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Results for “RNA, Nuclear”

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PubMed1979

The location of SV40 specific sequences in the heterogeneous nuclear RNA of transformed mouse cells.

Rapidly labelled nuclear RNA from an SV40 transformed mouse 3T3 line was isolated, and different molecular size classes pooled. The fractions were treated with Ehrlich ascites exonuclease III for various lengths of time, and the digestion of the 3' ends of the RNA measured by the appearance of TCA-soluble material. The resulting partially degraded RNA populations were then hybridised to SV40 DNA. The data suggest that a high proportion of the viral specific sequences are located near the 3' ends of heterogeneous nuclear RNA.

Animals↗

PubMed1999

The general transcription factors IIA, IIB, IIF, and IIE are required for RNA polymerase II transcription from the human U1 small nuclear RNA promoter.

RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.

Animals↗

PubMed1981

Nucleotide sequences of mouse genomic loci including a gene or pseudogene for U6 (4.8S) nuclear RNA.

We have isolated four clones which hybridize with U6 (4.8S) nuclear RNA, a mammalian small nuclear RNA(nRNA), from DNA of BALB/C mouse liver. Their restriction maps are totally different from each other, indicating that they derived from different loci in the mouse genome. The nucleotide sequences around the hybridizing region in the three clones have been determined. One clone gives a gene that is co-linear with the U6 RNA. There is a sequence TATAAAT beginning 31 nucleotides upstream of the gene, which may suggest that the U6 RNA is transcribed by RNA polymerase II. The other two clones contain a pseudogene for the U6 RNA which has 7 or 9 nucleotide changes from the RNA. The pseudogenes are surrounded by radically different sequences from those surrounding the gene, and they are closely linked to a pseudogene for another snRNA, 4.5S-I RNA, or a part of highly repetitive an interspersed sequence B1.

Animals↗

PubMed1982

Nuclear deprimerones (low molecular weight peptides controlling gene expression) are associated with DNA and nuclear RNA.

Isolated, deproteinized nucleic acids from calf thymus and rat liver nuclei were fractionated on Bio-Gel A-5m into DNA and RNA fractions. Extraction of deproteinized DNA and nuclear RNA with 80% ethanol at pH 9.5 yielded low molecular weight peptides (deprimerones) capable of inhibiting DNA transcription in a cell-free system. The extracted peptidic fractions were further fractionated on a Sephadex G-25 column and collected as a fraction of mol. wt. between 1 500 and 600. The yield of DNA deprimerones was about 30 microgram/mg DNA and of nuclear RNA deprimerones 150-200 microgram/mg RNA (measured by amino acid composition). The nucleoplasm contained a negligible amount of free, soluble peptides (about 18 microgram/g tissue). The DNA and RNA deprimerones were characterized by their amino acid composition and by two-dimensional thin layer chromatography on cellulose gel plates which yielded two major and two minor fractions. DNA deprimerones isolated in this way are very similar, if not identical, to the affinity DNA deprimerones isolated from a nuclear extract on DNA-cellulose but differ from RNA deprimerones. DNA and RNA deprimerones were also compared with the nuclear deprimerones extracted directly from the lysed nuclei. Their amino acid composition is different from both DNA and nuclear RNA deprimerones. Two-dimensional thin layer chromatography on cellulose plates yielded about ten fractions (spots) with various amino acid composition and inhibitory activity for cell-free transcription.

Amino Acids↗

PubMed1985

Brain "identifier sequence" is not restricted to brain: similar abundance in nuclear RNA of other organs.

A repeated 82 base pair sequence in genomic DNA of the rat was previously proposed as being a control element governing brain (neuron) specific genetic expression. This intronic sequence, termed the brain "identifier" (ID), is complementary to small RNA species localized in brain cytoplasm, and it was thought to be represented specifically in RNA produced by brain nuclei in vitro. The RNA blot analyses of total nuclear and polyadenylated heterogeneous nuclear RNA described in the present report show that this ID sequence is also present in the liver and kidney in abundances similar to those in the brain. This repeated sequence is not, therefore, restricted to transcripts produced in the brain as suggested from previous transcriptional "runoff" experiments. Measurements on rat and mouse nuclear RNA indicate that the abundance of ID sequence transcript is roughly proportional to the number of copies of this repeat in the respective genomes. This suggests a rather random genomic location and transcription of this sequence. From these results it seems improbable that the ID sequence functions as a transcriptional-level control element in genes expressed specifically in the brain.

Animals↗

PubMed1978

Transcription of the non-repetitive genome in liver hypertrophy and the homology between nuclear RNA of normal and 12 H-regenerating liver.

