket (Acheta domesticus) male accessory gland has been examined by sedimentation through a variety of step and linear sucrose gradients. After prolonged centrifugation there is a considerable decline in polysome content with a concurrent increase in monosomes. The extent of the reduction is more severe in step gradients, although the polysomes that remain show a typical profile on linear gradients.Evidence is presented which indicates that the reduction in polysome content is not due to nuclease action. The presence of detergents can affect the extent of disassembly but is not the principal cause. Comparison of [³H]leucine pulse-labelled gluteraldehyde-fixed and unfixed polysomes subjected to extended centrifugation reveals a release of nascent label near the top of the gradients in unfixed preparations. At least part of this displaced material is present as peptidyl-tRNA, suggesting that forced dissociation of polysomes rather than premature termination of nascent chains occurs as a consequence of sedimentation pressures. Comparison of the distribution of polyadenylic acid (poly(A)) sequences in sucrose gradients following short- and long-term centrifugation shows a shift of poly(A) containing RNA out of the polysome and into the pre-monomer region. It is concluded that sucrose gradient sedimentation results in the disassembly of a portion of the polysome population in the tissue examined. The implications with regard to the study of nonpolysomal messenger ribonucleoprotein and monomeric ribosomes are discussed.     

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [³⁵S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA_f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [³H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.     

An investigation of the stability of messenger RNAs in cell-free,translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells

The stabilities and translation of Ehrlich ascites tumor cell poly(A)-containing mRNA and mengovirus RNA in fractionated cell-free protein synthesizing systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells were studied. During incubation of the systems about 20% of the input RNA is reduced in size and associated with ribosomes engaged in polypeptide synthesis; the remainder is rapidly degraded by RNases. At the end of active translation, both mRNA and nascent proteins are bound to polysomes which are of the same size as those formed during active protein synthesis. The kinetics of protein synthesis closely follow those of RNA hydrolysis. The stabilities of mengovirus RNA and poly(A)-containing mRNA from Ehrlich ascites tumor cells are the same in both systems.     

Translation and characterization of poly(A)-lacking RNA from lupin root nodules

Total polysomal RNA from yellow lupin root nodules was fractionated by double oligo(dT)-cellulose chromatography. Poly(A)-containing and poly(A)-lacking RNA fractions showed considerable messenger activity in wheat germ and rabbit reticulocyte cell-free systems. The sizing of poly(A)-lacking RNA on sucrose-density gradient gives rise to separation of 14S mRNA from 22-24S mRNA species. A single polypeptide with molecular weight of 22,000 was coded for by 14S mRNA, while two polypeptides with an apparent mol. wt. of 90,000 and 87,000 were the main products of 22-24S mRNA fraction. High concentrations of unfractionated poly(A)-lacking RNA as well as the addition of poly(A) led to preferential synthesis of the 22,000 product. Preliminary results suggest the presence of m7GpppX cap structure at 5' terminus of the separated 14S and 22-24S mRNA species. This comes from the competition experiments with m7GMP and m7GTP as well as from the fact that the poly(A)-lacking RNA preparation was susceptible to methylation by methyl-transferase from vaccinia virus (methylated is the 2'-O-nucleotide adjacent to 7-methylguanosine). Digestion by T1 RNAase of methylated poly(A)-lacking RNA produced two short 5'-terminal oligonucleotides 10 and 17 nucleotides in length.     

The Role of the poly(A) sequence in mammalian messenger RNA

The poly(A) sequence is added to 3' termini of nuclear RNA segments destined to become part of the mRNA, and may play an essential role in the selection of these segments. It appears to be required for at least some of the splicing events involved in mRNA processing. In the cytoplasm, the poly(A) segment is the target of a degradation process which causes its gradual shortening, and leads to a heterogeneous steady-state poly(A)-size distribution. Complete loss of the poly(A) is probably followed by inactivation of the mRNA, since chains depleted of poly(A) do not accumulate in the cells. A role for this sequence in the promotion of mRNA stability is suggested by the behavior of globin mRNA depleted of poly(A) after injection into frog oocytes. The poly(A) shortening process may be part of the mRNA inactivation mechanism, as indicated by the greater sensitivity to degradation of the poly(A) of some short-lived mRNAs. However, the stochastic mRNA decay implies that new and old mRNA chains, with long and short poly(A) segments, respectively are equally susceptible to inactivation. The poly(A)-lacking histone mRNAs are stable only in cells engaged in DNA replication. Present knowledge favors a role for poly(A) in the control of mRNA stability. Loss of this sequence could be controlled through modulation of poly(A)-protein interactions or through masking of a sequence directly adjacent to the poly(A). In the nucleus, the poly(A) sequence could also serve as stabilizing agent, but, in addition, it might interact with the splicing machinery.     

