Messenger-like RNA Research Articles

Transcription during cell differentiation in Naegleria gruberi. Preferential synthesis of messenger RNA

Amebae of Naegleria gruberi undergo phenotypic transformation into flagellates when the aqueous environment is changed from axenic growth medium to non-nutrient buffer. This differentiation, which requires 80 min at 25 °C, is dependent on transcription during the first 40 min. The nature of the RNA synthesized during differentiation is the subject of this report. Net synthesis of RNA continues for the first 30 min after differentiation is initiated, but thereafter degradation exceeds synthesis leading to an overall decrease of 6 % in total RNA per cell by 90 min. [ 14C]-Uracil is incorporated into RNA during differentiation. Incorporation is linear until roughly 10 min before the appearance of flagella, at which time the rate of incorporation declines. Sucrose gradient analysis has been used to compare the RNA synthesized by cells differentiating in buffer versus amebae in growth medium. There is proportionally more synthesis of messenger-like RNA and less of rRNA in differentiating cells. The messenger-like RNA synthesized during differentiation is heterogeneous in size, but very large molecules (> 45 S) have not been found. This rapidlylabeled RNA is first present in the nucleoplasm, and moves rapidly to the cytoplasm, reaching equilibrium ( nucleus cytoplasm ) within 10 min. In the cytoplasm it is found associated with polysomes, where it has the EDTA and ribonuclease sensitivity expected of mRNA. These observations support the interpretation that the rapidlylabeled RNA is mRNA. It is not yet known whether this mRNA includes the RNA essential for differentiation.

RNA metabolism during the development of cotyledons of Pisum sativum L.

Changes in RNA synthesis in cotyledons of Pisum sativum L. during seed development and maturation were studied. The bulk of the total RNA accumluated by 21 days after flowering preceding by nearly 1 week the maximum accumulation of protein which occurred at 27 days. Gel electrophoresis of total RNA indicated that exogenously supplied [ 32P]orthophosphate was incorporated into heavy and light rRNAs and the 4–5-S RNA component but incorporation into these fractions became restricted as the seed matured. At maturity [ 32P]orthophosphate was incorporated increasingly into a non-nucleic acid contaminant in addition to the accumulation of radioactive material into an apparently novel RNA component. Ribosomes occurred in polysomic configurations in young cotyledons but at seed maturity there was a preponderance of monosomes. These changes presumably reflected changes in the mRNA population. Analysis of low molecular weight messenger-like RNA species associated with ribosomes indicated that both quantitative and qualitative changes occurred in these components during seed development.

Synthesis of RNA during Myxospore Induction in Myxococcus xanthus

SUMMARY: RNA was isolated from vegetative Myxococcus xanthus FB and myxospores. Organisms were labelled with [32P]orthophosphate during encystment and the metabolic stability of the radioactive RNA was followed by fractionating purified RNA from ‘pulse-chased’ cultures by polyacrylamide gel electrophoresis. Much of the label was in heterodisperse (messenger-like) RNA but the stable RNA (present in the mature myxospores) was largely high-molecular weight ribosomal RNA and its precursors. The role of RNA synthesis in the morphogenetic cycle of Myxococcus xanthus is discussed in the light of these data and of the properties of the ribosomes and ribosomal proteins from this organism.

Mitochondrial protein synthesis: RNA with the properties of Eukaryotic messenger RNA.

A heterogeneous RNA fraction with properties resembling those of messenger RNA was identified in mammalian mitochondria. Synthesis of contaminating RNA of nuclear origin was suppressed by treatment with camptothecin. Labeling of the messenger-like RNA is completely inhibited by ethidium bromide, a specific inhibitor of mitochondrial functions.Although mitochondrial protein synthesis resembles that of prokaryotes in several regards, the messenger-like RNA is covalently linked to poly(adenylic acid) [poly(A)]. Poly(A) has thus far been found only in eukaryotic cells. The poly(A) segment has a gel electrophoretic mobility of about 4 S, corresponding to a length of 50-80 nucleotides, and thus resembles in size the poly(A) found in some mammalian viral RNAs. The messenger RNA can be released from the mitochondrial protein-synthesizing structure by treatment with puromycin.