The percentage of the non-repetitive genome transcribed and the complexity of nuclear RNA were estimated in normal and 12 h-regenerating rat liver. Nuclear RNA from normal or 12 h-regenerating liver hybridizes with approximately 6.1% of non-repetitive DNA (12.2% of the single-copy genome, assuming assymetric transcription). The estimated complexity of either of these nuclear RNA populations is 7.6 . 10(10) daltons, which is approximately 7 times higher than that calculated for polysomal mRNA. Cross hybridization experiments did not show differences between the nuclear RNA populations of normal and 12 h-regenerating liver. The results indicate that liver hypertropy (without hyperplasia) may be brought about without a large increase in the proportion of the non-repetitive genome transcribed.

Animals↗

PubMed1982

Further evidence that the majority of primary nuclear RNA transcripts in mammalian cells do not contribute to mRNA.

Nuclear RNA from Chinese hamster ovary cells was effectively separated into polyadenylic acid [poly(A)]-containing [poly (A)+] and non-poly(A)-containing [poly(A)-] fractions so that -90% of the poly(A) was present in the (A)+ fraction. Only 25% of the 5'-terminal caps of the large nuclear molecules were present in the (A)+ class, but about 70% of the specific mRNA sequences (assayed with cDNA clones) were in the (A)+ class. It appears that many long capped heterogeneous nuclear RNA molecules are of a different sequence category from those molecules that are successfully processed into mRNA.

Animals↗

PubMed2001

7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes.

The transcription of eukaryotic protein-coding genes involves complex regulation of RNA polymerase (Pol) II activity in response to physiological conditions and developmental cues. One element of this regulation involves phosphorylation of the carboxy-terminal domain (CTD) of the largest polymerase subunit by a transcription elongation factor, P-TEFb, which comprises the kinase CDK9 and cyclin T1 or T2 (ref. 1). Here we report that in human HeLa cells more than half of the P-TEFb is sequestered in larger complexes that also contain 7SK RNA, an abundant, small nuclear RNA (snRNA) of hitherto unknown function. P-TEFb and 7SK associate in a specific and reversible manner. In contrast to the smaller P-TEFb complexes, which have a high kinase activity, the larger 7SK/P-TEFb complexes show very weak kinase activity. Inhibition of cellular transcription by chemical agents or ultraviolet irradiation trigger the complete disruption of the P-TEFb/7SK complex, and enhance CDK9 activity. The transcription-dependent interaction of P-TEFb with 7SK may therefore contribute to an important feedback loop modulating the activity of RNA Pol II.

Binding Sites↗

PubMed2001

The Drosophila U1 and U6 gene proximal sequence elements act as important determinants of the RNA polymerase specificity of small nuclear RNA gene promoters in vitro and in vivo.

Transcription of genes coding for metazoan spliceosomal snRNAs by RNA polymerase II (U1, U2, U4, U5) or RNA polymerase III (U6) is dependent upon a unique, positionally conserved regulatory element referred to as the proximal sequence element (PSE). Previous studies in the organism Drosophila melanogaster indicated that as few as three nucleotide differences in the sequences of the U1 and U6 PSEs can play a decisive role in recruiting the different RNA polymerases to transcribe the U1 and U6 snRNA genes in vitro. Those studies utilized constructs that contained only the minimal promoter elements of the U1 and U6 genes in an artificial context. To overcome the limitations of those earlier studies, we have now performed experiments that demonstrate that the Drosophila U1 and U6 PSEs have functionally distinct properties even in the environment of the natural U1 and U6 gene 5'-flanking DNAs. Moreover, assays in cells and in transgenic flies indicate that expression of genes from promoters that contain the "incorrect" PSE is suppressed in vivo. The Drosophila U6 PSE is incapable of recruiting RNA polymerase II to initiate transcription from the U1 promoter region, and the U1 PSE is unable to recruit RNA polymerase III to transcribe the U6 gene.

Animals↗

PubMed1979

Protein coding sequences in newly synthesized nuclear RNA from chicken immature red blood cells.

The contents of globin gene transcripts and other protein coding agene transcript (preferentially expressed in liver) within nuclear RNA from chicken immature red blood cells were analyzed by the method of cDNA hybridization. A comparison of the hybridization values obtained from steady state nuclear RNA and pulse labeled newly synthesized nuclear RNA demonstrated, that (a) there is a vast excess of globin gene transcripts over other individual protein coding transcripts and (b) the ratio of globin gene transcripts to the amount of other protein gene transcripts is nearly the same within both RNA species. We conclude, that in chicken immature red blood cells the globin genes are transcribed either at a much higher rate than other protein coding genes or that most of the transcription products of other genes transcribed at higher rates must be degraded during or very shortly after transcription. Moreover, an accumulation of both types of transcripts was observed, as the contents of these RNA molecules was higher within steady state nuclear RNA than within the newly synthesized RNA.

