scholarly journals Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells

1984 ◽  
Vol 218 (2) ◽  
pp. 583-590
Author(s):  
N K Chatterjee ◽  
C Tuchowski ◽  
G E Eagan ◽  
T M Haley
Keyword(s):  
Hela Cells ◽  
Sucrose Gradient ◽  
Actinomycin D ◽  
Rna Molecules ◽  
Filter Hybridization ◽  
Cytoplasmic Rna ◽  
Blot Technique ◽  
Nuclear Rna ◽  
Infected Cells ◽  
Two Populations

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.

scholarly journals THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS

1966 ◽  
Vol 28 (2) ◽  
pp. 263-275 ◽  
Author(s):  
John Seed
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Hela Cells ◽  
Nuclear Protein ◽  
X Rays ◽  
Normal Cells ◽  
Human Carcinoma ◽  
Physical Measurements ◽  
Cytoplasmic Rna ◽  
Nuclear Rna

Interferometric and photometric measurements have been made on HeLa cells, a strain of cells originally derived from a human carcinoma. From a study of the relations between successive physical measurements on individual cells, it was confirmed that, whereas the net syntheses of nuclear RNA and nuclear protein are closely associated during interphase, they are dissociated from DNA replication to a significant extent. These results on nuclear metabolism agree with others previously reported in cell strains derived from tumors; they contrast with results from freshly prepared normal cells, where the net syntheses of DNA, nuclear RNA, and protein are closely associated during interphase. Cytoplasmic measurements on HeLa cells showed that much of the net synthesis of cytoplasmic RNA is associated with DNA replication as in normal cells, and they failed to detect transfer from the nucleus of a stable RNA component synthesized independently from DNA replication. In auxiliary experiments, an inhibition of the onset of DNA synthesis was produced by a dose of X-rays; under these conditions it was shown that the major part of the accumulation of nuclear protein was independent of DNA replication and that the accumulation of nuclear RNA was equivalent to or slightly less than that of nuclear protein. About half the accumulation of cytoplasmic RNA was inhibited when DNA synthesis was blocked.

scholarly journals Mechanism of mda-5 Inhibition by Paramyxovirus V Proteins

2008 ◽  
Vol 83 (3) ◽  
pp. 1465-1473 ◽  
Author(s):  
K. S. Childs ◽  
J. Andrejeva ◽  
R. E. Randall ◽  
S. Goodbourn
Keyword(s):  
Helicase Domain ◽  
Type I ◽  
Rna Helicases ◽  
V Protein ◽  
Rna Molecules ◽  
Cytoplasmic Rna ◽  
Dsrna Binding ◽  
Infected Cells ◽  
Self Association ◽  
Inducible Gene

ABSTRACT The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.

Translational Control of Protein Synthesis in Eat Anterior Pituitary by N-2′-O-dibutyryl adenosine 3′,5′-monophosphate

1973 ◽  
Vol 51 (6) ◽  
pp. 913-919 ◽  
Author(s):  
Roger Boucher ◽  
Marie Gauthier ◽  
Paul Jolicoeur ◽  
Fernand Labrie
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Short Term ◽  
Nucleotide Pools ◽  
Cytoplasmic Rna ◽  
Nuclear Rna ◽  
Preferential Incorporation ◽  
Stimulation Of

Actinomycin D, at doses (25 and 50 μg/ml) that block RNA synthesis to less than 3% of the control rate, inhibits the incorporation of [3H]leucine into adenohypophyseal proteins and the release of newly synthesized proteins by 50 and 60%, respectively, of the control rates. Despite this lowering of basal levels of total protein synthesis and release in presence of the antibiotic, the percentage of stimulation of both protein synthesis and release by 5 mM N6-2′-O-dibutyryl adenosine 3′5′-monophosphate (dbcAMP) is not depressed by actinomycin D. When rat hemipituitaries are incubated with [3H]uridine, dbcAMP does not stimulate the labeling of total cytoplasmic RNA or the preferential labeling of any cytoplasmic RNA species resolved on sucrose gradient. There is no stimulatory effect of dbcAMP on total labeling or preferential incorporation into nuclear RNA species extracted at 24 °C or at 65 °C. Labeling of the nucleotide pools was unchanged up to 1 h of incubation but was increased (40–70%) during the last [Formula: see text] of incubation. These data suggest that the short-term stimulatory effects of dbcAMP on total adenohypophyseal protein synthesis and release are exerted at the transiational level.

