Adrenocorticotropic hormone regulation of adrenal RNA polymerases. Stimulation of nuclear RNA polymerase III.
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Adrenal Glands↗
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Results for “RNA, Nuclear”
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Antibody against a stimulatory factor of RNA polymerase II inhibits nuclear RNA synthesis.
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Animals↗
Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.
The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.
Animals↗
Migration of newly synthesized RNA during mitosis. 3. Nuclear RNA in the cytoplasm of metaphase cells.
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Animals↗
Heterogeneous nuclear RNA secondary structure: oligo (U) sequences base-paired with poly (A) and their possible role as binding sites for heterogeneous nuclear RNA-specific proteins.
HeLa cell heterogeneous nuclear RNA derived from high-molecular-weight nuclear ribonucleoprotein (RNP) particles contains oligo(U) sequences of 15-50 nucleotides base-paired with poly(A). These duplexes are resistant to pancreatic RNase at 0.5 M NaCl in native RNP, remain so after chemical deproteinization of the RNP digests, and then copurify with poly(A) on oligo(dT)-cellulose chromatography. Oligo(dT)-cellulose binding capacity of the oligo(U)-poly(A) duplexes is abolished by prior titration of the nonduplex poly(A) regions with excess poly(U). The oligo(dT)-purified fraction is 97.5 mole % A + U and the [3H]uridine-labeled component is resistant to redigestion by pancreatic RNase at 0.5 M NaCl but not at 0.01 M NaCl. After thermal denaturation, the [3H]uridine-labeled chains become RNase-sensitive at 0.5 M NaCl. Electrophoresis of [3H]adenosine- or [3H]uridine-labeled material in polyacrylamide gels containing 99% formamide confirms that the oligo(U) sequences are not covalently linked to poly(A). Controls establish that the A-U duplexes are not formed artifactually during isolation of heterogeneous nuclear RNP or subsequent fractionation. The oligo(U)-poly(A) duplexes appear to be associated with protein in native heterogeneous nuclear RNP, as reflected by the differential pancreatic RNase sensitivity of the duplexed oligo(U) in RNP (resistant) and RNA (sensitive), measured at physiological ionic strength.
Base Sequence↗
RNA primers and the role of host nuclear RNA polymerase II in influenza viral RNA transcription.
Influenza viral RNA transcription in the infected cell is inhibited by alpha-amanitin, a specific inhibitor of the host nuclear RNA polymerase II. Because viral RNA transcription in vitro catalysed by the virion-associated transcriptase is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by RNA polymerase II. In addition, because we did not detect any capping and methylating enzymes in virions, we have proposed that the 5' terminal methylated cap found on in-vivo viral messenger RNA (mRNA) is derived from the putative primer RNAs. Our recent experiments have proved these two hypotheses. Purified globin mRNAs were shown to stimulate viral RNA transcription in vitro very effectively. The resulting transcripts directed the synthesis of all the non-glycosylated virus-specific proteins in cell-free systems. Other eukaryotic mRNAs were also active as primers. The presence of a 5' terminal methylated cap structure in the priming mRNA was required for its priming activity. Thus, with globin mRNA, removal of the cap eliminated essentially all of its priming activity, and much of this activity could be restored by enzymically recapping the globin mRNA. Using globin mRNA containing 32P only in its cap, we demonstrated that the 5' cap of the globin mRNA primer was physically transferred to the viral RNA transcripts during transcription. Gel electrophoretic analysis suggested that, in addition to the cap, about 10-15 other nucleotides were also transferred from the globin mRNA to the viral RNA transcripts. A mechanism for the priming of influenza viral RNA transcription by globin mRNA is proposed. Initial experiments strongly suggest that priming by capped host mRNAs also occurs during the synthesis of viral mRNA in vivo.
Amanitins↗
A comparison between heterogeneous nuclear RNA and polysomal messenger RNA in HeLa cells by RNA-DNA hybridization.
Heterogeneous nuclear RNA (HnRNA) and mRNA from cytoplasmic polyribosomes of HeLa cells have been compared by RNA-DNA hybridization tests. 1 microg of HeLa cell DNA binds 0.05-0.10 microg of either HnRNA or mRNA. In addition, HeLa DNA that is preexposed to unlabeled HnRNA was found to have a reduced capacity to bind either HnRNA or mRNA. The results are compatible with considerable sequence similarity in the two types of RNA but, as is discussed, firm conclusions are precluded by imperfections of the hybridization reaction as presently employed.
Cell Nucleus↗
Pre-messenger RNA splicing of transcripts synthesized from human small nuclear RNA gene promoters.
