Synthesis Of Heterogeneous Nuclear RNA Research Articles

Aldosterone Modulates Steroid Receptor Binding to the Endothelin-1 Gene (edn1)

Aldosterone and endothelin-1 (ET-1) act on collecting duct cells of the kidney and are important regulators of renal sodium transport and cardiovascular physiology. We recently identified the ET-1 gene (edn1) as a novel aldosterone-induced transcript. However, aldosterone action on edn1 has not been characterized at the present time. In this report, we show that aldosterone stimulated edn1 mRNA in acutely isolated rat inner medullary collecting duct cells ex vivo and ET-1 peptide in rat inner medulla in vivo. Aldosterone induction of edn1 mRNA occurred in cortical, outer medullary, and inner medullary collecting duct cells in vitro. Inspection of the edn1 promoter revealed two putative hormone response elements. Levels of heterogeneous nuclear RNA synthesis demonstrated that edn1 mRNA stimulation occurred at the level of transcription. RNA knockdowns corroborated pharmacological studies and demonstrated both mineralocorticoid receptor and glucocorticoid receptor participated in this response. Aldosterone resulted in dose-dependent nuclear translocation and binding of mineralocorticoid receptor and glucocorticoid receptor to the edn1 hormone response elements. Hormone receptors mediated the association of chromatin remodeling complexes, histone modification, and RNA polymerase II at the edn1 promoter. Direct interaction between aldosterone and ET-1 has important implications for renal and cardiovascular function.

Inhibition of Transcription Selectively Reduces the Level of Ubiquitinated Histone H2B in Chromatin

The effect of inhibiting transcription and/or replication on the steady state levels of the ubiquitinated histone isoforms was investigated. We show that treatment of chinese hamster ovary (CHO), monkey kidney (COS), human endometrial carcinoma (Hec-50 and Ishikawa) cells with actinomycin D and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the levels of ubiquitinated (u) H2B, but not uH2A, uH2A.Z, polyubiquitinated H2A or a novel ubiquitinated histone species, in the chromatin of these cells. The level of the ubiquitinated histones was not affected when synthesis of DNA was arrested. These results show that, in general, maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription.

Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription

The relationship between transcription and ubiquitination of the histones was investigated. Previous studies have shown that ubiquitinated (u) histone H2B and, to a lesser extent, mono- and polyubiquitinated histone H2A are enriched in transcriptionally active gene-enriched chromatin fractions. Here, we show that treatment of T-47D-5 human breast cancer cells with actinomycin D (10 micrograms/mL) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the level of uH2B, but not uH2A, uH2A.Z, or polyubiquitinated H2A, in chromatin. Treatment of the cells with low levels (0.04 micrograms/mL) of actinomycin D slightly reduced the level of uH2B, suggesting that inhibition of ribosomal RNA synthesis does not have a profound effect on the level of uH2B in chromatin. The level of the ubiquitinated histones was not affected by treating the cells with inhibitors of DNA synthesis (sodium butyrate or aphidicolin), but heat-shock treatment resulted in the loss of all the monoubiquitinated histone species. These results demonstrate that maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription, particularly the synthesis of hnRNA. Thus, histone H2B would be ubiquitinated when the nucleosome was opened during transcription. Ubiquitination of histone H2B may impede nucleosome refolding, facilitating subsequent rounds of transcription.

Alterations in polyadenylic acid-containing messenger RNA synthesis of brain polysomes after seizures of seizure-susceptible E1 mice.

