Isolation and partial characterization of poly (A)-containing 7.5S messenger RNA from rat liver mitochondria.

Poly(A)-containing low molecular weight (7.5S) messenger RNA was isolated in a highly purified form from both polyribosomes and post-polysomal supernatant of rat liver mitochondria. Both mRNA's contain rather short poly(A) tracts (40-70 mononucleotides) according to a profile of their elution from poly(U)-Sepharose column with a gradient of formamide concentration. Both mRNA's when added to a preincubated mitochondrial lysate programmed the synthesis of a hydrophobic polypeptide of a molecular weight about 9000 daltons which was soluble in the neutral chloroform-methanol mixture.     

Quantitation and characterisation of poly(A)-containing messenger RNAs from mouse neuroblastoma cells

Comparison of several isolation procedures for neuroblastoma poly(A)-containing mRNAs shows that the highest percentage recovery of undegraded and biologically active messenger RNAs is obtained using proteinase K prior to phenol extraction. The messenger RNAs thus isolated comprise approximately 1.5% of the total ribosomal RNAs and have negligible contamination with 18 and 28 S RNAs. On denaturing polyacrylamide gels they have an average molecular weight of 6.5-10(5) with a range from 2.2-10(5) to 1.53-10(6). The messenger RNAs have an average poly(A) content of 154 nucleotides. They are highly active in wheat germ in vitro protein synthesizing systems, giving as much as 4.3 pmol [35S]methionine incorporation into total protein per mol of mRNA. This is almost as active as a control globin mRNA preparation.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

Methylated constituents of Aedes albopictus poly (A)-containing messenger RNA.

Poly (A)-containing mRNA prepared from cultured mosquito (Aedes albopictus) cells was found to contain methylated 5'-terminal "caps" as well as internal m6A residues. Both type I [m7G(5')ppp(5')Xmp] and type II [m7G(5')ppp(5')XmpYmp] caps were present, at molar ratio of ca five to one. All four common RNA bases were represented in the second position (Xm) of the caps, adenine being the most abundant and N6-methyladenine being absent. The four bases were also represented in the third position (Ym), but here uracil was the predominant base. There was approximately one internal m6A residue for every three caps. These studies demonstrate that mRNA from an invertebrate source can have a methylation pattern comparable with that of mammalian cells in it complexity.     

Characterization and messenger activity of poly(A)-containing RNA from Chlorella.

Poly(A)-containing RNA was isolated by cellulose column chromatography from total RNA extracted from Chlorella fusca var. vacuolata 211/8p. RNA retained by the column was identified as poly(A)-containing RNA because it contained ribonuclease-resistant tracts, 25 to 55 nucleotides in length, from which not less than 80% of base was found to be adenine after acid hydrolysis. The base composition of poly(A)-containing RNA differed from that of RNA (largely ribosomal) which did not adsorb to cellulose, having a higher adenine content and a lower guanine content. Poly(A)-containing RNA was polydisperse including molecules with mobilities from 10S to 40S with a mean of about 20S. In an in vitro system derived from wheat-germ, protein synthesis was stimulated by adding poly(A)-containing RNA from Chlorella. Optimum conditions were established in this system with respect to the amount of poly(A)-containing RNA added and the concentration of KCl and Mg-2+. It is proposed that, in Chlorella, poly(A)-containing RNA includes cytoplasmic mRNA as has been shown for some other eucaryotic organisms.     

Series of 4.5S RNAs associated with poly(A)-containing RNAs of rodent cells.

Uninfected mouse kidney cells and mouse leukemia cells L1210 in cluture contained a series of 4.5S RNAs which was structurally identical to the series of 4.5S RNAs associated with genomic RNAs of murine retroviruses and poly(A)-containing RNAs from virus infected cells. Normal rat kidney cells and baby hamster kidney cells in culture also contained a series of 4.5S RNAs. The strucutre of the 4.5S RNAs from mouse, rat and hamster cells were very similar, but not identical. These 4.5S RNAs were not found in cultured cells of other vertebrates, such as human, monkey, cat, mink, rabbit and chicken cells.     

Fractionation and characterization of rat liver poly(A)-containing RNA.

