Polyadenylated RNA isolated from the archaebacterium Halobacterium halobium.

Polyadenylated [poly(A)+] RNA has been isolated from the halophilic archaebacterium Halobacterium halobium by binding, at 4 degrees C, to oligo(dT)-cellulose. H. halobium contains approximately 12 times more poly(A) per unit of RNA than does the methanogenic archaebacterium Methanococcus vannielii. The 3' poly(A) tracts in poly(A)+ RNA molecules are approximately twice as long (average length of 20 nucleotides) in H. halobium as in M. vannielii. In both archaebacterial species, poly(A)+ RNAs are unstable.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

A modified nucleotide in the poly(A) tract of maize RNA

poly(A)+ RNA was isolated from maize by affinity chromatography on columns of oligo(dT)-cellulose. A modified nucleotide ('X') was detected in ribonuclease T2 digests of the RNA as part of a resistant dinucleotide. The dinucleotide was detected by means of the polynucleotide kinase-mediated transfer of a radioactive phosphate atom from adenosine triphosphate to the 5'-OH position of the dinucleotide. Intact poly(A) tracts were released from poly(A)+ RNA by digestion with ribonuclease T1 and A in a high salt buffer and were isolated by oligo(dT)-cellulose chromatography. The poly(A) preparation was found to consist of a series of polyadenylate fragments which varied in chain length from approximately 17 to greater than 70. The modified nucleotide was shown to occupy an internal position in these poly(A) tracts.     

POLYADENYLIC ACID-CONTAINING RNA FROM RAT BRAIN

A portion of rat brain RNA contains poly(A) sequences and binds to oligo(dT)-cellulose. Young rats have a greater amount of brain RNA which contains poly(A) than do adult animals. The length of the poly(A) sequence from the brain RNA of young animals was shown to be somewhat longer than that from the RNA of adults. The RNA which bound to the oligo(dT)-cellulose was found to be large and heterogeneous, and to be almost free of ribosomal or of small mol. wt. RNAs. When the polysomal RNA which bound to the oligo(dT)-cellulose columns and that which did not were used to prime a cell-free protein synthesizing system there was a noticeable difference in their‘messenger’activity; the RNA which bound to the oligo(dT)-cellulose was much more active than the unbound material. However, in the case of the nuclear RNA there was not as great a difference between the material which was bound and that which was not bound.     

Structure of nuclear ribonucleoprotein: identification of proteins in contact with poly(A)+ heterogeneous nuclear RNA in living HeLa cells

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA- protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.     

Isolation of messenger RNA from polysomes by chromatography on oligo(dT)-cellulose.

A method for the isolation of messenger RNA (mRNA) from polysomes is described. Polysomes are dissolved in a solution containing 0.5 m NaCl and Na dodecyl sulphate and applied to an oligo(dT)-cellulose column. RNA species containing poly(A) sequences are retained by the column, whereas ribosomal proteins and other RNA species are washed off. The column is then eluted with a buffer not containing NaCl. mRNA from HeLa cells and from duck reticulocytes has been fractionated in this way. When fractionated on sucrose gradients, 10 s globin mRNA is obtained in addition to a 20 s component, which can be translated in a cell free system into duck globin. This 20 s RNA is an aggregate of mRNA, which can be disaggregated. Experiments with HeLa cells have shown that the only mRNA species which is not retained by oligo(dT)-cellulose is histone mRNA; this mRNA does not contain a poly(A) sequence.     

Failure to detect "cap" structures in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells.