The effect of seizures on synthesis of the polyadenylic acid (poly(A]-containing messenger RNA (mRNA) isolated from brain polysomes in a genetically seizure-susceptible E1 mouse was studied in vivo. The seizure in the E1 mice was induced by tossed-up stimulation. Immediately after the seizure ceased, the labeled orotic acid was injected into the brain. The incorporation rates of labeled orotic acid into poly(A)-containing mRNA isolated from polysomes are represented as the specific radioactivity (SR) (dpm/mg RNA) and the relative specific radioactivity (RSR) (dpm/mg RNA/dpm/mumoles of acid soluble uridine-5'-monophosphate (UMP]. Both the rates were reduced to 70% in SR and 65% in RSR at 1 h after the seizures. This reduction was gradually recovered to the level of interictal E1 mice at 6 h. The seizure-induced alterations are not attributable to the difference in the uridine nucleotide pool because the SR of UMP was not significantly affected by the seizure. The peak of labeled poly(A)-containing mRNA by analysis of gel electrophoresis displaced towards a lower molecular weight at 1 h after the seizures. The RNA showed a higher ratio of AMP and UMP per GMP and CMP in nucleotide composition, implying that this RNA is identical with DNA. These results suggest that the temporary decrease found in cytoplasmic mRNA synthesis induced by the seizures of E1 mice appears to be a result of impaired transcriptional processes in heterogeneous nuclear RNA synthesis and that the smaller mRNA coding for protein associated with seizures is newly synthesized during the postictal period.

Effect of convulsions of the synthesis of heterogeneous nuclear RNA associated with polyadenylate and oligoadenylate sequences from El mouse brain as a convulsive strain

Animals from the El (susceptible to seizures) and ddy (nonsusceptible) mouse strains were subjected to vestibular stimulation by tossing. After convulsions in the El mice, both the stimulated El mice and ddY mice were intracranially injected with [ 14C]- and [ 3H]adenosine, respectively. In the control experiment, nonstimulated El and ddY mice received radioactive adenosines in the same manner. The rate of incorporation of adenosine into brain nuclear RNA, expressed as a percentage of the 3H: 14C ratio, was reduced to an average of 68% at 15 min after convulsions, then increased and reached a control value at 5 h. This reduction in nuclear RNA synthesis was not due to alteration of the adenosine triphosphate pool. Gel electrophoresis of RNA revealed no obvious differences in the labeling distribution between El and ddY mice, but the synthesis of RNA species larger than 35S in heterogeneous nuclear RNA (HnRNA) was impaired in convulsed El mice. Nuclear resistant segments of HnRNA with both T 1 RNase and RNase A, were chromatographed with poly(U)-Sepharose followed by urea-polyacrylamide gel electrophoresis. The ologo(A) and poly(A) segments consisted of 29, 19, and 11, and 203, 135, and 69 nucleotides, respectively. The convulsions of El mice reduced the incorporation of radioactive adenosines into oligo(A) and poly(A) segments, suggesting that they inhibited transcription as well as polyadenylation within HnRNA.

Effect of size of poly(A) segments on inhibition of heterogeneous nuclear RNA synthesis in the rat brain by cordycepin

Effect of size of poly(A) segments on inhibition of heterogeneous nuclear RNA synthesis in the rat brain by cordycepin

Serum enhances the cycling and survival of HeLa cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

We have defined the growth kinetics of HeLa cell populations by determining the frequencies of mitoses and deaths and the lengths of intermitotic intervals. This was done by time-lapse cinemicrography. Untreated control cells proliferated at closely similar rates in medium enriched with 5% or 15% fetal calf serum, with an average of 4% dividing and less than 0.1% dying per hr. The mean intermitotic interval was 16 hr during exponential growth of the control populations. In contrast, in cultures treated with 40 or 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a selective inhibitor of heterogeneous nuclear RNA synthesis, the frequency of mitoses was markedly and directly dependent on serum concentration, whereas the frequency of deaths was inversely dependent. DRB prolonged the intermitotic interval in cells cycling in the presence of the drug, but the effect was less in 15% than in 5% serum. After prolonged treatment of HeLa cells with DRB, the inhibition of heterogeneous nuclear RNA synthesis by DRB appeared to be reduced, which was not due to inactivation of DRB in the culture medium.

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Compartmentation with respect to the synthesis of heterogeneous nuclear RNA and precursors to ribosomal RNA.

