mRNA-dependent Reticulocyte Lysate Research Articles

Partial purification of a developmentally regulated messenger RNA from Dictyostelium discoideum by thermal elution from poly(U)-Sepharose

We have demonstrated that thermal elution from poly(U)-Sepharose is useful for separating newly synthesized messenger RNAs from steady-state mRNAs isolated from vegetative cells of Dictyostelium discoideum (Palatnik et al., 1979,1980). Because we were able to enrich between ten- and 60-fold for new transcripts, we suggested that this technique might be applicable to the purification of developmentally regulated mRNAs. The utility of the technique would, however, depend on the differential rate of synthesis of the particular mRNAs. For example, in vegetatively growing Dictyostelium amoebae the relative distribution of translation activities in newly synthesized mRNA is similar to that found in steady-state mRNA (Palatnik et al., 1979), and the technique does not lead to substantial purification of any particular mRNA species. In order to test the applicability of the method, we have studied, as a model system, changes in actin mRNA synthesis during early D. discoideum development. It has been shown that the relative concentration of actin mRNA changes during Dictyostelium development. Experiments with inhibitors of RNA synthesis have suggested that changes in the concentration of this mRNA are mediated at the transcriptional level. This conclusion is supported by the experiments of this paper: newly synthesized poly(A)-containing RNA from growing and developing cells was translated in mRNA-dependent reticulocyte lysates and the translation products were analyzed by two-dimensional polyacrylamide gel electrophoresis. At two hours of development, when the relative concentration of actin mRNA is at its peak, we find that its translation activity is also substantially enriched in the fraction of RNA that is newly synthesized. At five hours of development, when the percentage of total translatable actin mRNA has begun to decline, actin represents a much smaller percentage of the translation products coded for by this RNA fraction. The physical isolation and characterization of the newly synthesized mRNA fraction thus provides evidence that changes in the concentration of actin mRNA during Dictyostelium development occur at the level of messenger RNA synthesis. Furthermore, the results dramatically illustrate the usefulness of thermal elution from poly(U)-Sepharose for enriching for mRNAs induced by alterations in developmental or metabolic states.

Comparison of the NH2-Terminal sequences of chick Type I preprocollagen chains synthesized in an nRNA-dependent reticulocyte lysate.

RNA was extracted from 17-day-old chick embryo calvaria and translated by an mRNA-dependent reticulocyte lysate. Procedures were developed for purifying intact pro alpha 1(I) and pro alpha 2 translation products from the lysate. These products were identified by comparing tryptic peptide elution patterns of the translation products with those obtained from secreted pro alpha chains. Partial sequence data from the amino terminus of each translation product demonstrated that each chain begins with a sequence that is different from that of the corresponding pro alpha chain, and that the two sequences are not the same. Also, the bacterial collagenase-resistant peptide from the amino terminus of prepro alpha 1(I) had an apparent molecular weight which was 10 000 greater than the peptide from pro alpha 1(I).

Primary structure of the signal peptide of tropoelastin b.

Elastin is a major protein of compliant connective tissue and is characterized by an amino acid composition abundant in nonpolar residues. The soluble precursor to elastin tropoelastin, is extractable in organic solvents and possesses an extensive clustering of nonpolar amino acid residues in the immediate NH2-terminal region (Foster, J. A., Shapiro, R., Voynow, P., Crombie, G., Faris, B., and Franzblau, C. (1975) Biochemistry 14, 857-864). It was, therefore, of special interest to determine whether tropoelastin requires a hydrophobic signal peptide for vectorial transport of the nascent polypeptide. The possibility that the initial tropoelastin translation product possesses a short signal peptide was examined in a cell-free translation system. Total RNA, isolated from aortae of 1-day-old chicks, was translated in an mRNA-dependent reticulocyte lysate translation assay. The translation products were then immunoprecipitated and subjected to automated radiosequencing. Comparison of the NH2-terminal sequence of tropoelastin b synthesized in the cell-free system versus that synthesized in organ culture demonstrated the presence of a signal peptide 24 amino acid residues in length. The signal peptide sequence is as follows: Met-Arg-Gln-Ala-Ala-Ala-Pro-Leu-Leu-Pro-Gly-Val-Leu-Leu-Leu-Phe-Ser-Ile-Leu-Pro -Ala-Ser-Gln-Gln. The preponderance of hydrophobic amino acid residues as well as the polar residues adjacent to the initiator methionine and the carboxyl termini found in the signal peptide is similar to that reported for other secreted proteins.