Effect of Light on Ribonucleic Acid Metabolism in Greening Maize Leaves

The effect of illumination on the incorporation of labeled precursors into RNA of dark-grown maize (Zea mays) leaves was studied using either (32)P-phosphate or double labeling with (14)C- and (3)H-uridine. In the dark, label was preferentially incorporated into etioplast ribosomal RNAs. Incorporation into this fraction and into lower molecular weight fractions was strongly and preferentially stimulated by light during the first 2 hours of illumination. The effect persisted after illumination was terminated. The possibility that light-induced alterations in plastid ribosomal RNA metabolism may not be required for chlorophyll accumulation in maize is discussed.Sucrose density gradient analyses of ribosomes and of extracted RNA did not reveal light-induced incorporation of label into messenger-like RNA associated with polyribosomes during brief illumination. However, newly produced RNA which seems to be neither ribosomal RNA nor transfer RNA is detectable after illumination for 2.5 hours or longer.

Polyribosome-bound and free-cytoplasmic-hemoglobin-messenger RNA in differentiating avian erythroblasts.

Cytoplasmic messenger‐like RNA not associated with ribosomes has been investigated in immature duck erythrocytes and compared with polyribosome‐bound messenger RNA. It is shown that 9‐S RNA of the same electrophoretic mobility as the polyribosomal 9‐S globin messenger is present free in the cytoplasm. Its rate of synthesis and decay is consistent with a role as a precursor of polyribosomal 9‐S globin messenger.mRNA species different from 9‐S RNA, among them a predominant 12‐S species, are found associated with polyribosomes as well as in the free cytoplasmic pool. The free cytoplasmic mlRNA pool is more heterogeneous compared to the polyribosomal mRNAs, suggesting a translational control qualitatively and quantitatively selecting among the free cytoplasmic mlRNAs for those to be translated.

Isolation of messenger-like RNA from immunochemically separated polyribosomes

A method for the isolation of a given messenger RNA from mammalian cells has been developed. This method consists of (i) specific immunochemical precipitation of polyribosomes synthesizing the protein, and (ii) extraction of the mRNA coding for this protein from the polyribosome/antigen/antibody complex. From a study of the kinetics of 32P incorporation into the cytoplasmic RNA of MOPC 149 mouse plasmacytoma cells, which secrete an immunoglobulin κ-type light chain, it was concluded that within 30 minutes two species of RNA were predominantly labelled, i.e. transfer RNA and a class of polydisperse RNA considered to be mRNA. Polyribosomes labelled with 32P were isolated from MOPC 149 plasmacytomas and treated with an excess of F(ab′) 2 fragments prepared from either a specific (rabbit anti-MOPC 149 L-chain) or a control (rabbit anti-goat IgG) antiserum. Optimum amounts of the respective antigens were added to the soluble complexes as carriers to yield maximum precipitation. A comparison of the 32P activity in the precipitates revealed that L-chain-bearing polyribosomes were specifically isolated. A rapidly labelled species of RNA, having a sedimentation coefficient of 11 to 12 s and a base composition of 48 moles of G + C per cent, was isolated from the precipitated polyribosomes. These properties can be directly correlated with the size and composition of the MOPC 149 L-chain and suggest that this RNA molecule is the mRNA coding for this L-chain.

Proteins associated with globin messenger RNA in avian erythroblasts: Isolation and comparison with the proteins bound to nuclear messenger-likie RNA

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Synthesis of lens protein in vitro V. Isolation of messenger-like RNA from lens by high resolution zonal centrifugation

Recently Lingrel described the isolation of a 9 S fraction from mouse reticulocytes which exhibited messenger activity in a cell-free system from rabbit reticulocytes [ 1,2]. Also from other eukaryotic systems RNA with template activity could be isolated. Heywood described the isolation of messenger-like RNA from muscle polyribosomes. He was able to show that with this messenger and 80 S ribosomes from reticulocytes a protein could be synthesized which has the characteristics of myosin [3] . An RNA fraction isolated from myeloma appeared to function as template for the synthesis of a mouse Ig light chain in a heterologous cell-free reticulocyte system [4]. The lens is a unique tissue which predominantly produces a class of highly specific proteins: the crystallins. Like mRNA from reticulocyte [5-71 the lens messenger has been reported to be stable [8] In the present paper a high resolution zonal centrifugation technique is described which allows the isolation of two species of lens messengers differing by a number of criteria from the bulk of ribosomal RNA and tRNA.