Control of breakdown of the polyadenylate sequence in mammalian polyribosomes: role of poly(adenylic acid)-protein interactions

The poly(adenylic acid) [poly (A)] segment in mouse sarcoma polysomes in not hydrolyzed by snake venom exonuclease under conditions which cause extensive degradation of the poly(A) in deproteinized polysomal RNA. The protecting effect of polysomes is presumably caused by the interaction between the poly(A) sequence and the protein known to be associated with it. This protection is reduced at low KCl concentration, but addition of exogenous RNA restores the protecting effect. The poly(A) segment also becomes susceptible to exonuclease after fragmentation of the polysomes by mild ribonuclease treatment. The latter treatment releases the poly(A) in association with protein. The poly(A) sequence in polysomes in readily degraded by a cytoplasmic extract of S-180 cells. Partial purification leads to a preparation active against the poly(A) in polysomes under conditions where no fragmentation of the messenger RNA is observed. Snake venom exonuclease increases the activity of the cytoplasmic preparation against poly(A) in polysomes. The active cytoplasmic factor appears to interfere with the poly(A)-protein interaction, thus rendering the polynucleotide susceptible to degradation by exonuclease. The poly(A) sequences in polysomes and in free cytoplasmic nucleoprotein particles are hydrolyzed to the same extent. The results suggest that the poly(A) sequence is normally protected from nucleases by virtue of its association with protein. The slow reduction in poly(A) size in cytoplasmic mRNA can be accounted for by a factor capable of interfering with the poly(A)-protein interaction. The latter interaction seems also dependent on the structural integrity of the polysomes or messenger ribonucleoproteins. It is suggested that a polynucleotide segment adjacent to the poly(A) can modulate the affinity of the protein for the latter sequence, thus permitting control of poly(A) stability in individual messenger RNAs.     

The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by differential centrifugation of lysed spheroplasts. The preparation, a modified 100,000 x g supernatant fraction, contains ribosomes and monosomes, ribosomal subunits, translation factors, and aminoacyl-tRNA synthetases, but no polysomes. After removal of small amounts of remaining mRNA with micrococcal nuclease, protein synthesis is stringently dependent on the addition of mRNA, as well as amino acids and an energy-generating system. The 5'-cap analogue, 7-methylguanosine 5'-phosphate, inhibits translation of several natural mRNAs, but has no effect on chain elongation. Incubation of the polysome-free extract with natural mRNA leads to the formation of protein-synthesizing polysomes and eventually, to the release of protein; the molecular weight of the protein synthesized in the presence of BMV (brome mosaic virus) RNA is consistent with that of BMV coat protein.     

Extensive homology between poly(A)-containing mRNA and purified nominal poly(A)-lacking mRNA in mouse kidney

To investigate poly(A)-lacking mRNA in mouse kidney, we studied a fraction of renal mRNA that does not bind to oligo(dT)-cellulose but can be purified by benzoylated cellulose chromatography. Nominal poly(A)-lacking mRNA and poly(A)-containing mRNA have complete nucleotide sequence homology, suggesting that kidney does not contain mRNAs that are not represented in the polyadenylated RNA fraction. Translation products directed by nominal poly(A)-lacking mRNA and poly(A)-containing mRNA are qualitatively and quantitatively similar in one-dimensional polyacrylamide gels. [3H]cDNA transcribed from poly(A)-containing mRNA hybridizes with its template and with nominal poly(A)-lacking mRNA to the same extent (95%) and with the same kinetics; reaction of [3H]cDNA to nominal poly(A)-lacking mRNA with the two mRNA populations gives the same result. The extensive homology these two mRNA populations share is important to the interpretation of mRNA lifetime and to the analysis of authentic poly(A)-lacking mRNAs.     