scholarly journals STABILITIES OF NUCLEAR AND MESSENGER RNA MOLECULES IN SEA URCHIN EMBRYOS

1972 ◽  
Vol 53 (2) ◽  
pp. 474-482 ◽  
Author(s):  
Bruce P. Brandhorst ◽  
Tom Humphreys
Keyword(s):  
Steady State ◽  
Sea Urchin ◽  
Half Life ◽  
Messenger Rna ◽  
Cell Fractionation ◽  
Rna Molecules ◽  
Sea Urchin Embryos ◽  
Cytoplasmic Rna ◽  
Nuclear Rna ◽  
Kinetics Of

The kinetics of accumulation of radioactive adenosine in adenosine triphosphate and in RNA of nuclear, cytoplasmic, and polysomal fractions of sea urchin embryos have been analyzed. 85% of the RNA synthesized decays in the nucleus with an apparently uniform half-life of about 7 min. The remaining 15% goes to the cytoplasm, mostly entering polysomes, and decays with a quite uniform half-life of about 75 min. The nuclear RNA accounts for one-third and the cytoplasmic RNA accounts for two-thirds of the total unstable RNA which accumulates at steady state in the embryo. The size distribution of short-labeled nuclear RNA is very similar to that of long-labeled messenger RNA, when both are extracted directly from the cells without a previous cell fractionation.

scholarly journals The nucleus-limited Hsr-omega-n transcript is a polyadenylated RNA with a regulated intranuclear turnover.

1994 ◽  
Vol 125 (1) ◽  
pp. 21-30 ◽  
Author(s):  
N C Hogan ◽  
K L Traverse ◽  
D E Sullivan ◽  
M L Pardue
Keyword(s):  
Heat Shock ◽  
Actinomycin D ◽  
Cell Types ◽  
Rna Turnover ◽  
Simple Diffusion ◽  
New Class ◽  
Transcription Site ◽  
Cytoplasmic Rna ◽  
Rapid Changes ◽  
Nuclear Rna

The Drosophila Hsr-omega puff, one of the largest heat shock puffs, reveals a very unusual gene, identified by heat shock but constitutively active in nearly all cell types. Surprisingly, Hsr-omega yields two transcription end-products with very different roles. The larger, omega-n, is a nuclear RNA with characteristics suggesting a new class of nuclear RNAs. Although it neither leaves the nucleus nor undergoes processing, omega-n RNA is polyadenylated, showing that polyadenylation is not limited to cytoplasmic RNA, but possibly has a function in the nucleus. The amount of omega-n within the nucleus is specifically regulated by both transcription and turnover. Heat shock and several other agents cause rapid increases in omega-n. A rapid return to constitutive levels follows withdrawal of the agents. Degradation of omega-n is inhibited by actinomycin D, suggesting a novel intranuclear mechanism for RNA turnover. Within the nucleus, some omega-n RNA is concentrated at the transcription site; however, most is evenly distributed over the nucleus, showing no evidence of a concentration gradient which might be produced by simple diffusion from the site of transcription. Previous studies suggested that omega-n has a novel regulatory role in the nucleus. The actinomycin D-sensitive degradation system makes possible rapid changes in the amount of omega-n, allowing the putative regulatory activities to reflect cellular conditions at a given time. Omega-n differs from the best studied nuclear RNAs, snRNAs, in many ways. Omega-n demonstrates the existence of intranuclear mechanisms for RNA turnover and localization that may be used by a new class of nuclear RNAs.

scholarly journals Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells

1993 ◽  
Vol 106 (1) ◽  
pp. 11-22
Author(s):  
L.F. Jimenez-Garcia ◽  
S.R. Green ◽  
M.B. Mathews ◽  
D.L. Spector
Keyword(s):  
Protein Kinase ◽  
Hela Cells ◽  
Post Infection ◽  
Ribosome Biogenesis ◽  
Actinomycin D ◽  
Double Stranded Rna ◽  
Interphase Cells ◽  
Infected Cells ◽  
Fibrillar Region ◽  
Polymerase I

We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2)-infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with alpha-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10–14 hours post-infection as fine dots in the nucleoplasm. By 18–24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.

Sign in / Sign up

Export Citation Format

Share Document