Adenosine-rich sequences in rapidly hybridizing messenger-like RNA and their possible significance for reiterated base sequences in eukaryotic DNA

FEBS LettersVolume 17, Issue 1 p. 68-72 Full-length articleFree Access Adenosine-rich sequences in rapidly hybridizing messenger-like RNA and their possible significance for reiterated base sequences in eukaryotic DNA Klaus Scherrer, Klaus Scherrer Swiss Institute for Experimental Cancer Research, Lausanne, SwitzerlandSearch for more papers by this author Klaus Scherrer, Klaus Scherrer Swiss Institute for Experimental Cancer Research, Lausanne, SwitzerlandSearch for more papers by this author First published: September 15, 1971 https://doi.org/10.1016/0014-5793(71)80565-3Citations: 14AboutPDF 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 K. Scherrer, L. Marcaud, J. Cell Physiol., 72, (1968), 181– Suppl.1 2 K. Scherrer, G. Spohr, N. Granboulan, C. Morel, J. Grosclaude, C. Chezzi, Cold Spring Harbor Symp. Quant. Biol., 35, (1970), 539– 3 K. Scherrer, L. Marcaud, F. Zajdela, B. Breckenridge, F. Gros, Bull. Soc. Chim. Biol., 48, (1966), 1037– 4 J. Kates, Cold Spring Harbor Symp. Quant. Biol., 35, (1970), 743– 5 M. Edmonds, M.H. Vaughan Jr., Federation Proc., 29, (1970), 2397– 6 L. Lim, E.S. Canellakis, Nature, 227, (1970), 740– 7 R.J. Britten, D.E. Kohne, Science, 161, (1968), 529– 8 P.M.B. Walker, Nature, 219, (1968), 228– 9 B. McCarthy, Biochem. Genet., 2, (1968), 37– 10 R.J. Britten, E.H. Davidson, Science, 165, (1969), 349– 11 G.P. Georgiev, J. Theoret. Biol., 25, (1969), 473– 12 K. Scherrer, personal communication Gordon Conf., (1966), 13 K. Scherrer, K. Habel N.P. Salzman Fundamental Techniques in Virology (1969), Academic Press New York 413– 14 A. Hiby, B. Kröger, J. Chromat., 26, (1967), 545– 15 S. Penman, K. Scherrer, Y. Becker, J.E. Darnell, Proc. Natl. Acad. Sci. U.S., 49, (1963), 651– 16 K. Scherrer, L. Marcaud, Bull. Soc. Chim. Biol., 47, (1965), 1697– 17 K. Scherrer, L. Marcaud, F. Zajdela, I.M. London, F. Gros, Proc. Natl. Acad. Sci. U.S., 56, (1966), 1571– 18 R. Soiero, H.C. Birnboim, J.E. Darnell, J. Mol. Biol, 19, (1966), 362– 19 G. Spohr, N. Granboulan, C. Morel, K. Scherrer, European J. Biochem., 17, (1970), 296– 20 Ch. Coutelle, A.P. Ryskov, G.P. Georgiev, FEBS Letters, 12, (1970), 21– 21 A.P. Ryskov, V.L. Mantieva, A.R. Avakain, G.P. Geogiev, FEBS Letters, 12, (1971), 141– 22 E.M. Southern, Nature, 227, (1970), 794– 23 C. Morel, B. Kayibandan, K. Scherrer, Experientia, 27, (1971), 22– 24 J. Marbaix, personal communication (1971). 25 M.-E. Mirault and K. Scherrer, European J. Biochem., submitted. Citing Literature Volume17, Issue1September 15, 1971Pages 68-72 ReferencesRelatedInformation

Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin-kieselguhr.