The structure of the 5'-termini has been investigated in mitochondrial DNA-coded poly(A)-containing RNA from HeLa cells. For this purpose, mitochondrial RNA isolated from cells labeled for 3 hours with [32P]orthophosphate in the presence of 20 microgram/ml camptothecin, and selected for poly(A) content by two passages through oligo(dT)-cellulose, was digested either with the nuclease P1 or with a mixture of RNases: the digestion products were then fractionated by two-dimensional electrophoresis. No "cap" structures were detected under conditions where the presence of such structures in one out of five to ten RNA molecules would have been recognized. It is, therefore, likely that "cap" structures are completely absent in HeLa cell mitochondrial poly(A)-containing RNA.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

Oligo(A) and double-stranded segments in polyadenylated and non-polyadenylated RNA from cytoplasm and nuclei of chick embryo

Chick embryonic RNA was fractionated by affinity chromatography on oligo(dT)-cellulose and poly(U)-Sepharose into three classes: poly(A)+RNA containing poly(A) segments of 100 and more residues, poly(A)-oligo(A)+RNA containing oligo(A) segments of about 25 residues, and poly(A)-oligo(A)-RNA which bound to neither of the beds used and which contained double-stranded segments of 300 and more base pairs. These three classes of RNA were found in cytoplasmic as well as in heterogeneous nuclear RNA. Double-stranded segments in hnRNA, unlike those in cytoplasmic RNA, were intermolecular in nature; this may explain the occurrence of "giant" molecules in hnRNA.     

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2alpha, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.     

Aberrant c-myc RNAs of Burkitt's lymphoma cells have longer half-lives.

BL67 and BL18 are Burkitt's lymphoma cell lines with t(8;14) translocations (the breakpoint is in the first exon and first intron, respectively) in which the mu-heavy chain switch region is fused to the c-myc gene in head to head orientation. In both cell lines only aberrant c-myc RNAs are found. BL67 cells contain two c-myc RNA species of 2.4 and 3.5 kb. The 2.4-kb RNA is initiated at several cryptic promoters in the first intron. The 3.5-kb RNA is transcribed from the immunoglobulin heavy chain anti-sense strand across the breakpoint of the translocation into the first exon of the c-myc gene and is then normally spliced using the physiological splice donor and acceptor sites of the c-myc gene. BL18 contains c-myc RNA of 2.4 kb initiated at cryptic promoters in the first intron and additional RNAs of 0.90 kb and 0.74 kb transcribed from the dual c-myc promoters on the reciprocal fragment of the translocation. The cytoplasmic turnover of these RNAs differs significantly from that of the normal c-myc message. The 3.5-kb RNA of BL67 cells and the 0.90-kb and 0.74-kb RNAs of BL18 cells, which are both hybrid molecules consisting of c-myc and immunoglobulin sequences, have a half-life of several hours in contrast to the normal c-myc message with a half-life of 15 min. The aberrant 2.4-kb c-myc RNAs of BL67 and BL18 cells are also more stable than the normal c-myc message and disappear with a half-life of 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells.

Ribonucleoprotein particles containing either heterogeneous nuclear RNA or polyribosomal messenger RNA were isolated from growing HeLa cells in order to compare their respective protein components. The major obstacle to analysing the proteins bound to HeLa cell mRNA proved to be the cosedimentation of a large fraction of the mRNP² particles with ribosomal subunits following puromycin or EDTA disassembly of polyribosomes. This was circumvented by oligo(dT)-cellulose chromatography, in which essentially all of the ribosomal subunits passed through the column without retention, while approximately 80% of the pulse-labeled, poly(A)-containing mRNP became bound and could be eluted with formamide. Polyacrylamide gel electrophoresis of the non-bound fraction (ribosomal subunits) revealed polypeptides between 15,000 and 55,000 molecular weight, with no detectable components greater than 55,000. The oligo-(dT)-bound mRNP contained a much simpler protein complement, consisting of three major components having molecular weights of 120,000, 76,000 and 52,000.In the case of the nuclear ribonucleoprotein particles that contain heterogeneous nuclear RNA, oligo(dT)-cellulose chromatography revealed two classes of particles. The first contained 10 to 20% of the hnRNA, did not bind to oligo(dT)-cellulose in 0.25 m-NaCl, 10 mm-sodium phosphate buffer, pH 7.0 (4 °C), and contained primarily a single polypeptide component having an estimated molecular weight of 40,000 (“informofers”). A second population of hnRNP particles comprised approximately 80% of the hnRNA, displayed strong binding to oligo(dT)-cellulose at 0.25 m-NaCl, and contained a very complex population of proteins, having molecular weights between 40,000 and 180,000, the same as unfractionated hnRNP. The results indicate that, at the resolution of gel electrophoresis and at the sensitivity of Coomassie blue dye, the proteins bound to HeLa cell hnRNA are qualitatively distinct from those bound to polyribosomal mRNA and, in addition, that the hnRNP proteins are the more complex of the two. These results are discussed in relation to the possible nucleotide sequence elements in hnRNA and mRNA to which these specific proteins are bound.     