In order to explore the coupling of transcription with splicing in mammalian cells we have prepared hybrid genes in which either the human U2 promoter, recognized by RNA polymerase II, or the human U6 promoter, recognized by RNA polymerase III, was fused to an intron-containing gene segment. Neither human small nuclear RNA gene contains an intron although U6 genes from some species of yeast contain a short intervening sequence. Following transfection of human cells and analysis of specific RNAs by primer extension we found that the chimeric U2 promoter-derived transcript was efficiently spliced but the RNA polymerase III transcript driven by the U6 promoter remained unspliced. Hence, the splicing apparatus differentiates between transcripts produced from two closely related promoters that are distinguished by RNA polymerase selectivity.
Base Sequence↗
Preparative polyacrylamide gel electrophoresis of nuclear RNA.
A method is described for the separation of nuclear RNA by preparative polyacrylamide gel electrophoresis. Separation is performed on a slab gel. The elution chamber system is rectangular. Total nuclear RNA can be fractionated into the prominent RNA species 4 - 7S, 18S and 28S RNA. The less prominent RNA, especially the high molecular weight precursor molecules of ribosomal RNA 45S (Mr = 4.1 X 10(6)) and 32S (Mr = 2.1 X 10(6)) can be separated from the total nuclear RNA without contamination by other RNA. The time needed for a preparative run is 260 min.
Animals↗
Heterogeneous nuclear RNA promotes synthesis of (2',5')oligoadenylate and is cleaved by the (2',5')oligoadenylate-activated endoribonuclease.
Heterogeneous nuclear RNA contains double-stranded regions that are not found in mRNA and that may serve as recognition elements for processing enzymes. The double-stranded regions of heterogeneous nuclear RNA prepared from HeLa cells promoted the synthesis of (2',5')oligoadenylate [(2',5')oligo(A) or (2'5')An] when incubated with (2',5')An polymerase. This enzyme is present in elevated levels in interferon-treated cells, and labeled heterogeneous nuclear RNA incubated with extracts of these cells is preferentially cleaved, since mRNA included in the same incubations is not appreciably degraded. The cleavage of heterogenous nuclear RNA is caused by the synthesis of (2'5')An and by a "localized" activation of the (2',5')An-dependent endonuclease, since it was enhanced by ATP, the substrate of the (2',5')An polymerase, and inhibited by 2'-dATP and ethidium bromide. Both of these compounds suppress the synthesis of (2',5')An, the first by competitive inhibition and the latter by intercalating into double-stranded RNA. The possible role of double-stranded regions and of the (2',5')An polymerase-endonuclease system in the processing of heterogeneous nuclear RNA is discussed.
2',5'-Oligoadenylate Synthetase↗
Transcription of a gene for human U1 small nuclear RNA.
Transcription of U1 small nuclear RNA from a 592 bp fragment of human DNA was analyzed in vivo and in vitro. When injected into Xenopus laevis oocyte nuclei, the cloned DNA is transcribed by RNA polymerase II to make human U1 snRNA. Thus the sequences of this fragment are sufficient for expression of the U1 snRNA gene. Moreover, injection of templates carrying deletions of flanking sequences demonstrates that the DNA sequences required for in vivo transcription are located at least 100 nucleotides upstream from the point corresponding to the 5' end of mature U1 snRNA. In vitro transcription in a HeLa cell extract leads to synthesis not of mature U1 snRNA, but of a larger molecule starting 183 nucleotides upstream from the site corresponding to the 5' end of mature U1 snRNA. Transcription from this upstream promoter also is catalyzed by RNA polymerase II, and is comparable in efficiency with the very strong major late promoter of adenovirus 2. We propose that U1 snRNA is synthesized in vivo as a precursor that is processed by an enzyme or enzymes missing from our extracts.
Cloning, Molecular↗
Cloning, characterization and transient expression of the gene encoding a rice U3 small nuclear RNA.
A rice U3 small nuclear RNA (snRNA)-encoding gene has been isolated. The coding region of this gene contains all five conserved sequence boxes common to plant U3 snRNAs. The upstream and downstream regions of the gene harbour characteristic sequence elements required for transcription with RNA polymerase III (pol III), as well as three monocot-specific promoter (MSP) elements [Connelly et al., Mol. Cell Biol.14 (1994) 5910-5919], two of them comprising a palindromic G+C-rich segment. The sequence (TTTAAAA) of the TATA-box in this gene does not fit the established consensus [Marshallsay et al., Plant Mol. Biol. 19(1992) 973-983], making this gene unique among reported snRNA-encoding genes of plants. An RNase protection assay showed that the gene is expressed properly in cucumber protoplasts. We thus suggest that, with other promoter elements present, the TATA-box for RNA pol III-specific snRNA-encoding genes may not be as well conserved as that for RNA pol II-specific genes.
Base Sequence↗
Sequences more than 500 base pairs upstream of the human U3 small nuclear RNA gene stimulate the synthesis of U3 RNA in frog oocytes.