Three fractions of poly(A)-containing RNA were separated from total rat liver RNA using poly(U)-Sepharose 4B affinity chromatography. The poly(A)-containing RNA fractions were released by thermal elution. Fraction 1, eluted under the mildest conditions, and had poly(A) tracts of approx. 200 AMP units in length which appeared to be associated with poly(U) sequences of 20-50 UMP in length. Fraction 1 appeared to be present mainly in the nucleus and, its size distribution was similar to that of fractions 2 and 3. Fractions 2 and 3 eluted at higher temperatures and were associated mainly with polysomal and microsomal fractions. Poly(U) sequences were absent in fractions 2 and 3 while their poly(A) sequences had a size distribution characteristic of those reported in the mRNA of other organisms.     

The size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

Nucleotide sequences of 4.5S RNAs associated with poly(A)-containing RNAs of mouse and hamster cells.

The nucleotide sequences of 4.5S RNAs associated with poly-(A)-containing RNAs of mouse and hamster cells were determined. These RNAs have 91 to 94 nucleotides, a high content of G (almost 40%) and no modified nucleoside. The 5'-termini are pppG, but the 3'-termini lack uniformity in the number of uridylate residues. These molecules contain two sets of repeating sequences, and a central purine-rich sequence. There is only one base exchange between mouse and hamster 4.5S RNAs. Possible binding sites of these RNAs to poly(A)-containing RNAs are discussed.     

Non-polysomal poly(A)-containing messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina

Non-polysomal poly(A)-containing messenger ribonucleoprotein (mRNP) of Artemia salina has been isolated by thermal chromatography on oligo(dT)-cellulose in moderate (250 mM) and low (50 mM NaCl and 5 mM MgCl2) ionic strength. The purified particles sedimented between 5 S and 30 S and banded at a density of 1.38-1.40 g/cm3 and 1.26-1.27 g/cm3 in CsCl and sucrose isopycnic centrifugation, respectively. The translatability of the mRNP in a cell-free system depended on the conditions of isolation. The protein composition of the free mRNP is independent of the conditions used in oligo(dT)-cellulose chromatography. The proteins have Mr of 87,000, 76,000, 65,000, 50,000, 45,000, 38,000 and 23,500. A specific set of proteins is associated wtih different ribonucleoproteins, although some proteins are present on multiple particles. The main 17 +/- 2-S particle is composed of proteins with Mr of 87,000, 76,000, 45,000 and 38,000. Approximately the same proteins were present on free mRNP and mRNP isolated from non-polysomal mRNP-ribosome complexes. Poly(A)-binding proteins have Mr of 38,000 and 23,500. The 38,000-Mr protein comprised at least 60% of the total mRNP protein. Poly(A)-binding proteins with Mr of 38,000 and 76,000 are also present in a free state in the cytoplasm. A relation between the main poly(A)-binding mRNP protein and the helix-destabilizing protein HD40 [Marvil, D. K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472] is discussed.     

The majority of calf muscle cell messenger RNAs contain poly(A)

Previous studies from our laboratory have investigated messenger RNA metabolism in calf muscle cells in tissue culture. The analysis of mRNA was based on its poly(A) content. We have now examined directly the proportion of mRNA which contains poly(A) in these cells. After separation of poly(A)+ -and poly(A) - -RNA on oligo(dT) -cellulos, the two fractions were translated in a reconstituted, heterologous cell-free protein-synthesizing system and the products were compared with those from the translation of total RNA. The great majority of mRNA form either prefusion or postfusion cultures was poly(A)- containing; quantitative determinations show that about 70-90% of the actin mRNA is poly(A)-containing. In order to determine if a large fraction of the calf muscle mRNA can be translated by a heterologous cell-free system, [3H]-POLY(A)+ -RNA was added to reticulocyte lysates and the formation of initiation complexes was followed. These experiments suggest that the bulk of calf muscle cell mRNA would be utilized in such a system and justify the use of cell-free systems to examine the poly(A) content of total mRNA. Thus, differential polyadenylation does not seem to be an important aspect of mRNA metabolism in cultured muscle cells. The previous study of mRNA in these cells, based on poly(A) content, is apparently a valid measure of overall mRNA metabolism.     

Isolation of poly(A)-containing RNA on synthetic fluorophlogopite (Mica).