An immunochemical method for mRNA purification. Application to messenger RNA encoding trypanosome variable surface antigen.

A simplified procedure for isolation of specific mRNA using Staphylococcus aureus protein A immunoadsorbent chromatography has been developed. This procedure has been applied to the mRNA encoding Trypanosoma brucei variable surface antigen. Trypanosome polyribosomes were reacted with antibodies isolated from an anti-variable antigen-specific serum by protein A-Sepharose column chromatography. Antibody-bound variable antigen-synthesizing polyribosomes were then separated from unbound polyribosomes also by protein A-Sepharose column chromatography. This simple protocol gave a greater than 50% yield of variable antigen-specific mRNA which appeared to be very highly purified as determined by translation in an mRNA-dependent reticulocyte lysate assay. The mRNAs encoding three different T. brucei surface antigens have been purified. The procedure described here should be useful in purification of other mRNAs.

Isolation and characterization of the antifreeze protein messenger RNA from the winter flounder.

The messenger RNA coding for the serum antifreeze protein of the winter flounder (Pseudopleuronectes americanus) was extracted from the liver polysomes of fish caught in November and was purified by oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The length of the mRNA was 520 nucleotides measured by agarose gel electrophoresis in the pesence of methyl mercury. It sedimented between trout protamine mRNA (6 S) and rabbit globin globin mRNA (9-10 S) on sucrose density gradients. The purified antifreeze protein mRNA assayed in the mRNA-dependent reticulocyte lysate translation system incorporated 10 times as much alanine as arginine into a single translation product. the molecular weight of the primary translation product was 11,700 estimated by sodium dodecyl sulfate-gel electrophoresis. Antifreeze protein mRNA was the only major mRNA species present in liver polysomes of fish caught in November but was absent in the liver polysomes of fish caught in April. This finding emphasizes the seasonal nature of antifreeze protein production in the winter flounder. A 32P-labeled cDNA prepared from the antifreeze protein mRNA was hybridized against total cellular RNAs from flounder liver. These RNA excess hybridizations showed that antifreeze protein mRNA is several orders of magnitude more abundant in liver in mid-November than in late August. This suggests that transcriptional control is involved in the seasonal expression of the antifreeze protein gene.

Preferential translation of mRNAs in an mRNA-dependent reticulocyte lysate.

Messenger RNA competition experiments were performed in an mRNA-dependent reticulocyte lysate using three kinds of mRNA: rabbit globin mRNA, calf eye lens mRNA and RNA of turnip yellow mosaic virus. Our results indicate that at supersaturating concentrations of mRNA preferential translation of certain mRNA species can be observed. Furthermore, the pattern of mRNA selection by the translational apparatus suggests that the rate of translation of different mRNA species is limited by different components of the reticulocyte lysate. Our observations n the cell-free system are strikingly different from our previously published mRNA competition experiments in Xenopus oocytes using the same preparations of lens and globin mRNA, in which no selective translation was observed [Asselbergs et al., Eur. J. Biochem. 94, 249-254 (1979)]. The restraints on mRNA translation in vitro are apparently different from those in vivo, i.e. in oocytes.

In vitro translation of virion RNA from Moloney murine sarcoma virus

Virion RNA of Moloney murine sarcoma virus (Mo-MSV) was translated in the mRNA-dependent reticulocyte lysate and the translation products were analyzed by gel electrophoresis. The major products had apparent molecular weights of 62,000, 37,000, 33,000, 24,000, 18,000, and 15,000. None of these proteins was derived from the helper virus RNA present in our Mo-MSV virion preparations. Immunoprecipitation studies showed that the 62K protein was related to the gag gene product of Moloney murine leukemia virus (Mo-MLV). The 37K, 33K, 24K, 18K, and 15K proteins, on the other hand, were found to be unrelated to either the gag gene or env gene products of Mo-ML V. Tryptic peptide mapping showed that the 37K, 33K, 24K, and 18K proteins had overlapping amino acid sequences. By this criterion the 37K family of proteins was not related to the 62K protein nor to the 15K protein. Likewise, the 15K protein was not related to the 62K protein. Translation of naturally occurring polyadenylated fragments of virion RNA showed that the 62K protein was derived from RNA of genomic length (30 S). The 15K protein was made from RNA of about 25 S, while the 37K, 33K, 24K, and 18K proteins were synthesized from RNAs ranging from 20 to 17 S. On the basis of these results, we have proposed a model for the expression of the genome of Mo-MSV in vitro. The 62K protein would arise from the gag gene at the 5′ end of the Mo-MSV genome. The 15K protein would originate from one of the two pol gene fragments in the Mo-MSV genome. The 37K, 33K, 24K, and 18K proteins would be derived from the src gene of Mo-MSV.