The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7–45

The effects of incubation of yeast spheroplasts at elevated temperature (40°C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells (wt A364A) and a temperature-sensitive mutant (ts 7–45) derived from it. With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: (1) the ribosomal subunit-polysome pattern; (2) the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; (3) the translation of poly(U) in postpolysomal extracts; (4) the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; (5) the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and (6) the formation of eIF-2·GTP·Met-tRNA_f ternary complex in the cytosol. With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations. Preincubation of temperature-sensitive spheroplasts at 40°C resulted in: (1) a further decrease in the level of 40 S subunits; (2) disaggregation of polysomes; (3) loss of ability to translate natural mRNA but not poly(U); (4) decreased ability to form 40 S preinitiation intermediates; and (5) production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly. The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.     

Translation in micrococcal nuclease-treated cell-free extracts from Ehrlich ascites tumor cells: Stimulation by initiation factor eIF-2B

Translation of exogenous mRNAs in micrococcal nuclease-treated extracts from Ehrlich ascites tumor cells is greatly stimulated by the addition of crude initiation factors or initiation factors eIF-2B and eIF-2 containing eIF-2B. The requirement for exogenous eIF-2B in micrococcal nuclease-treated extracts does not result from either loss or enhanced phosphorylation of eIF-2 during incubation.     

Characterization of messenger RNA by direct translation from agarose gels

A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed. The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease. A number of parameters which affect the efficiency of translation in this system have been examined. These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin. The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.     

Cellular protein synthesis shutoff by mengovirus: translation of nonviral and viral mRNA''s in extracts from uninfected and infected Ehrlich ascites tumor cells.

The mechanism whereby picornaviruses inhibit host protein synthesis while their own synthetic processes proceed unabated has remained elusive. One of our approaches to this problem was to study the ability of cell-free extracts derived from uninfected and mengovirus-infected Ehrlich ascites tumor cells to translate viral and nonviral mRNA's under various conditions of incubation. Our results indicate that viral messengers (from mengovirus and encephalomyocarditis virus) and cellular messengers [L cell and Ehrlich ascites tumor poly(A)-containing mRNA's, rabbit globin mRNA, and chicken embryo lens crystallin mRNA] are translated equally well in both extracts. We also examined the simultaneous translation of viral and nonviral mRNA's in extracts from uninfected Ehrlich ascites tumor cells. Our results indicate that under certain conditions mengovirus RNA can suppress completely the translation of globin mRNA. The significance of these results in terms of the shutoff of host protein synthesis is discussed.     

Size of poly (A) +-mRNA in Saccharomyces cerevisiae polysomes.

The size of poly (A) +-mRNA in different classes of yeast polysomes is estimated. The average molecular weight of long-term labelled polysomal poly (A) +-mRNA is about 0,65 x 10(6) daltons. Approximately 60% of the poly (A) +-mRNA polynucleotide chains located at the 5' end, are unprotected by ribosomes and degraded by nucleases upon incubation of cell lysates, to yield a population of poly (A) +-mRNA with an average molecular weight of 0,25 x 10(6) daltons.     

Mechanism of interferon action: inhibition of pppA(2'p5'A)n-dependent ribonucleases activity in micrococcal nuclease-treated mouse L cell-free extracts

The ability of nonpreincubated as compared to micrococcal nuclease-treated mouse L cell-free extracts supplemented with 2′-deoxythymidine-3′, 5′-diphosphate (pTp) and ethyleneglycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) to catalyze the 2′,5′-oligoadenylate-dependent degradation of reovirus [³H]mRNA was investigated. 2′,5′-Oligo A tetramer enhanced degradation in nonpreincubated but not micrococcal nuclease-treated extracts. Neither pTp nor EGTA significantly affected the 2′,5′-oligo A-dependent degradation in nonpreincubated extracts. The presence of both micrococcal nuclease and calcium was required to establish the subsequent reduction in both 2′,5′-oligo A-dependent and -independent degradation observed in micrococcal nuclease-treated extracts containing pTp and EGTA.     