The incorporation of [14C]orotate and [14C]uridine into UMP residues of hnRNA (heterogeneous nuclear RNA) and pre-rRNA (precursors to rRNA) of Eharlich ascites-tumour cells was compared: orotate was incorporated at a markedly higher rate into hnRNA. On the other hand, the rate of incorporation of uridine into pre-rRTNA was even somewhat higher than into hnRNA. The ratio of specific radioactivities of CMP to UMP residues in pre-rRNA and hnRNA was studied. At all times of labelling this ratio was similar for both RNA species independently of the precursor used. On addition of excess unlabelled uridine, the CMP/UMP labelling ratio in both pre-rRNA and hnRNA rose. However, this increase was much more pronounced with hnRNA. It is concluded that nuclear pyrimidine nucleotide pool for RNA synthesis is compartmentalized. The synthesis of hnRNa is supplied preferentially by the large and the small compartment, respectively. A detailed model for the cellular compartmentation of uridine nucleotide precursors to RNA is proposed.U

Dimethyl-10,12-benz(a)acridine: Evidence for differential effects on the synthesis of RNA of mammalian or avian fibroblasts and some RNA viruses

We have studied the differential effect of dimethyl-10,12-benz(a)acridine (DMBAcr) on the synthesis of RNA of chicken or mouse fibroblasts in culture and that of some RNA-containing viruses such as Rous sarcoma virus and Mengovirus. DMBAcr at low concentrations blocks the cell multiplication of both normal and Rous sarcoma virus-transformed chicken fibroblasts in culture; it affects transformed cells more than normal ones. The cell growth inhibiting effect of DMBAcr is reversible after short periods of incubation. DMBAcr depresses the synthesis of cellular DNA and RNA in parallel. Concurrently the synthesis of protein proceedes at a relatively high rate in DMBAcr-treated cultures. Its inhibitory effect on cellular RNA synthesis is mostly due to a block in the formation of 28 S and 18 S ribosomal RNA species; in contrast, the synthesis of 45 S ribosomal RNA precursor is proceeding at almost control rate. Also, the synthesis of heterogeneous nuclear RNA is not blocked by DMBAcr. The production of Rous sarcoma virus in transformed fibroblasts is not affected by DMBAcr. Since this is correlated with persisting high rates of protein and heterogenous nuclear RNA synthesis, the effects of DMBAcr suggest that the synthesis of Rous sarcoma virus-RNA shares the specificity of messenger and heterogeneous nuclear RNA. DMBAcr inhibits the synthesis of viral RNA of Mengovirus under conditions where the synthesis of total cellular RNA is not appreciably depressed, suggesting its differential effect on the DNA-directed and the RNA-directed RNA synthesis.

The absolute frequency of labeled N-6-methyladenosine in HeLa cell messenger RNA decreases with label time

When HeLa cells were labeled with either [ 3H]adenosine or H 3 32PO 4, the absolute frequency of m 6A ( N-6-methyladenosine) decreased 2 to 2.5-fold between a short and long label. The frequency of caps (m 7GpppXmpYp) remained constant at a level which suggests that most if not all mRNAs are capped in either a short or a long label. Since the inhibition of heterogeneous nuclear RNA synthesis with either actinomycin D or 5,6-dichloro-I-β- d-ribofuranosyl-benzimidazole halted the labeling of both m 6A and caps within 10 to 15 minutes and no direct cytoplasmic addition of m 6A could be detected, m 6A must be lost faster than caps from the cytoplasmic mRNA. This preferential loss could be due to one or more of the following: (1) a m 6A demethylase; (2) excision of m 6A containing sequences followed by splicing of the remaining parts of the mRNA; or (3) a minority of the mRNA with a high frequency of m 6A leaving the cytoplasmic compartment rapidly, presumably by being degraded.

Oestriol-stimulated synthesis of ribonucleic acid in the uterus of the immature rat.

Oestradiol-17beta and oestriol both sequentially stimulate the synthesis of heterogeneous nuclear RNA and rRNA in vivo. However, the stimulation by oestradiol-17beta, particularly of rRNA synthesis, is very much greater than that elicited by oestriol.

