Nuclear RNA Species Research Articles

Subcellular location of precursors to small nuclear RNA species C and D and of newly synthesized 5 S RNA in HeLa cells

The nuclear-cytoplasmic partition of newly synthesized C, D and 5 S RNAs of HeLa cells was studied with aqueous and non-aqueous methods of cell fractionation. The level of briefly labeled 5 S RNA in cytoplasmic fractions prepared by a non-aqueous method is lower than in cytoplasmic fractions obtained by an aqueous method. The reverse is true in nuclear fractions. At the same time, with both aqueous and non-aqueous cell fractionation, the level of briefly labeled precursors to RNAs C and D is similarly high in cytoplasmic fractions and low in nuclear preparations. This suggests that the previously reported finding that RNA species C and D are cytoplasmic during the first minutes of their lifetime, may represent an in vivo phenomenon, instead of an artifactual elution from nuclei during fractionation.

Rat liver nuclear skeleton and small molecular weight RNA species.

Small molecular weight RNA species (smwRNAs) were studied in rat liver nuclei with and without chromatin as well as with and without nuclear envelope and nucleoplasm. From all the species identified, only two, N5 and 5Sb, were related to ribosomes. The others were localized exclusively in the nuclear skeleton or the spongelike network that was described in the preceding communication. This network or protein matrix contains a less abundant but exclusive set of molecules designated 5Sa, N1, and 4.5S, as well as other more abundant molecules which also exist in rat liver endoplasmic reticulum but not in polysomes or postribosomal RNP complexes. The smwRNAs behave like HnRNA; they remain located in the nuclear skeleton when nuclei are deprived of nucleoplasm and chromatin. With the information presently available, it is not possible to know whetherer both species are in the same or different RNP complexes and whether some of the smwRNAs contribute to the architecture of the nuclear skeleton. Distinct from any other nuclear RNA species, smwRNAs have two unique properties: facility of extraction, and resistance to nuclear ribonuclease digestion.

Potentiation by 2'-deoxycoformycin of the inhibitory effect of xylosyladenine on nuclear RNA synthesis in L1210 cells in vitro

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), on the inhibitory effect of 9-β- d-xylofuranosyladenine (XA) on nuclear RNA synthesis was examined in L1210 cells in vitro. Pretreatment of cells for 15 min with a 100 per cent inhibitory dose (1 × 10 −6 M) of dCF resulted in approximately a 3- to 8-fold reduction in the 50 per cent inhibitory dose ( id 50) of XA for [ 3H]uridine and [ 3H]thymidine incorporation into RNA and DNA respectively. The id 50 for XA for RNA synthesis vs DNA synthesis was 5-fold lower in the absence of dCF and 20-fold lower in the presence of dCF, indicating the greater sensitivity of RNA synthesis to this inhibitor. Fractionation of nuclear RNA into rRNA, non-poly(A) heterogeneous RNA and poly(A)heterogeneous RNA revealed the latter species of RNA to be less sensitive to XA in the absence of dCF; however, in the presence of dCF, all three species of nuclear RNA showed similar sensitivities. Nuclear polyadenylic acid synthesis was among the most sensitive RNA fractions to XA, and was also inhibited to a greater degree by pretreatment of cells with dCF. These results indicate that XA is potentiated markedly by inhibition of adenosine deaminase, and that deamination serves as a major catabolic route for this drug.

Sequence diversity studies of rat brain RNA: effects of environmental complexity on rat brain RNA diversity.