Encephalomyocarditis virus RNA: variations in polyadenylic acid content and biological activity.

Encephalomyocarditis (EMC) viral RNA was isolated from purified virus grown in Ehrlich ascites tumor cells. The viral RNA was found to contain polyadenylic acid [poly(A)] regions that were very heterogeneous in length. Chromatography of the EMC viral RNA on oligo(dT)-cellulose columns separated the RNA into three distinct fractions (peaks 1 to 3). Approximately 20% of the EMC viral RNA appeared as peak 1, 40% as peak 2, and 40% as peak 3. The RNA in each fraction appeared to be intact as shown by co-sedimentation with 35S unfractionated EMC viral RNA in SDS-sucrose density gradients. Approximately 95 to 100% of peaks 1 and 3, and 60 to 70% of peak 2, reappeared at the same elution position after rechromatography on oligo(dT)-cellulose. The RNA in peak 1 contained poly(A) with an average length of 16 nucleotides, peak 2 contained poly(A) with an average of 26 nucleotides, and peak 3 contained an average of 74 nucleotides in its poly(A) region. The distribution in the three fractions, as well as the average length of the poly(A) moieties, was relatively unaffected by changes in the cell suspension medium used during infection. Finally, each of the three viral RNA fractions was assayed for biological activity using an infectious RNA assay on L-cell monolayers. Infectivity of the viral RNA was found to increase with poly(A) length, with peak 3 viral RNA being approximately 10 times more infectious than peak 1 viral RNA.     

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts.

An abrupt concommitant increase in total cellular RNA and protein was observed as cultured human diploid fibroblasts entered the senescent phase of their in vitro lifespan. DNA content remained stable from early to final passages. Fractionation of cellular RNAs by polyacrylamide gel electrophoresis demonstrated an increase in both 28S and 18S ribosomal and 4S transfer RNAs in these senescent cells. Separation of poly(A) RNA (mRNA) by oligo(dT)-cellulose chromatography suggests an increase in this group of RNAs. However, the ratios of 28S to 18S rRNAs, tRNA to rRNA, and mRNA to total cellular RNA were not significantly different in cells before and after senescence, indicating that the overall increases in total cellular RNA was not due to an accumulation of a single RNA class.     

Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns.

A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion.     

Selection of prosomes and prosomal RNA by immobilized viral RNAs

Viral messengers were used to select and purify prosomes and prosomal RNA from subribosomal fractions of HeLa cells and mouse erythroblasts. Adenovirus mRNA immobilized on oligo(dT)-cellulose and tobacco mosaic virus RNA (TMV) sedimenting in sucrose gradients associated strongly with prosomes at high salt conditions forming intermolecular RNA-RNA hybrids between prosomal RNA and viral RNA. Hybrid selection of small cytoplasmic RNAs with immobilized TMV-RNA revealed a RNA species migrating at the same position as prosomal RNA. The possible existence of a box-like sequence involved in hybridization will be discussed.     

Characterization of foot-and-mouth disease virus types O and Asia 1 RNA

Poly (A) RNA was isolated from foot-and-mouth disease virus-infected cells by oligo (dT)-cellulose chromatography. One-dimensional oligonucleotide mapping of virus-induced poly (A) RNA indicated major differences between virus types O and Asia 1. Base composition analysis of virus-induced RNA showed no significant differences between types O and Asia 1.