Immunochemical isolation of prolactin messenger RNA.

Prolactin mRNA has been isolated using immunochemical techniques. Initial experiments demonstrated that 125I-labeled prolactin antibody was able to bind to pituitary polysomes but not to liver polysomes, suggesting that the binding is specific. Prolactin-synthesizing polysomes were immunoprecipitated by reaction with antiprolactin followed by anti-antibody. Immunoprecipitated polysomal RNA was chromatographed on oligo(dT)-cellulose, and the poly(A) RNA was sedimented through a sucrose gradient. This procedure resulted in a 320-fold purification of prolactin mRNA as determined by translation in a mRNA-dependent reticulocyte lysate assay. Translation analysis also suggested that the isolated prolactin mRNA is greater than 95% pure. The molecular weight of prolactin mRNA determined by electrophoresis on agarose gels containing 10 mM mercury hydroxide was 350,000. Purified prolactin mRNA was used to synthesize full-length cDNA by means of avian myeloblastosis reverse transcriptase. Use of this cDNA as a hybridization probe demonstrated that estrogen is able to increase the concentration of prolactin mRNA sequences in the pituitary.

Cell-free translation and regulation of Candida lipolytica acetyl-coenzyme-A carboxylase messenger RNA.

Messenger RNA from Candida lipolytica directs the synthesis of complete acetyl-coenzyme-A carboxylase in the mRNA-dependent reticulocyte lysate cell-free translation system. The identity of the translation product is evidenced by the following results: first, it is immunoprecipitated with antibody to acetyl-CoA carboxylase and competes with authentic acetyl-CoA carboxylase for binding to the antibody; secondly, it co-migrates with authentic acetyl-CoA carboxylase (Mr 230000) upon dodecylsulfate/polyacrylamide gel electrophoresis; finally, the peptide fragments formed by its partial proteolysis with papain or alpha-chymotrypsin are identical with those formed from authentic acetyl-CoA carboxylase. With the use of this assay system, it has been demonstrated that the level of acetyl-CoA carboxylase mRNA activity in C. lipolytica cells decreases with increasing concentrations of oleic acid in culture medium and that the changes in the mRNA activity parallel those in the cellular level of acetyl-CoA carboxylase. This finding, in conjunction with our previous study, indicates that the diminished synthesis of acetyl-CoA carboxylase in cells grown in the presence of fatty acid is due to a reduced level of the mRNA coding for the enzyme.

In vitro translation of avian erythroblastosis virus RNA: Identification of two major polypeptides

RNA of avian erythroblastosis virus (AEV) pseudotyped with RAV-1, AEV(RAV-1), and its fragments containing poly(A) were translated in an mRNA-dependent reticulocyte lysate. p74 and 46 were found to be AEV RNA specific out of several main products, pr76, p74, p46, and p37. p74 was translated from 30 S AEV RNA and contained some peptides homologous to pr76, but most peptides as specific. On the other hand p46 was translated from 20 S AEV RNA containing poly(A). However, no relation to known viral proteins was established. This preliminary observation suggests that the 5′-half of AEV genome encodes p74 and the 3′-half p46.

Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA

An mRNA-dependent reticulocyte lysate has been used to translate foot-and-mouth disease virus RNA in vitro. Polypeptides P16, P20a, and P88, which have been shown to be derived from the 5' end of the RNA by pactamycin mapping experiments with infected cells, were preferentially synthesized in vitro. Removal of VPg, the small protein covalently linked to the 5' end of the genome RNA, had no effect on the translation of the RNA. The two RNA fragments (L and S) produced by specific digestion of the polycytidylic acid [poly(C)] tract with RNase H were also translated in vitro. The L fragment, consisting of RNA to the 3' side of the poly(C) tract and including the polyadenylic acid [poly(A)] tract, directed the synthesis of the same products as those made by full-length RNA. However, no small defined products were produced when the S fragment, which contains the 5' end of the RNA, was translated. These results show that the major initiation site for protein synthesis on foot-and-mouth disease virus RNA is to the 3' side of the poly(C) tract. Furthermore, the use of N-formyl [35S]methionine tRNAfMet as a label for the initiation peptides showed that the major polypeptide labeled in lysates primed with both full-length RNA and the L fragment was P16, i.e., the protein nearest the initiation site for translation as deduced from pactamycin mapping experiments. Fragments of RNA were also translated in vitro. Those containing the poly(C) tract gave products similar to those produced when full-length RNA was translated. The polypeptides synthesized when fragments containing the poly(A) tract were used, however, did not resemble those made from full-length RNA.