Abstract—The sequence complexities of rat brain RNAs were measured by RNA‐driven hybridization reactions with nonrepetitive rat DNA. The total sequence complexity of rat brain HnRNA was estimated to be 6.61 x 108 nucleotides while rat brain poly(A)‐mRNA sequence complexity was 1.32 x 108 nucleotides. Up to 33.7% of the total transcribable nonrepetitive DNA was expressed in the nuclear RNA. The nuclear RNAs reacted with complex kinetics over at least 4.5 decades of equivalent Rot (product of RNA concentration and time), with an apparent division into three major RNA abundance classes. The abundances of average nuclear RNA species in these classes ranged from 2.9 x 109 copies per brain (18 copies per cell) to 2.4 x 105 copies per brain (1.5 x 10−3 copies per cell). Poly(A)‐mRNA diversity was sufficient to code for 8.8 x 104 polypeptides of 50,000 daltons. There were also three distinguishable abundance classes of poly(A)‐mRNA with frequencies which ranged from 8.9 x 108 copies per brain (5.5 copies per cell) to 3.2 x 105 copies per brain (2 x 10−3 copies per cell). Evidence for compartmentalization of expressed RNA sequences supports the concept that the extensive morphological and physiological specialization evident in brain parallels extensive transcriptional specialization at the cellular level.Brain and liver RNA diversities were measured under double‐blind experimental conditions in three experiments with rats raised in experientially enriched (EC) or impoverished (IC) environments. Liver RNA diversity of EC animals was not different from that of IC animals. Brain total RNA of EC animals, at equivalent R0ts of 184,000‐212,000, hybridized to 10.6% of rat unique DNA (mean of 11 separate groups of rats). The average hybridization of brain RNA from 11 groups of IC animals in the same range of equivalent Rot was 8.2% of the unique DNA. The difference was statistically significant at P < 0.02. Of 10 groups of 3 littermate pairs (paired across EC and IC groups) brain RNA diversity was greater in EC animals in 8 cases. A least squares fit of the kinetics of hybridization to a pseudo first order reaction showed that, at saturation, the RNA from brains of EC animals was complementary to 16.4% of the unique DNA while that from IC animals was complementary to 9.1%. This difference was found in the least abundant class of rat brain RNA. These changes in sequence diversity reflected either an increase in the number of diverse RNA species present or an increase in the number of copies of certain RNA species in the rats raised in an enriched environment. A change in brain RNA populations of this magnitude may reflect a significant difference in brain function between EC and IC animals.

A new class of small nuclear RNA molecules synthesized by a type I RNA polymerase in hela cells

A new class of previously undetected small RNA molecules with a range of discrete sizes between 6S and 10S has been identified in HeLa cell nuclei. They differ in size and location from the previously described small nuclear RNA species (snRNA). These RNA molecules were initially found by selective RNA labeling in vitro in isolated nuclei. The in vitro products migrate in gel electrophoresis in the region from 6–10S with predominant components between 8S and 10S. They are labeled in the presence of very high concentrations of α-amanitin (150–400 μg/ml), suggesting they are synthesized by a type I polymerase. Unlike the major polymerase I product, ribosomal precursor RNA, however, these molecules are found in the nucleoplasm and their labeling is not affected by pretreatment of cells with low concentrations of actinomycin D (0.04 μg/ml). Their formation by a presumptive polymerase I type of enzyme is the basis of their tentative designation as small nuclear polymerase I (snPI) RNAs. The snPI RNA molecules appear to be associated with chromatin and the nuclear matrix. They can be selectively eluted from nuclei leaving most of hnRNA behind. This association is used as the basis of fractionation procedures which separate these molecules from hnRNA and permit the demonstration of the synthesis of at least the most predominant of these RNA molecules in vivo. w

Nuclear ribonucleoprotein particles in the cellular slime mold Dictyostelium discoideum.

The nuclear ribonucleoprotein (RNP) particles containing rapidly labeled RNA were isolated from interphase cells of the cellular slime mold Dictyostelium discoideum and characterized. The size of the isolated RNP particles was small (10S to 50S) in comparison with that of nuclear RNP particles found in higher eukaryotes. These small RNP particles do not seem to be artifacts due to degradation during the preparation of nuclear extracts. The rapidly labeled RNA of the nuclear RNP particles was heterogeneous in size and a considerable amount contained polyadenylic acid sequences. Synthesis of RNA in the nuclear RNP particles was resistant to a relatively high concentration of actinomycin D. The protein component of the RNP particle consists of at least four proteins with molecular weights of 80,000, 66,000, 60,000, and 42,000. Thus it is suggested that almost all of the nuclear RNP particles containing rapidly labeled RNA in interphase cells are RNP complexes consisting of Heterogeneous nuclear RNA and several protein species.

The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke.

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 X 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 X 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 X 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 X 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.

Synthesis methylation, and capping of nuclear RNA by a subcellular system.

A subcellular system is described which is capable of in vitro synthesis of large nuclear RNA and the formation of both cap I [m7G(5')pppXmpYp] and capII [m7G(5')-pppXmpYmpZp] structures. This system, which consists of partially purified intact nuclei and residual cytoplasmic tags, carries out both guanosine addition, utilizing GTP, and the appropriate methylation reactions, utilizing S-adenosylmethionine as the methyl donor. The general structure of the caps was verified by analyses of methylated derivatives recovered after RNase T2 hydrolysis and after digestion with P1 nuclease, bacterial alkaline phosphatase,and nucleotide pyrophosphatase. Cap formation in large nuclear RNA species was found to be closely associated with transcription, as indicated by alpha-manitin sensitivity and a requirement for the presence of all four nucleoside triphosphates. Recovery of a class of cap II structures, in which only the methyl group at position Y is labeled, as well as capII structures in which all methylated constituents are labeled, indicates the presence of at least two independent methylation events in the in vitro system.

