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At least 127 records · Page 7Linked to original sources
PubMed1983

[Intercellular information transfer in the process of immunogenesis. IX. The induction of the synthesis of antiphage antibodies in lymphosarcoma cells of rats as affected by different fractions of immune nuclear RNA].

Various fractions of the immune nuclear RNA were isolated from spleens of phage T2 immunized rats. The fractions were compared for their ability to induce anti-phage T2 antibody synthesis in transplantable lymphosarcoma cells. The most active proved to be the nuclear sap RNA and its subfraction with sedimentation constant of 10 S. The 4S and 26S subfractions RNA were less stable and in some experiments failed to induce antibody synthesis.

Animals↗
PubMed1975

The inhibition of nuclear RNA synthesis by the rifampicin derivative AF/013 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-beta-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, alpha-amanitin. 5,6-Dichloro-1-beta-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.

Animals↗
PubMed1994

Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures.

Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.

Base Sequence↗
PubMed1996

Evidence of post-transcriptional regulation of U6 small nuclear RNA.

Mechanisms regulating the intracellular level of endogenous U6 small nuclear RNA were studied by transient transfection of ectopic U6 gene constructs into immortalized normal and malignant human cell lines. Transfection and expression of a modified U6 gene containing native promoter, capping, and termination sequences but lacking all highly conserved internal spliceosome sequences produced dose-dependent effects on endogenous U6 gene expression. At low transfection doses, no significant changes in endogenous U6 RNA levels or half-life were noted. However, as the dose of the transfected gene and its expression increased, native U6 RNA levels dramatically decreased in association with an apparent decrease in U6 RNA half-life. Down-regulation of native U6 RNA levels was transient, with recovery noted within 48-96 h in conjunction with declining expression of the ectopic gene. These modulatory effects appeared specific to endogenous U6 transcripts, because no changes were noted in 7sk, U1, U3, or 5S RNA levels or half-lives. Transfection with an unmodified U6 gene did not alter total U6 transcript levels but did produce a similar dose-dependent decrease in U6 RNA half-life. These studies suggest a hitherto unrecognized U6-specific intracellular regulatory mechanism, through which over-accumulation of U6 small nuclear RNA is prevented.

Cell Line↗
PubMed1983

Selective expression of cloned middle-repetitive sequences in nuclear RNA of mouse organs.

Mouse middle-repetitive sequences have been cloned in the bacterial plasmid pBR322. The DNA sequences, expressed only in nuclei, were screened by filter hybridization using nuclear RNA as probes. Several clones were expressed predominantly in the nuclear RNA in certain types of organs. The copy numbers of typical middle-repetitive sequence clones in genomic DNA were analyzed quantitatively and results showed that 10(3)-10(4) copies exist per haploid DNA, indicating that they belong to a relatively abundant class of repeated sequences. Homology within the family of each middle-repetitive sequence ranged from 81% to 94%. The expression of cloned middle-repetitive sequences was analyzed by liquid-phase hybridization with nuclear RNAs from particular organs. The content of transcripts of the middle-repetitive sequence in nuclear RNA of a particular organ was at least 1000 times higher than that in other organs. In steady-state nuclear RNA these transcripts were calculated to amount to 10(2)-10(4) copies per nucleus. Most of these transcripts seem to be retained in the nuclei and do not appear in the cytoplasm.

Animals↗
PubMed1994

Measurement of gene-specific transcription by nuclease protection of pulse-labeled nuclear RNA.

A gene-specific transcription assay was developed that is based on pulse-labeled incorporation of [3H]uridine into nuclear RNA. Transcription is quantified by scintillation counting of [3H]uridine incorporated into nuclear RNA that is protected from S1 nuclease digestion by hybridization with cold gene probes. This assay was dependent upon partial degradation of nuclear RNA and optimization of hybridization and nuclease digestion conditions. To validate this assay, transcription of beta-actin and c-myc genes was measured in two different human cell lines using the incorporation assay in parallel with the nuclear run-off assay. Transcription kinetics of the beta-actin and c-myc genes in serum-stimulated fibrosarcoma HT-1080 cells determined by [3H]uridine incorporation were comparable to that determined by the nuclear run-off method. For beta-actin, there was an approximate 2-fold increase in transcription rate within two hours of stimulation that declined to basal levels by 20 h. The c-myc gene response followed a similar kinetics as for the beta-actin gene except that maximal enhancement was greater at 6-9-fold. The relative transcriptional activities of the beta-actin gene to that of the c-myc gene were virtually identical using the two assay methods. Comparable transcription results using both methods were also observed when beta-actin and c-myc gene transcription were measured in log-phase HL-60 leukemia cells.

Actins↗
PubMed1985

Transcription boundaries of U1 small nuclear RNA.

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly.

Base Sequence↗
PubMed1974

Topology of repeated sequences: relationship of nuclear RNA to the repeated sequences of the main and satellite DNA in mouse plasmocytoma cells.