Chromatography on methylated albumin-kieselguhr of RNA from Saccharomyces cerevisiae was used to separate stable RNA from a tenaciously bound DNA-like RNA fraction. The tenaciously bound RNA, which was eluted with a dilute solution of sodium dodecyl sulphate, was characterized as messenger-like RNA by its sedimentation behaviour, nucleotide composition, lack of methylated bases and labelling kinetics. Chromatography of purified ribosomal RNA indicated a minor contamination of the tenaciously bound fraction with ribosomal RNA. On the other hand, a large portion of pulse-labelled polyribosomal RNA from protoplasts of Saccharomyces cerevisiae was tenaciously bound to the columns. The ;chase' of isotopic label from the messenger-like RNA was found to be retarded during inhibition of protein synthesis both by cycloheximide and by starvation for a carbon source.

Adenine-rich polymer associated with rabbit reticulocyte messenger RNA.

POLYPURINES have been isolated from subcellular fractions of mouse liver by a method which involves a limited nuclease digestion of the RNA, followed by chromatography of the digestion products on polystyrene columns1–3. These polypurines consist of clusters of adenylic acid and guanylic acid in which the A/G ratio is 2–10; the materials isolated from the rough endoplasmic reticulum have sedimentation coefficients of 3–5S, but a molecule containing twenty nucleotides can be obtained from the free polysomes4. The latter polymer is part of a rapidly labelled RNA species that sediments between the 32S subunit and 5S RNA when EDTA-treated polysomes are centrifuged on a sucrose density gradient. With longer times of labelling with 32P the polymer is also found in association with the 32S subunit. It seems likely that the adenine-rich polymer is found in close association with messenger-like RNA. These obssrvations prompted us to investigate the polypurine content of a better characterized mammalian messenger RNA. Such an RNA is the haemoglobin messenger RNA which has been isolated from rabbit reticulocytes5,6, and purified by polyacrylamide gel electrophoresis7. This species of RNA8 and a similar RNA from mouse reticulocytes9 have been demonstrated to initiate specific globin synthesis in vitro. We now describe the isolation of an adenine-rich polymer from purified messenger RNA obtained from rabbit reticulocyte polysomes and its relationship to the size of the ribosomes from which the messenger RNA is derived.

Thyroid cytoplasmic ribonucleoprotein particles of very high size containing messenger-like and ribosomal ribonucleic acids.

Cytoplasmic giant‐size ribonucleoprotein particles has been purified from the deoxycholate treated 15000 ×g supernatant of sheep thyroid slices incubated in the presence of [3H]uridine for 0.5 to 8 hours. After treatment of unclarified polyribosomes with 10 mM EDTA, 400 to 1000 S ribonucleoprotein particles have been isolated free of ribosomal subparticles by differential sedimentation. They are heterogeneous in size and buoyant density in CaCl gradients (from 1.38 to 1.52 g/ml, with maxima at 1.52, 1.46, and 1.42).Polyribosome‐associated 400 to 1000 S ribonucleoprotein particles deprived of free ribosomal subparticles contain messenger‐like and ribosomal RNA as shown by the rate of labelling and sucrose gradient centrifugation, polyacrylamide gel electrophoresis and base composition.Particle proteins obtained by 3 M LiCl‐6 M urea treatment separate in polyacrylamide gel electrophoresis into 3 to 4 basic components which disclose the same electrophoretic mobility as some ribosomal proteins. These 3 to 4 protein bands are distinct from total thyroid soluble proteins and from histones. Similar protein species are present in ribonucleoprotein particles diffusing spontaneously from thyroid nuclei in vitro.The artifactual formation of these structures during EDTA exposure or slice incubation has been rendered unlikely by appropriate controls. Although tentative, the possible physiological significance of these particles is briefly discussed.

Nuclear and Cytoplasmic Messenger-like RNA and Their Relation to the Active Messenger RNA in Polyribosomes of HeLa Cells

Nuclear and Cytoplasmic Messenger-like RNA and Their Relation to the Active Messenger RNA in Polyribosomes of HeLa Cells

RNA metabolism in mammalian cells at elevated temperature.