The function of the 5' cap of mRNA and nuclear RNA species.

The function of the 5' cap of mRNA and nuclear RNA species.

Nucleo-cytoplasmic relationships of high-molecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain.

The metabolism of high-molecular-weight RNA in the nuclear and cytoplasmic fractions of newborn and adult rat brain was investigated after the intracranial administration of [32P]Pi. In young brain, a considerable proportion of the newly synthesized radioactive RNA is transferred to the cytoplasm, in contrast with the adult brain, where there appears to be a high intranuclear turnover. Electrophoretic analysis of the newly synthesized RNA showed that processing of the rRNA precursor to yield the 28S and 18S rRNA may be more rapid in the adult than in the young, although most of the adult rRNA in the nucleus is not transferred to the cytoplasm. In young brain, processing is probably tightly coupled to transport of rRNA into the cytoplasm, so that 28S and 18S rRNA are not subjected to possible degradation within the nucleus. Polyadenylated RNA turns over in concert with high-molecular-weight RNA in the nuclei of the adult rat brain. In the cytoplasm the polyadenylated RNA has a higher turnover rate relative to rRNA. In the young brain the polyadenylated RNA is transferred to the cytoplasm along with rRNA, although polyadenylated RNA is transported into the cytoplasm at a faster rate. The nuclear and cytoplasmic polyadenylated RNA species of young brain are larger than their corresponding adult counterparts. These results suggest that there are considerable changes in the regulation of the nucleo-cytoplasmic relationship of rRNA and polyadenylated RNA during the transition of the brain from a developing replicative phase to an adult differentiated and non-dividing state.

Differential subnuclear distribution of polyadenylate-rich ribonuclei acid during induction of egg-yolk protein synthesis in male Xenopus liver by oestradiol-17 beta.

A 4-8-fold increase in the rate of hepatic nuclear RNA synthesis occurred within 11 h after a single injection of oestradiol-17 beta to male Xenopus to induce egg-yolk protein synthesis. 2. By using a gentle procedure for fractionating nuclei into their major structurally different components [J. R. Tata& B. Baker (1974) Exp. Cell Res. 83. 111-124], it was found that the hormone-induced increase in the total amount of newly made RNA was associated with a 2-10-fold increase in the poly(A) content of nuclear RNA. 3. When the poly (A) content of nuclear RNA was determined by hybridization to poly[3H](U) or specific binding to oligo(dT)-cellulose, most of the increase (10-fold) in poly (A) content of newly synthesized RNA was associated with the euchromatin fractions, whereas the increase was less marked in the other subnuclear fractions. 4. Resolution of nuclear RNA into poly (A)-poor and poly(A)-rich RNA species by chromatography on oligo(dT)-cellulose, followed by polyacrylamide-gel electrophoresis with sodium dodecyl sulphate or in the pressence of 99% formamide, revealed that the hormone caused a preferential enhancement of high-molecular-weight (25S-60S) poly (A)-rich HnRNA (heterogeneous nuclear RNA,) much of which was associated with euchromatin and not with the nuclear sap. 5. Induction of vitellogenin in male frogs was in particular characterized by the appearance of a high-molecular-weight polyadenylated component exhibiting a peak at 35-36S, i.e. a molecular weight of approx. 2.05x10(6)+/-0.15x10(6). Although there is no evidence as yet that such a polyadenylated high-molecular-weight nuclear RNA species contains sequences corresponding to vitellogenin mRNA, it is possible that a high proportion of the most stable form of the putative nuclear precursor to vitellogenin mRNA induced by oestrogen in male Xenopus liver may be only marginally bigger than the cytoplasmic mRNA, and may at any one time be predominantly associated with the euchromatin fraction.

Ribosomal and informational ribonucleoprotein complexes of animal cells. Study on rat liver ribonucleic acids as constituents of ribonucleoprotein complexes by chromatography on nucleoprotein-celite columns.

A novel method of RNA fractionation has been developed. Nuclear and cytoplasmic rat liver RNA species were fractionated as constituents of corresponding ribonucleoprotein particles, which were previously adsorbed on a Celite-column by their protein component. The fractionation is based on a dissociation of the particles (linear concentration gradient of LiCl and urea with subsequent temperature gradient), which results in a gradual release of the RNA molecules from ribonucleoprotein complexes. Thus the fractionation is in accordance with the tightness of the RNA-protein bonds. A gradient elution of RNA from a nucleoprotein-Celite column permitted fractionation of both ribosomal and rapidly labelled non-ribosomal RNA. The latter, both nuclear and cytoplasmic, could be separated by chromatography on nucleoprotein-Celite columns into two main fractions (components I and V). In cytoplasmic RNA components I and V presumably correspond to mlRNA (messenger-like RNA of free cytoplasmic particles) and mRNA (template RNA associated with ribosomes) respectively.