An improved method for the isolation of polysomes from synchronous macroplasmodia of Physarum polycephalum

An improved method for the isolation of polysomes from synchronous macroplasmodia of Physarum polycephalum is described. The procedure involves preincubation and homogenization of the macroplasmodia in high ionic strength media supplemented with high concentrations of Mg²⁺ and ethylene glycol bis (β-aminoethyl ether) N,N′-tetraacetic acid. The polysomes recovered from plasmodial postmitochondrial lysates or heavy particle fractions prepared in this way are sensitive to RNase and EDTA and more than 90% of the total single ribosome population is present as polysomes. The polysomes obtained could also be utilized as a beginning point for the isolation of poly(A)-containing RNA which showed a mass average sedimentation value of 18 S. The development of a satisfactory procedure for the isolation of intact polysomes and poly(A)-containing RNA from macroplasmodia should facilitate studies on mRNA metabolism and translational activity during a naturally synchronous mitotic division cycle.     

Free,cytoskeletal-bound and membrane-bound polysomes isolated from MPC-11 and Krebs II ascites cells differ in their complement of poly(A) binding proteins

A three-step detergent/salt extraction procedure (Vedeleret al., Mol Cell Biochem 100: 183–193, 1991) was used to isolate free polysomes (FP), cytoskeletal-bound polysomes (CBP) and membrane-bound polysomes (MBP) from MPC-11 and Krebs II ascites cells. Polysomes were pelleted, washed with high salt buffer and re-pelleted. Proteins in the dialysed high-salt extracts were subjected to poly(A) Sepharose chromatography and poly(A) binding and non-binding proteins were separated by SDS-PAGE. In MPC-11 cells the FP fraction contains thirteen poly(A) binding proteins and four non-poly(A) binding proteins while the corresponding fraction in Krebs II ascites cells has four poly(A) binding proteins and six proteins which do not bind poly(A). The CBP fraction isolated from MPC-11 cells has a complement of ten poly(A) binding proteins, four which are non-poly(A) binding, and a protein of 105 kDa which has both poly(A) binding and non-poly(A) binding properties. In the CBP fraction prepared from Krebs II ascites cells a protein band at 32 kDa exhibits both poly(A) binding and non-poly(A) binding properties. In this fraction there are six poly(A) binding proteins and an additional eight which do not bind poly(A). Of the total number of proteins eight of these have a molecular weight below 40 kDa. The MBP fraction in MPC-11 cells contains three poly(A) binding proteins and eleven with non-poly(A) binding properties. In contrast this fraction in Krebs II ascites cells has a complement of thirteen poly(A) binding and ten non-poly(A) binding proteins. The results show differences in the poly(A) binding properties of the proteins in the three polysome fractions and that the complements of polysome-associated proteins are different in the two cell lines. This may be related to the differences in the growth characteristics of MPC-11 and Krebs II ascites cells.     

The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7-45

The effects of incubation of yeast spheroplasts at elevated temperature (40 degrees C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells (wt A364A ) and a temperature-sensitive mutant (ts 7-45) derived from it. With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: the ribosomal subunit-polysome pattern; the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; the translation of poly(U) in postpolysomal extracts; the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and the formation of eIF-2 X GTP X Met-tRNAf ternary complex in the cytosol. With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations. Preincubation of temperature-sensitive spheroplasts at 40 degrees C resulted in: a further decrease in the level of 40 S subunits; disaggregation of polysomes; loss of ability to translate natural mRNA but not poly(U); decreased ability to form 40 S preinitiation intermediates; and production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly. The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.     

Effect of polyadenylic acid on the functional half-life of encephalomyocarditis virus RNA during translation.

The translation of poly(A)-rich and poly(A)-poor populations of Encephalomyocarditis viral RNA was studied in Ehrlich ascites cell-free extracts. The poly(A)-rich viral RNA was translated 2–3 times more efficiently than the poly(A)-poor RNA. Both viral RNA populations were found to be similar with respect to susceptibility to nuclease attack as well as their ability to initiate and carry out protein synthesis early in the translation reaction. Later in the reaction, however, translation of the poly(A)-poor RNA was markedly reduced whereas translation of the poly(A)-rich RNA continued. These results are consistent with the hypothesis that poly(A) plays a role in the re-utilization and longevity of mRNA.