RNA synthesis and metabolism were investigated in Hela cells heated to 42°. At this temperature celluar growth is arrested. After prolongued periods of preheating the initial rate of uridine incorporation into RNA is only slightly affected but no new stable RNA accumulates. After a shift down to 37° the normal pattern of RNA formation resumes.45 S precursor to rRNA is still formed but its normal metabolism to functional rRNA is blocked. No new rRNA appears in cytoplasmic ribosomes. On the contrary, the synthesis of nascent messenger‐like RNA is only slightly affected and non‐ribosomal RNA appears in the cytoplasm at a normal rate. The temperature sensitive event seems thus to be selectively located in the metabolic chain producing functional rRNA.

Rapidly-labelled ribonucleic acid-protein complexes of the thyroid tissue.

The existence of non-ribosomal rapidly-labelled ribonucleoprotein particles has been examined by isopycnic and sucrose gradient centrifugation in nuclear and cytoplasmic subcellular fractions of sheep thyroid tissue. These particles have been described in the saline extract (pH 8.0) of purified nuclei, in EDTA-treated polyribosomes and in postribosomal regions of deoxycholatetreated postmitochondrial supernatant. They have been identified as a special class of ribonucleoproteins rich in proteins by the following criteria: low buoyant densities in CsCl gradients (from 1.36 to 1.41 g/ml, with a predominance at 1.41), DNA-like base composition of their constitutive RNA, increase or decrease of their buoyant densities after trypsin or ribonuclease digestion. Although the specific nature of the constitutive protein(s) is not yet determined, it appears that both nuclei-associated and cytoplasmic (postribosomal and polyribosomal) rapidlylabelled ribonucleoproteins are complexes of messenger-like RNA and protein in a proportion of about 15 to 30% RNA, 85 to 70% protein by weight. RNA of cytoplasmic postribosomal ribonucleoprotein particles is heterogeneous and composed of species ranging from about 6 S to 22 S. It stimulates protein synthesis in the E. coli cell-free system whereas the corresponding ribonucleoproteins are poorly stimulatory. The relationships between these particles and their possible role in messenger RNA function are discussed.

Messenger RNA in avian erythroblasts at the transcriptional and translational levels and the problem of regulation in animal cells

The RNA metabolism in immature duck erythrocytes has been investigated in order to determine the characteristics of messenger RNA (mRNA) in a highly differentiated animal cell. mRNA-like fractions were obtained from polysomes, on the one hand, and from pulse-labeled total cells or isolated nuclei, on the other, and were characterized by sedimentation, labeling kinetics, base composition, and hybridization to homologous DNA. At the translational level, the pulse-labeled RNA from polysomes consists of a predominant species sedimenting with about 9S and of a class of polydisperse material sedimenting between 6 and 28S. Very little ribosomal RNA (rRNA) is synthesized. The 9S RNA has been purified. Its base composition is relatively high in G + C (but different from rRNA or transfer RNA) – as determined, after alkaline hydrolysis, by 32P distribution or spectrophotometric analysis. The polydisperse RNA has a base composition characterized by relatively high proportions of U and A and is similar in this respect to nuclear RNA. Total polysomal RNA hybridizes to homologous DNA. The biological activity tested in a cell-free protein-synthesizing system of Escherichia coli is highest in the 16 to 18S zone of polysomal RNA. The rapidly labeled RNA synthesized at the transcriptional level in the nuclei sediments predominantly in the 30 to 80S zone. Base-composition analysis of this RNA reveals the presence of a predominant fraction of high-U-type RNA and of a small amount of 45 and 32S rRNA precursors. The former fraction — tentatively termed nascent, messenger-like RNA (nascent mlRNA), with respect to its base composition and capacity of selective hybridization — is metabolically more unstable than the precursor rRNA. Hybridization experiments demonstrate that up to 7% of the DNA is homologous to the nascent RNA fractions. Polysomal RNA hybridizes to a much smaller extent and competes only slightly with the heavy nuclear fractions. The significance of this heavy, nascent mlRNA and its eventual role in the regulation of protein synthesis in animal cells is discussed. We conclude that in a highly differentiated cell many more mRNA species are produced than would be expressed phenotypically through protein synthesis in the polysome. A surprisingly large part of the genome is activated, but an important fraction of the transcription products never reaches the sites of protein synthesis. Thus, the spectrum of functional RNA is not defined through synthesis only, but is restricted during metabolism. Under these conditions, control of differentiation is probably not limited exclusively to the transcription of the genome, but is subject to regulation mechanisms operating at the intermediate or translational level.