Small nuclear RNA (snRNA) genes contain strong promoters capable of initiating transcription once every 4 s. Studies on the human U1 snRNA gene, carried out in other laboratories, showed that sequences within 400 bp of the 5' flanking region are sufficient for maximal levels of transcription both in vivo and in frog oocytes [reviewed in Dahlberg and Lund (1988)]. We studied the expression of a human U3 snRNA gene by injecting 5' deletion mutants into frog oocytes. The results show that sequences more than 500 bp upstream of the U3 snRNA gene have a 2-3-fold stimulatory effect on the U3 snRNA synthesis. These results indicate that the human U3 snRNA gene is different from human U1 snRNA gene in containing regulatory elements more than 500 bp upstream. The U3 snRNA gene upstream sequences contain an AluI homologous sequence in the -1200 region; these AluI sequences were transcribed in vitro and in frog oocytes but were not detectable in HeLa cells.
Animals↗
Are globin coding sequences expressed in nuclear RNA of chick embryo brain?
The content of globin coding sequences in nuclear RNA from chicken embryo red blood cells and chicken embryo brain was determined by hybridization with globin cDNA to be 0.27% for embryonic red blood cells and 0.001% for embryo brain, i.e. 0.37% of globin coding sequences relative to embryonic red blood cells. By transfusion of [3H]uridine labelled red blood cells it could be shown that the brain nuclear RNA preparation was contaminated by 0.41% RNA originating from blood cells. As this value is in the same range as the hybridization rate there is no evidence for globin transcripts in brain nuclear RNA. It has also not been possible to detect a short-lived globin mRNA precursor in pulse labelled brain nuclear RNA. In further experiments the RNAs were translated in an Ehrlich ascites cell-free system. No globin synthesis could be observed with brain RNA.
Animals↗
Evidence for splicing of interrupted immunoglobulin variable and constant region sequences in nuclear RNA.
Evidence is presented that the mouse light-chain coding sequence is interrupted in a 27S nuclear RNA species, whereas the sequence is continuous in both a 13S nuclear RNA and in cytoplasmic mRNA. The discontinuity of coding regions in the 27S nuclear RNA parallels the situation found in myeloma DNA and indicates, therefore, that the removal of interruptions in the V and C regions occurs at the level of nuclear RNA.
Animals↗
Diversity of sequences in total and polyadenylated nuclear RNA from Drosophila cells.
Complementary DNA was synthesized using polyadenylated nuclear RNA of cultured Drosophila cells as template. The kinetics of hybridization of this cDNA with nuclear RNA indicated that the complexity of this RNA population is five to ten times greater than that of cytoplasmic mRNA. The same difference in the fraction of DNA represented was obtained when nuclear and cytoplasmic RNA were hybridized with labeled unique sequence DNA. The fraction of the DNA sequences represented in total number of polyadenylated nuclear RNA is much higher than that represented in cytoplasmic RNA.
Base Sequence↗
The synthesis and processing of a nuclear RNA precursor to rat pregrowth hormone messenger RNA.
A recombinant DNA plasmid, pBR322-GH1, which contains about 80% of the sequences of rat pregrowth hormone (pGH) mRNA, allowed an analysis of nuclear RNA from GH3 cells for possible precursors of cytoplasmic pGH mRNA. A single 20-22S RNA SPECIES ABOUT 2-3 TIMes larger than pGH mRNA was detected in nuclear RNA from GH3 cells labeled for 5 min. with 3H-uridine. After longer label times a 12S RNA indistinguishable in size from cytoplasmic 12S pGH mRNA became the predominant labeled RNA complementary to the plasmid pBR322-GH1. Both of these nuclear RNA species contained poly (A). Kinetic analysis of the labeling of nuclear and cytoplasmic pGH mRNA sequences showed that the 20S and 12S nuclear RNA molecules were labeled before significant labeling of cytoplasmic pGH mRNA was detected, and also indicated that there is complete conservation of nuclear pGH mRNA sequences in the production of cytoplasmic pGH mRNA. These results indicate that cytoplasmic pGH mRNA is generated by nuclear processing of a larger nuclear RNA molecule.
Animals↗
Size of murine RNA tumor virus-specific nuclear RNA molecules.
About 1% of the total RNA of cell lines producing murine leukemia virus is virus-specific RNA. About one-third of the virus-specific RNA is located within the nucleus. The size distribution of virus-specific RNA was determined before and after denaturation. Before denaturation, virus-specific RNA sequences sedimented as a heterogeneous population of RNA molecules, some of which sedimented very rapidly. After denaturation, most of the virus-specific RNA had a sedimentation coefficient of 35S or lower, but a small fraction of the nuclear virus-specific RNA sedimented more rapidly than 35S RNA even after denaturation.
Cell Line↗