Synthetic fluorophlogopite, an aluminosilicate of the same structure as naturally occurring mineral mica in which potassium ions on the basal surface have been replaced by aluminum ions, has the ability to retain polynucleotides irreversibly. This property of Al³⁺-mica was used for irreversible adsorption of poly(U) and subsequent selective adsorption of poly(A)-containing RNA from rabbit reticulocyte polysomes at high salt concentration and its elution by 50% dimethylsulfoxide. The properties of RNA isolated on poly(U)-Al³⁺-mica were studied by sucrose density gradient centrifugation and by stimulation of globin synthesis in an in vitro protein synthesizing system from wheat germ and from Krebs II-ascites cells. The preparation contained 9s RNA species which corresponds to rabbit globin messenger RNA, and under optimal conditions it stimulated protein synthesis more than 100-fold. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that synthesized product was identical with rabbit globin.     

'Cap' structures in maize poly(A)-containing RNA.

Poly(A)-containing RNA was isolated from maize embryos by chromatography on columns of oligo(dT)-cellulose and exhaustively digested with ribonucleases T2, T1, and A. Fractionation of the digests by two-dimensional electrophoresis revealed the presence of three 7-methylguanosine-terminated 'cap structures' of the type m7GpppNp.     

Separation technique for messenger RNAs by use of schizophyllan/poly(A) tail complexation

Schizophyllan (SPG) is one of the water soluble beta-1,3-glucans and has a peculiar molecular recognition capability, namely, the single stranded SPG (s-SPG) can form a stoichiometric complex with certain polynucleotides such as poly(C) and poly(A), although it cannot bind poly(G) and poly(dC) at all. In this paper, we prepared an s-SPG-appended column and made an attempt to separate polynucleotides on the bases of this molecular recognition capability. The s-SPG-appended column trapped only such RNAs that could form the complex with s-SPG but eluted other RNAs which did not form the complex. Encouraged by the results in the model system, we extended the s-SPG-appended column into separation of native messenger RNAs (mRNAs) from a RNA mixture (total RNA) obtained from yeast. Since eukaryotic mRNAs have a poly(A) tail with 150-300 bases, we supposed that the tails would be trapped by the s-SPG-appended column. The results indicate that mRNAs were separated from total RNA in good yield and with high purity. It should be emphasized that this is the first device to separate natural mRNAs without using a dA/dT Watson-Crick-type interaction.     

The effect of age on the properties of poly(A)-containing messenger RNA in Physarum polycephalum

The effect of ageing on the properties of polysomal poly(A)-containing messenger RNA [poly(A)+ mRNA] of Physarum polycephalum has been investigated. Using poly(U)--Sepharose affinity chromatography it was shown that shortening of the poly(A) tract occurred as the age of the mRNA population increased. Analysis of the poly(A) segments by use of polyacrylamide gel electrophoresis, after digestion of polysomal poly(A)+ mRNA molecules with RNAase A and RNAase T1, revealed that their lengths ranged from 140 to 220 nucleotide residues. A reduction in the efficiency of utilization of mRNA for translation as the age of the mRNA population increased was demonstrated by measuring the proportion of poly(A)+ mRNA present in the polysomal fraction as compared with post-polysomal material.     

Characterization of protein components of poly(A)-containing messenger ribonucleoproteins from cryptobiotic gastrulae of Artemia salina

Conditions for the isolation of intact poly(A)⁺mRNP from cryptobiotic gastrulae ofA. salina are described. In the presence of Mg²⁺ ions nucleolytic cleavage occurs in vitro in the vicinity of the 3-poly(A) segment of mRNP during the isolation procedure. The resulting two parts of poly(A)⁺mRNP complex are separated by thermal elution from oligo(dT)-cellulose affinity column. Analysis by SDS-gel electrophoresis of protein components associated with intact poly(A)⁺mRNP has revealed the existence of 20–30 S RNP complex containing five major proteins with M_r 68,000, 53,000, 50,000, 45,000 and 38,000, respectively, but completely lacking the poly(A)-specific M_r 76,000 protein.     

Poly(A)-containing RNA from Petroselinum hortense: Isolation,properties and messenger function in vitro

Cell suspension cultures from parsley (Petroselinum hortense Hoffm.) were labelled in vivo with [2-3H] adenosine. The RNA isolated from the ribosomal pellet was fractionated on an oligo(dT)-cellulose column. Approximately 1.5% of the RNA, representing about 15% of the total radioactivity, was retained at high salt concentrations and eluted at low ionic strength. As determined by two independent methods, this fraction contained poly(A) segments with an average length of about 80 nucleotides. It was active as template in a cell-free system from wheat germ, directing the synthesis of peptides ranging in molecular weight from about 4000-40000 daltons.