New method for the purification of polypeptide synthetases. VII

A new method for purification of “polypeptide synthetase” from Alcaligenes faecalis is described in which nucleic acids are removed by purification on DEAE‐cellulose with a recovery of 60–75% of the initial enzymatic activity.The enzyme preparation catalyses the binding of the naturally occuring l‐amino acids to a messenger‐like RNA whose properties have been previously described. The reaction requires the addition of any one of the four nucleoside triphosphates (ATP, GTP, CTP or UTP), which is degraded to the corresponding nucleoside diphosphate and orthophosphate. For certain amino acids, however, the requirement for a nucleoside triphosphate is limited to two of the four.The purified enzyme preparation, unlike crude extracts, does not catalyse an amino dependent ATP‐[32P]PPi exchange. Neither crude extracts, nor purified preparations show detectable exchange between [32P]PPi and GTP, CTP, or UTP in the presence or in the absence of l‐amino acids.The presence of more than one enzyme in the purified preparation was demonstrated by further substantial fractionation of [14C]leucine binding activity and separation of this from [14C]alanine binding activity. However, no fractionation, which was dependent on a specific nucleoside triphosphate, could be achieved.

Gel filtration heterogeneity of Escherichia coli soluble RNA

Gel filtration heterogeneity (Sephadex G100) is observed for sRNA preparations obtained from various strains of Escherichia coli by phenol extraction and elution from DEAE-cellulose using M-sodium chloride. These preparations contain, in addition to the transfer RNA which accounts for 75% of the soluble RNA, ribosomal and messenger-like RNA, both of which are soluble in cold (4°C) M-sodium chloride, plus an RNA containing 122 ± 10% nucleotides per chain having a major nucleotide composition similar to transfer RNA, but unable to accept amino acids under the usual in vitro conditions. Moreover, it is devoid of methylated nucleotides, pseudouridylic acid, reduced pyrimidine nucleotides, and is terminated at each end by a uridylic acid residue, with the 5′ and 3′ termini being phosphorylated and unesterified, respectively.

On the relationship between a post-microsomal fraction and polynucleotide-directed amino acid incorporation by rat-liver ribosomes

1. 1. A fraction (X) obtained by high-speed centrifugation of the post-microsomal supernatant of rat-liver homogenates was shown to contain, besides activating and transfer enzymes, a factor which stimulates the incorporation of amino acids by rat-liver ribosomes in the absence and presence of poly-U. Similarly, the stimulatory effect of polyuridylic acid (poly-U) on the uniformly 14C-labelled phenylalanine incorporation was greatly enhanced by X. The effect of X on the poly-U-stimulated phenylalanine incorporation depended on Mg 2+ concentration and the amount of poly-U added. Evidence is presented indicating that X does not contain messenger-like RNA nor a releasing factor activity or a ribonuclease (EC 2.7.7.16) inhibitor. 2. 2. When freshly prepared ribonucleoprotein particles were preincubated or treated with ribonuclease, their ability to incorporate amino acids was reduced. Optimal reactivation of the treated ribosomal preparations by poly-U was possible only when the incorporation system was supplemented with X. 3. 3. The time course of the poly-U-directed amino acid incorporation indicated that the stimulatory effect of X was progressive and time-dependent. 4. 4. Essentially similar results as regard the X-dependency of the poly-U-mediated phenylalanine incorporation and the time course of this effect were obtained with a polysome and an 80-S fraction isolated from preincubated ribonucleoprotein particles by sucrose density gradient centrifugation. 5. 5. It is suggested that during protein synthesis in vitro X might initiate and/or stabilize the interaction between released free ribosomes and messenger RNA (poly-U or endogenous) resulting in the formation of complexes which are active in incorporating amino acids. The possibility that X may be involved in the process of re-attachment of free ribosomes to polyribosomes is also discussed.