Structural and transcriptional features of the mouse spermatid genome.

A whole-mount electron microscope technique has allowed direct visualization of the transcription process in mouse spermatids. Thes observations have been supported by light and electron microscope autoradiographic techniques that employ [3H]uridine and [3H]arginine in attempts to clarify mechanisms of RNA synthesis and their relationship to nuclear histone changes throughout spermiogenesis. Early spermatid genomes are dispersed almost completely, whereas in later spermiogenic steps the posterior or flagellar nuclear region is readily dispersed and the anterior or subacrosomal nuclear region remains compact. Display of genome segments permits identification of regions where transcription complexes, presumably heterogeneous nuclear RNA species, are seen related to chromatin. These complexes appear as ribonucleoprotein chains, some of them of considerable length, decreasing progressively in number in late spermiogenic steps. This decrease coincides with diminishing rates of [3H]uridine incorporation. Two distinct patterns of chromatin have been identified: a beaded chromatin type associated with transcription complexes encounterd in early spermatids; and a smooth chromatin type not involved in transcriptive activity observed in advanced spermiogenic genomes. Protein particles staining densely with phosphotungstic acid become apparent in nuclei of spermatids after [3H]arginine incorporation becomes significant. There is no structural or autoradiographic evidence for the presence of nucleoli during spermiogenesis. From these data and from previous experimental findings, we conclude that: (a) spermatogonia, spermatocytes and Sertoli cells are transcriptionally expressed into heterogeneous nuclear RNA and preribosomal RNA species whereas transcription in spermatids is predominantly heterogeneous nuclear RNA; and (b) the modification of the chromatin patterns in late spermiogenic steps indicates a stabilized genome that restricts transcriptive functions.

Distribution of 3H-uridine-5 into brain RNA species of rats exposed to various training tasks—An electrophoretic analysis

Operant schedules were used to isolate component parts of a training task and specific activities were determine for nuclear and cytoplasmic RNA species separated by polyacrylamide disc gel electrophoresis. Rats exposed to a stimulus or schedule change incorporated more radioactivity into nuclear rRNA and mRNA and cytoplasmic rRNA and tRNA than littermates exposed to no change from baseline training. Rats developing a change in response probability to a stimulus in the environment incorporated more radioactivity into cytoplasmic mRNA.

Synthesis of RNA containing polyadenylic acid in resting and stimulated human lymphocytes

Human peripheral lymphocytes have been cultured with and without a mitogen, leucoagglutinin, and the labelling of nuclear and cytoplasmic RNA selected by oligo(dT) cellulose have been studied. Leucoagglutinin stimulates incorporation of [ 8H]thymidine in DNA and [ 8H]uridine in RNA by about 7-fold. 1. 1. Nuclear RNA of lymphocytes contains about 6% species having an attached poly(A) segment while the figure for cytoplasmic RNA is 4%. Stimulation does not change these figures. 2. 2. Labelling of nuclear and cytoplasmic RNA with a poly(A) segment is about 3 times greater than that of RNA lacking poly(A). The relative labelling rates remain roughly equal on stimulation. 3. 3. The proportion of radioactivity in poly(A) containing nuclear RNA species remains unchanged as compared to the total radioactivity in nuclear RNA, about 20%, between incorporation times of 45 min and 12 h. In contrast, the relative radioactivity in poly(A) containing cytoplasmic RNA decreases from 45 to 18%. Stimulation does not affect these proportions. 4. 4. Agarose-acrylamide gels of radioactive RNA samples reveal quantitative differences between fractions obtained from resting or stimulated cells. These data suggest that activation of lymphocytes with mitogens leads to increased incorporation of radioactive uridine into mRNA and rRNA in equal proportions.

Nucleotide sequence homology between a virus-specific 6S RNA and various RNA species from adenovirus-2-infected KB cells.

To investigate the metabolic fate of a 6S RNA (VA-RNA), which appears in human cell lines only after infection with adenovirus, various species of RNA from the cells at different stages of infection were tested for the presence of nucleotide sequence homologous to this RNA. RNA extracted from subvellular fractions of the cells was further fractionated by sucrose gradient centrifugation and analysed by acrylamide gel electrophoresis or by competition against 32P-labeled VA-RNA in hybridization with adenovirus DNA. The results pointed to the following conclusions. (1) VA-RNA is synthesized in the cells throughout the entire period after infections, starting before the onset of viral DNA synthesis. (2) VA-RNA does not participate in protein synthesis as a messenger RNA or a precursor of transfer RNA. (3) The nucleotide sequence homologous to VA-RNA exists as a part of certain species of nuclear high molecular weight RNA in the infected cells.