Synthesis of heterogeneous nuclear RNA in full-grown oocytes of xenopus laevis (Daudin)

At various times following injection of either 3H-GTP or 32PO 4 into full-grown (stage 6) Xenopus laevis oocytes, RNA has been extracted and fractionated on polyacrylamide gels. Based on size, base composition and incorporation data, we have defined the kinetics of synthesis and accumulation of ribosomal RNA (40S, 28S, 18S), heterogeneous RNA of high molecular weight (>40S) and heterogeneous RNA migrating with molecular weights of from 4S to 40S. Nuclear isolations have been performed to determine the cellular distribution of these classes of RNA as a function of time. Evidence is presented which shows that stage 6 oocytes synthesize RNA which by virtue of its size, base composition, rapid turnover and nuclear location is equivalent to the heterogeneous nuclear RNA observed in somatic cells. In addition, the data suggest synthesis of a class of nuclear RNA with a half-life of several hours. A small fraction (5%) of the nuclear RNA is stable, enters the cytoplasm and may represent RNA added to the stockpile of maternal transcripts known to be present in stage 6 oocytes.

Effects of ionizing radiation on RNA metabolism in cultured mammalian cells: III. Specific reduction in mRNA synthesis during resumed division and plateau phases following exposure to 800 rads of X irradiation

The cell growth response of cultured Chinese hamster ovary cells, line CHO, to 800 rads of X irradiation involves a period of division delay, followed by a period of resumed division which terminates in a plateau phase. Over 95% of the cells die eventually. No direct effects on RNA or protein metabolism are evident during the delay period. During the resumed division and beginning of plateau phases, however, a specific and relatively constant reduction in mRNA synthesis relative to messenger-like RNA and heterogeneous nuclear RNA synthesis is evidenced. The ratio of mRNA to messenger-like RNA synthesis ranges from 0.8 to 0.65 during these phases. The effect is not due to altered cell-cycle distribution, and evidence is presented to indicate that it is probably not a compensatory response to the unbalanced growth that occurs during the division delay period.

Initiation inhibition and reinitiation of the synthesis of heterogenous nuclear RNA in living cells.

THE nucleoside analogue 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) inhibits the synthesis of heterogeneous nuclear RNA (hnRNA) at the initiation level in explanted salivary glands of Chironomus tentans, but does not interfere with the synthesis of ribosomal RNA and transfer RNA (refs 1–4). When the radioactive RNA precursors and the inhibitor are coincidently added to the glands the labelling of hnRNA transcripts produced by smaller transcriptional units is suppressed before that of the larger ones2,3. If, however, the glands are pretreated with DRB, and the time of preincubation is sufficiently long, labelling of small as well as large hnRNA transcripts is equally eliminated, and the DNA template becomes depleted of nascent hnRNA chains2–4. Important questions, arising from these findings, are whether DRB can be washed out from the cells, and whether a reinitiation of hnRNA synthesis can subsequently be achieved. This paper presents data which show that the initiation of the RNA synthesis resumes when glands pretreated with DRB are washed and replenished with inhibitor-free medium.

Preferential inhibition of the synthesis of 45S ribosomal RNA precursor by N-acetoxyacetylaminofluorene in rat liver epithelial cultures

After exposing a line of rat liver epithelial cells to a single dose of the carcinogen N-acetoxy-2-acetylaminofluorene ( N-acetoxy-AAF), a dose-dependent decrease in [ 3H]uridine incorporation into total cellular RNA was found. Approx. 50% inhibition occurred with 0.5 μg/ml of the compound. The kinetics of the response, the effects of actinomycin D, and the fractionation of the newly synthesized RNA by polyacrylamide gel electrophoresis indicated preferential inhibition of the synthesis of 45S ribosomal RNA precursor and a relative sparing of the synthesis of heterogeneous nuclear RNA.

Estrogen-induced changes in the ribonucleic acid metabolism of chicken livers

In response to estrogen, chicken livers synthesize egg-yolk materials, including phosvitin. When roosters are treated with estrogen, the response is accompanied by the following changes in the RNA metabolism: 1. Two hours after estrogen-treatment there is increased synthesis of heterogeneous nuclear RNA, as shown by analysis of the RNA on methylated albumin kieselguhr and by RNA synthesis in isolated nuclei. 2. Changes in the rate of RNA synthesis are paralleled by changes in the template activity of purified chromatin, as measured by Escherichia coli RNA polymerase. 3. Four to six hours after estrogen treatment the synthesis of phosvitin becomes temporarily insensitive to inhibition by actinomycin D, although the actual synthesis of phosvitin does not begin until about 18 h after estrogen treatment.