Malate synthase messenger RNA in relation to enzyme derepression in Euglena gracilis

The production of poly (A)+ RNA was determined over the cell-cycle in Euglena cultures division-synchronized by 14 h light periods alternating with 10 h dark periods. The amount of poly (A)+ RNA per culture aliquot was constant from the beginning until near the end of the light phase of the cell-cycle when it increased in a sharp burst after the time of DNA replication. Poly (A)+ RNA from all stages of the cell-cycle promoted protein synthesis with equal efficiency in a mRNA-dependent reticulocyte lysate translation system. Malate synthase mRNA was detected using malate synthase antibody to precipitate nascent malate synthase protein in the translation system, binding the IgG-antigen complex to Staphylococcus aureus, concentrating, eluting the IgG-antigen complex and analysing the products by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Malate synthase mRNA was absent from phototrophic cells and was only produced in darkened cultures following the addition of acetate.

An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA

SE 21Qlb is a recombinant avian oncovirus which is continuously produced by a line of transformed quail cells. These cells produce no detectable infectious viral progeny. Virions of SE 21Q1b contain 1–2% of the normal amounts of RSV-specific RNA found in PR-RSV-C virions and substantial quantities of heterogeneously sedimenting nonviral RNA ( M. Linial, E. Medeiros, and W. S. Hayward, 1978b, Cell, 15:1371–1381). RNA extracted from purified virions of SE 21Q1b is as efficient a template for amino acid incorporation in a mRNA-dependent reticulocyte lysate as oligo(dT)-cellulose purified cellular mRNA. Virion RNA from SE 21Q1b serves as a template in vitro for a small amount of the precursor to the internal structural proteins ( gag proteins). But unlike RNA from other avian oncoviruses, SE 21Q1b RNA makes, in addition, a number of proteins whose sizes on sodium dodecyl sulfate (SDS)-polyacrylamide gels resemble those of a number of uninfected quail fibroblast proteins. The proteins range in size from 15,000–220,000 daltons. Immunoprecipitation of proteins synthesized in vitro from SE 21Qlb virion RNA with antisera against the gag, pol, and env gene products shows that this RNA serves as a template for only one viral gene product, the recombinant gag gene precursor of 72,000 daltons (ΔPr76) ( R. Shaikh, M. Linial, J. Coffin, and R. Eisenman, 1978, Virology 87, 326–338). Addition of lysed virus to proteins made in vitro from PR-C RNA or RNA from PR-E-95c, a recombinant whose gag gene structure is similar to that of PR-E-21Qlb, results in the cleavage of gag protein precursors ( K. von der Helm, 1977, Proc. Nat. Acad. Sci. USA 74, 911–915) into several of the internal structural proteins. On the other hand, products of translation of PR-E-21Q1b RNA are not detectably cleaved into the internal structural proteins, suggesting that a very low amount of PR-E-21Q1b translation products are gag related. A protein of 37,000 daltons constitutes about 15% of the total protein synthesized from SE 21Qlb RNA. Data from serological and peptide mapping studies indicate that this protein is unrelated to the virion gag, pol, or env proteins. However, the major tryptic peptide of this protein appears to be identical to the major peptide of a prominent quail cell protein having the same apparent molecular weight as the in vitro translation product. Thus, several lines of evidence suggest that SE 21Q1b virions contain substantial amounts of functional cellular messenger RNAs.

Translation of chick calvarial procollagen messenger RNAs by a messenger RNA dependent reticulocyte lysate

A method was developed for the extraction of RNA from chick embryo calvaria which should be generally applicable to other connective tissues. Total RNA prepared by this method was translated by a mRNA-dependent reticulocyte lysate into discrete pro alpha chains. Several criteria were used to identify these translation products, including (1) preferential labeling with [3H]proline, (2) appropriate migration on sodium dodecyl sulfate-polyacrylamide gels, (3) selective sensitivity to collagenase digestion, and (4) specific precipitability by two different antisera against procollagen. Data from the immunoprecipitation experiments indicated that the majority of the pro alpha chains contained the carboxy-terminal antigenic determinants. These results demonstrate that this translation system can be used as an assay for intact procollagen mRNAs and as a source of in vitro synthesized pro alpha chains for future structural analysis.