The topology of repeated sequences in mouse plasmocytoma DNA was studied by high-resolution CsCl density gradient centrifugation and heterogeneous nuclear RNA.DNA hybridization. Satellite region DNA of plasmocytoma cells contains additional components and hybridizes specifically with entire heterogeneous nuclear RNA molecules. A linkage is demonstrated between the A+T-rich satellite sequences and those hybridizing with heterogeneous nuclear RNA. Heavy DNA also hybridizes specifically with heterogeneous nuclear RNA molecules that show sequence similarity to heterogeneous nuclear RNA hybridized to satellite DNA. These results could suggest that part of satellite DNA became heavier after integration of some other DNA species, which could belong to a virus or to immunoglobulin repetitive genes. Dispersed, highly repetitive, short nucleotide sequences could constitute recognition sites for such a process.

Animals↗
PubMed1985

Deletion of a yeast small nuclear RNA gene impairs growth.

We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10- strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non-conditional.

Base Sequence↗
PubMed1977

Oligouridylate stretches in heterogeneous nuclear RNA.

Three classes of heterogeneous nuclear RNA (HnRNA) alpha, beta, and gamma, with different chemical and physical properties, can be identified in the early sea urchin embryo by hybridization with poly(U). The relative amounts of these classes vary as a function of embryonic development. It is demonstrated here that the adenyl-containing classes of HnRNA, alpha and beta, can be subfractionated by hybridization with poly(A)-agarose into species containing and lacking oligo(U)-enriched segments. These oligo(U) segments could not be detected in gamma HnRNA, which was previously shown to also lack adenylate segments. The relative proportions of these species undergo marked changes during development from early blastula (7 hr) to mesenchyme blastula (20 hr). I propose models to explain the possible effects of complementary sequences of adenylate and uridylate on the secondary structure of HnRNA, and speculate on the functional significance of such complexes.

Animals↗
PubMed1977

Secondary structure of heterogeneous nuclear RNA: two classes of double-stranded RNA in native ribonucleoprotein.

Heterogeneous nuclear RNA (hnRNA) from HeLa cells contains intramolecular duplexes. Since hnRNA is associated with protein in vivo, it is possible that the double-stranded regions observed in deproteinized hnRNA form spontaneously upon the release of protein from single-stranded but potentially complementary sequences. We show here that this is not the case for a class of double-stranded sequences that is defined by resistance to RNases A + T(1) at high ionic strength. Exposure of HeLa hnRNA.ribonucleoprotein (hnRNP) particles to Escherichia coli RNase III, a double-strand-specific endoribonuclease, destroys most of the sequences resistant to RNases A + T(1). This effect is completely blocked when hnRNP is exposed to RNase III in the presence of an excess of purified double-stranded RNA. In addition, we show that there exist two classes of double-stranded RNA in hnRNP at a salt concentration of 0.13 M. These are distinguished by their relative resistance to RNases A + T(1). The more stable double-stranded sequences, which are resistant to RNases A + T(1) at 0.13 M, comprise 1.0-1.1% of the nucleotides in hnRNP. The less stable double-stranded sequences comprise an additional 1.5-2.0% of the nucleotides in hnRNP. These are sensitive to RNase III at 0.13 M, but are not resistant to RNases A + T(1) unless the salt concentration is raised to 0.63 M. The demonstration that double-stranded sequences resistant to RNases A + T(1) exist in native ribonucleoprotein and are not artifacts of deproteinization now makes it appropriate to seriously consider their possible functional role in hnRNA metabolism, perhaps as binding sites for regulatory proteins involved in mRNA processing.

Cell Nucleus↗
PubMed1985

Purification of the three nuclear RNA polymerases from Neurospora crassa.

Nuclear RNA polymerases I, II, and III from Neurospora crassa have been purified 3,000-, 1,500-, and 10,000-fold, respectively, by a procedure that minimizes proteolysis of the 220-kDa subunit of polymerase II. The Neurospora enzymes resemble, in polypeptide composition, the corresponding polymerases isolated from other eukaryotes. The 220-kDa subunit of Neurospora polymerase II cross-reacts with antisera directed against the 220-kDa subunits of type II polymerases from Drosophila and wheat germ. However, the proteolyzed 180-kDa derivative of the Neurospora 220-kDa subunit fails to cross-react with the heterologous antisera, suggesting that the region removed by proteolysis contains or contributes to structural features of the enzyme that have been highly conserved during evolution. A 700-kDa complex of 12 polypeptides was found associated with polymerase II during purification. The complex was eventually separated from the enzyme, and its properties suggest that it might be associated with polymerase II in the nucleus. We describe two additional examples of polypeptides associated in variable amounts with Neurospora polymerase II.

Animals↗
PubMed1982

Association of cell lethality with incorporation of 5-fluorouracil and 5-fluorouridine into nuclear RNA in human colon carcinoma cells in culture.