The inhibition of nuclear RNA synthesis by the rif ampicin derivative [formula omitted] in living cells

The rifampicin derivative, AF 013 , completely inhibits synthesis of the nucleolar and chromosomal RNA in explanted salivary gland cells of Chironomus tentans. When the glands are preincubated in rifampicin AF 013 for a short period before the addition of the radioactive precursors, labelling of RNA is depressed in all size classes to the same extent. In contrast, if rifampicin is replaced by the nucleoside analogue, 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole, a tentative initiation inhibitor of heterogeneous nuclear RNA, the label is reduced preferentially in the lower molecular weight region of the heterogeneous nuclear RNA spectrum. In chase type experiments, when rifampicin AF 013 is added after an initial labelling period, the synthesis of heterogeneous nuclear RNA is suppressed equally in all size classes, a result analogous to that obtained with the elongation inhibitor, α-amanitin. 5,6-Dichloro-1-β- d-ribofuranosylbenzimidazole, under similar chase conditions, preferentially inhibits the labelling of smaller heterogeneous nuclear RNA molecules, but later on abolishes labelling of molecules with higher S values, also. Rifampicin AF 013 prevents or affects seriously the normal processing of the prelabelled preribosomal RNA in the nucleolus. It further interferes with the export of nuclear RNA to the cytoplasm, and/or promotes a non-physiological breakdown of cytoplasmic RNA. The experimental data suggest that rifampicin AF 013 acts on RNA synthesis in living cells by interference with chain elongation.

The Chanock Centre for Virology, Hebrew University. Hadassah Medical School, Jerusalem, Israel.

Summary The cytoplasmic deoxyvirus frog virus 3 (FV3) can replicate in HeLa cells at a permissive temperature (26 °C). The effect of virus infection on the host-cell RNA metabolism was studied at both permissive and non-permissive (37 °C) temperatures. The inhibition of precursor ribosomal RNA synthesis was shown to take place soon after infection at both temperatures. However, the inhibition of synthesis of heterogeneous nuclear RNA occurs only at the non-permissive temperatures.

Nuclear envelope-associated resumption of RNA synthesis in late mitosis of HeLa cells.

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G(1)) has been investigated in synchronized HeLa S(3) cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G(1) was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope.

Studies of Rous Sarcoma Virus: EFFECTS OF NUCLEOSIDE ANALOGUES ON VIRUS SYNTHESIS

We have studied the effects of two nucleoside analogues on the synthesis of Rous sarcoma virus and cellular RNA in monolayer cultures of chick embryo fibroblasts. The analogues are 5-fluorouridine and 5-bromotubercidin. 5-Fluorouridine irreversibly inhibits the synthesis of cellular RNA by more than 80%. Most of this inhibition is due to an irreversible block in the formation of ribosomal RNA. Protein synthesis continues at disproportionately elevated rates for several days, suggesting that messenger RNA synthesis also is maintained. The synthesis of heterogeneous nuclear RNA is not blocked by 5-fluorouridine; the same holds for the Rous sarcoma virus-RNA and Rous sarcoma virus-protein. Although normal quantities of Rous sarcoma virus are produced in cultures exposed to 5-fluorouridine, the infectivity is decreased; this is correlated with a profound inhibition of viral glycoprotein synthesis, and is in contrast to the effect of the aglycone, 5-fluorouracil, which does not decrease infectious virus yield under the same conditions. In contrast to the effects of 5-fluorouridine, 5-bromotubercidin reversibly blocks the synthesis of heterogeneous nuclear RNA as well as that of ribosomal RNA. Likewise 5-bromotubercidin blocks the synthesis of Rous sarcoma virus-RNA and, following a lag period of 8 to 10 hours, 5-bromotubercidin also inhibits the synthesis of viral proteins and mature virions. In relation to the action of the analogues, the synthesis of Rous sarcoma virus-RNA shares the specificity of heterogeneous RNA and mRNA.