Initiation factor eIF-4B (IF-M3)-dependent recognition and translation of capped versus uncapped eukaryotic mRNAs.

Translation of capped and uncapped eukaryotic mRNAs is stimulated by addition of eIF-4B to an mRNA-dependent reticulocyte lysate system. m7G5 ppp inhibits translation of capped but not uncapped mRNAs and reduces translation of capped vaccinia mRNA to the level obtained with uncapped vaccinia mRNA. Exogenous eIF-4B but no other initiation factor reverses inhibition of protein synthesis by m7G5'ppp. Both capped and uncapped mRNAs interact directly with eIF-4B to form a stable complex, which can be detected by a simple nitrocellulose filter binding assay. However, addition of a 5'-cap to beta-eliminated globin mRNA or satellite tobacco necrosis virus RNA (normally uncapped) increased binding affinity of these mRNAs for eIF-4B and causes binding of these mRNAs to become sensitive to inhibition by m7G5'ppp. These results indicate that the role of the mRNA 5'-cap in translation is related specifically to the function of eIF-4B in forming a complex with mRNA (prior to association of mRNA with the 40 S ribosomal subunit) and that both cap and non-cap sequences participate in this process.

Translation of Encephalomyocarditis Virus RNA in vitro Yields an Active Proteolytic Processing Enzyme

In contrast to other cell-free translation systems, the mRNA-dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome.

Dibutyryl cyclic AMP increases the amount of functional messenger RNA coding for tyrosine aminotransferase in rat liver.

The administration of N6,O2-dibutyryl cyclic AMP and theophylline to adrenalectomized rats results in an increase in the amount of functional mRNA coding for tyrosine aminotransferase that can be isolated from liver. The induction of this specific mRNA, as quantitated in a mRNA-dependent reticulocyte lysate system, and using poly(A)+ mRNA extracted from total tissue and polysomes, is very rapid. Within an hour after the intraperitoneal injection of the cyclic AMP derivative there is a 5- to 7-fold elevation of functional mRNA coding for tyrosine aminotransferase (mRNATAT), and by 3 h this has returned to basal levels. In contrast, the 4- to 5-fold induction of tyrosine aminotransferase catalytic activity is maximal at 2 h and is still significantly greater than the basal level at 5 h. In the basal state, tyrosine aminotransferase mRNA codes for 0.019 +/- 0.003% of the protein synthesized in the in vitro system, whereas after cyclic nucleotide treatment this value 0.115 +/- 0.015%, hence the increase in mRNATAT activity is relatively specific. Cordycepin, at a concentration which prevents the accumulation in cytoplasm of poly(A)+ mRNA, completely blocks the increase in both the catalytic and mRNA activity of this enzyme. The marked increase in functional mRNA, the requirement for continued synthesis of poly(A)+ RNA, and the rapid induction and deinduction suggest that the cyclic nucleotide is enhancing specific mRNA synthesis and/or, processing, however an effect on mRNA degradation cannot be excluded.

Translation of TYMV RNA into high molecular weight proteins

The wheat germ cell-free system leads to the translation of TYMV RNA into mature coat protein and several other polypeptides [l] , although not to a polypeptide whose size corresponds to the full translation of the viral genome. TYMV contains not only complete viral genomic RNA (2 X lo6 daltons), but also several other classes of RNA molecules ranging from 2-0.25 X 10” daltons [2,3] . The complete viral genome by itself is infectious [2], and therefore contains all the information necessary to generate new virus particles; in a wheat germ cell-free system, however, it directs the synthesis of only small amounts of coat protein molecules, if at all, thereby suggesting that the coat protein gene is masked in such a system. In order to determine whether TYMV RNA can direct the synthesis of high molecular weights proteins (>lOO 000 daltons), we turned to the use of the mRNA-dependent reticulocyte lysate which has been shown by Pelham and Jackson [4] to translate TMV RNA into one protein of 130 000 daltons. Our results show that in the reticulocyte system total TYMV RNA is efficiently translated into two high molecular weight proteins, one of 195 000 daltons, the other of 150 000 daltons, as well as into coat protein (20 000 daltons). The largest protein is