Transcription sites in spread meiotic prophase chromosomes from mouse spermatocytes.

Mouse spermatocytes at pachytene stage have been examined by whole-mount electron microscope techniques complemented with autoradiography as an approach for visualizing their transcriptive activity. Structural elements of meiotic bivalents, such as synaptonemal complexes and chromatin fibers, have been satisfactorily displayed in the total set of autosomal and sexual bivalents in single spermatocytes. Adequate preservation of the entire set of bivalents has provided a basis for recognition of sites where presumptive preribosomal RNA and heterogeneous nuclear RNA species are being transcribed at different segments of autosomal bivalents. Nucleoli attached to the basal knob region where nucleolar organizer cistrons are assumed to be located and ribonucleoprotein fibrils associated with distinct chromatin loops have been recognized. These structural findings have been correlated with display of [(3)H]uridine incorporation sites in thin-section and whole-mount electron microscopy autoradiographic preparations. A low transcriptive activity of the sexual bivalent contrasted with extensive gene expression in autosomal bivalents. Each sex chromosome shows a double axial core. A short region of pairing with a synaptonemal complex joins the two chromosomes at one end. We conclude that variations in the rate of RNA synthesis throughout meiotic prophase stages in the mouse are expressed as fluctuations in the amount and distribution of distinct RNA species at specific segments of the bivalents.

Stimulation of ribosomal and DNA-like RNA synthesis by α-methyltryptophan

Rapidly labeled nuclear RNA from rat liver was fractionated by methyl albumin kieselguhr chromatography into the ribosomal and DNA-like RNA components. Injection of 100 mg/kg of α-methyltryptophan into rats stimulated the labeling by [ 3H] orotic acid of both species of nuclear RNA; there was a greater effect on the ribosomal than on the RNA-like fraction. This stimulatory effect of α-methyltryptophan could not be accounted for by an increase in the uptake of the labeled nucleoside by the liver, nor by the increase in the specific activity of the nucleoside and nucleotide pool of the liver.

Globin messenger RNA synthesis and processing during haemoglobin induction in friend cells. I. Evidence for transcriptional control in clone M2

On exposure to 1.5–2% dimethylsulphoxide (DMSO) the M2 clone of the Friend erythroleukaemic cell is induced to form large amounts of haemoglobin. Complementary DNA (cDNA) synthesised on a template of mouse globin mRNA was used to measure the concentration of mRNA in RNA from different cell compartments. The globin mRNA content of polysomes increased 50- to 100-fold during induction whereas the globin mRNA content of nuclear RNA increased only 5- to 6-fold. The greatest increase, more than 8-fold, occurred in the largest nuclear RNA species. RNA made on chromatin templates was similarly tested. No globin mRNA sequences could be measured in this RNA from uninduced cells but significant amounts were found in chromatin-dependent RNA from induced cells. It is concluded that there is control of transcription of the globin gene in this cell and probably also control at a post-transcriptional level.

Low molecular weight nuclear RNA in human lymphocytes: A comparison of PHA-treated and control cells

Seven species of low molecular weight nuclear (LMN) RNA were identified in human peripheral lymphocytes. These were designated as A, B, C, D, D′, E and F. The same 7 species of LMN RNA were obtained from resting lymphocytes and from lymphocytes exposed to PHA for 16 and 60 h, respectively. Thus, no detectable qualitative changes were seen in the spectrum of LMN RNAs during PHA-induced lymphocyte transformation. However, the amount of species A, D-D′, E and F per nucleus in fully transformed cells was greater than in untreated lymphocytes. This increase had not yet occurred after 16 h treatment with PHA. 3H-Uridine was incorporated into all species of LMN RNA of resting and PHA-treated lymphocytes. Furthermore, all species of LMN RNA except C (5S RNA) were methylated in both resting and transformed cells. The 3H and 14C specific activities of LMN RNAs following an 8 h exposure to 3H-uridine and 14C-methyl methionine were higher in PHA-treated cells than in untreated lymphocytes. For several species of LMN RNA (A, D-D′, E, F) the highest 3H and 14C spec. act. were observed after 16 h exposure to PHA. The possibility that quantitative alterations in the synthesis and methylation of LMN RNAs may occur during lymphocyte transformation is discussed.