The cytokinetic and biochemical effects of 5-fluorouracil and 5-fluorouridine were examined in a human colon carcinoma cell line (HT-29) in culture. Logarithmically growing cells were approximately 100 times more sensitive to the lethal effects of 5-fluorouridine than 5-fluorouracil as measured by colony formation in soft agar medium. A 2-hr exposure of cells to 10(-3) M 5-fluorouracil or 10(-5) M 5-fluorouridine produced a 2-log reduction in colony formation, a 31--33% inhibition of [14C]deoxyguanosine incorporation into DNA, and 30--40% inhibition of [3H]adenosine incorporation into total RNA. Increasing the duration of drug exposure to 24 hr produced a proportional reduction in the drug concentration required to produce similar biochemical and cytocidal effects. However, cell lethality produced by either drug did not correlate quantitatively with inhibition of DNA or RNA synthesis. Examination of nuclear rRNA and 4 S RNA synthesis by agarose gel electrophoresis following 2-hr and 24-hr exposure to 5-fluorouracil or 5-fluorouridine indicated that processing of rRNA was not impaired, rRNA synthesis was inhibited by 10--40%, and 4 S RNA synthesis was unaffected. In contrast to these results, measurements of the incorporation fo [3H]5-fluorouracil or [3H]5-fluorouridine into nuclear RNA showed that a significant correspondence existed between the amount of drug incorporated into nuclear RNA and cell lethality. These results indicate that the primary determinant of cell lethality in HT-29 cells is the degree of fluoropyrimidine substitution in nuclear RNA and not inhibition of either DNA or RNA synthesis.

Cell Nucleus↗
PubMed1981

Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen cross-linking in vivo.

The psoralen derivative 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) reacts with base-paired regions of RNA and forms interstrand covalent cross-links. Since psoralens permeate living cells, they can be used to probe RNA-tRNA interactions in vivo. We used AMT to investigate whether small nuclear RNAs are base-paired with high molecular weight nuclear RNA in the cell. Intact HeLa cells were treated with AMT, and high molecular weight RNA was isolated under denaturing conditions from nuclei or from subnuclear fractions. The presence of base-paired snRNAs in the high molecular weight nuclear RNA was examined by electrophoresis after photochemical reversal of the cross-links. We found snRNA U3 and 5.8S rRNA to be cross-linked to nucleolar RNA. IN contrast, snRNA U1 was crosslinked to high molecular weight RNA in ribonucleoprotein particles containing hnRNA. The U1 base-paired to hnRNA was identified by its hybridization with a cloned U1 DNA sequence after reversal of the cross-links. These results demonstrate that U1 is base-paired with hnRNA in vivo, suggesting a role in mRNA processing.

Base Sequence↗
PubMed1994

RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements.

Transcription of U2 small nuclear RNA (snRNA) genes in eukaryotes is executed by RNA polymerase II and is dependent on extragenic cis-acting regulatory sequences which are not found in other genes. Here we have mapped promoter elements of the Trypanosoma brucei U2 snRNA gene by transient DNA expression of mutant constructs in insect form trypanosomes. Unlike other eukaryotic U2 snRNA genes, the T. brucei homolog is transcribed by an RNA polymerase III-like enzyme on the basis of its sensitivity to the inhibitors alpha-amanitin and tagetitoxin. Thus, the trypanosome U2 snRNA provides a unique example of an RNA polymerase III transcript carrying a trimethylated cap structure. The promoter of this gene consists of three distinct elements: an intragenic sequence close to the 5' end of the coding region, which is probably required to position the polymerase at the correct transcription start site; and two extragenic elements, located 110 and 160 nucleotides upstream, which are essential for U2 snRNA gene expression. These two elements closely resemble both in sequence and in distance from each other the A and B box consensus sequences of the internal control regions of tRNA genes.

Amanitins↗
PubMed1977

Adenovirus type 2 nuclear RNA accumulating during productive infection.

The viral-specific nuclear RNA which accumulates early and late during productive infection of HeLa cells by adenovirus-type 2 (Ad2) has been characterized with respect to its size and stability after denaturation by Me2SO. Early nuclear transcripts, under nondenaturing conditions, sediment in the range 28 to 45S, but treatment with Me2SO prior to sedimentation results in a shift to about 20S. Later nuclear RNA accumulates as a composite of two populations of molecules: one with a broad size distribution centering on 45S under nondenaturing conditions and less than 32S after denaturation and a second having a narrow size distribution around 35S which is quite stable to Me2SO. Analysis of late RNA by hybridization to Sma fragments of Ad2 DNA suggests that the 35S RNA species is derived from a limited portion of the left half of the viral genome.

Adenoviruses, Human↗
PubMed1977

Preparation in undenatured form of the main protein bound to heterogeneous nuclear RNA in liver and hepatoma cells.

Particles carrying heterogeneous nuclear RNA (30 S-particles) were prepared from rat liver and Zajdela hepatoma ascites cell nuclei after ultrasonic disruption. The ribonucleoprotein structures were disintegrated in the presence of 100mM spermidine. Using chromatography on Sepharose-polyadenylate a protein component has been obtained which possessed high affinity for heterogeneous nuclear RNA, polyuridylate and polyadenylate, and double-stranded DNA. This protein was the main species of the ribonucleoprotein studied; it showed bands with molcular weights of 37000 and 40000 respectively in SDS gel electrophoresis. The RNA-binding proteins isolated from liver and hepatoma had identical molecular weights and the same affinity for Sepharose-polyadenylate used in the isolation.

Animals↗