Animals↗

PubMed1983

Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo.

Mouse erythroleukemia cells were pulse-labeled with [3H]uridine and irradiated with 254-nm light to produce covalent crosslinks between RNA and proteins in close proximity to one another in vivo. Nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA were isolated and digested with nucleases, and the resulting proteins were subjected to gel electrophoresis. Proteins carrying covalently crosslinked [3H]uridine nucleotides were identified by fluorography. The results demonstrate that heterogeneous nuclear RNA is complexed in vivo with a set of six major proteins having molecular weights between 32,500 and 41,500. Analysis of chromatin fractions indicates that nascent heterogeneous nuclear RNA chains assemble with these six proteins as a very early post-transcriptional event. These data, and other results [Nevins, J. R. & Darnell, J. E. (1981) Cell 15, 1477-1493], lead us to propose the usual order of post-transcriptional events to be: heterogeneous nuclear RNA-ribonucleoprotein particle assembly leads to poly(A) addition leads to splicing.

Animals↗

PubMed1981

Determination of the nuclear RNA content of cells from donors of three different ages during in vitro senescence.

Diploid fibroblasts from male donors aged 8, 40 and 84 years were grown in vitro and their nuclear RNA content was determined at early and late doublings by absorption cytophotometry after staining with Azure B for 2 hours at 40 degree C and pH 4. It was found that the average nuclear RNA content of the early doublings was lower than that of the last doubling in all three strains. This difference became smaller as donor age increased. Also, as the age of the donor increased the percentage of early passage cells with a relative nuclear RNA content of less than 6 arbitrary units decreased, those with a relative nuclear RNA content of more than 6 units increased and the diversity in nuclear RNA content increased. The distribution pattern of relative RNA content for late passage cells was similar for the three donors.

Adult↗

PubMedDate not supplied

[Differences in transcription of nuclear RNA from rat liver with normal and malignant growth].

Total nuclear RNA was isolated under conditions of actinomycin D blocking ion-exchange chromatography on kieselguhr columns with methylated albumin detected differences in transpiration of rat liver nuclear RNA with intensive normal and malignant growth. Actinomycin D in doses blocking the appearance of peculiar proteins in the blood serum of rats with the regenerating liver and RS-1 hepatoma produces a different effect on the biosynthesis of nuclear DNA-like RNA of the rat liver. 24h after a partial hepatectomy the antibiotic inhibits considerably the biosynthesis of nuclear DNA-like RNA which is eluated during chromatographying with 0.2% solution of sodium dodecyl sulphate at 70 degrees C. With RS-1 hepatoma the actinomycin D effect is most pronounced with respect to nuclear DNA-like RNA of rats with a tumour which is washed off from the column with a 0.2% solution of sodium dodecyl sulphate at 37 degrees C.

Animals↗

PubMed1978

Immunoglobulin light chain mRNA is processed from large nuclear RNA.

Recombinant DNA probes, produced by the molecular cloning of immunoglobulin kappa light chain mRNA, have been used to analyze heterogenous nuclear RNA for presumptive precursors to cytoplasmic kappa light chain mRNA, Three discrete classes of nuclear RNA containing kappa mRNA sequences were detected after pulse-labeling of immunoglobulin-producing P3 myeloma cells. Two of these were substantially larger than kappa mRNA (approximately 10 and 4 times larger); the third was similar in size to kappa mRNA. Beginning with the largest, the sequential appearance of these three classes of nuclear RNA preceded the first appearance of newly synthesized kappa light chain mRNA in the cytoplasm. The results presented here suggest that immunoglobulin kappa light chain mRNA is generated by the stepwise cleavage and processing of a large nuclear RNA transcript.

Cell Line↗

PubMed1984

Structure and distribution of Alu family sequences or their analogs within heterogeneous nuclear RNA of HeLa, KB, and L cells.

We studied the distribution of repetitive sequence elements capable of forming double-stranded regions in nuclear RNA of HeLa, KB, and L cells. In human RNA populations, we called these regions duplex Alu family RNA (dAfRNA) because they represent transcripts of the highly reiterated family of DNA regions known as "Alu family DNA" (Rubin et al., Nature (London) 284:372-374, 1980). Although the dAfRNA populations of both human cell lines (HeLa and KB) have low sequence complexity, they represent 5% of the total heterogeneous nuclear RNA and have identical fingerprints; mouse L-cell dAf-like RNA (which has a similar complexity) represents only 2% of the total heterogeneous nuclear RNA and has an entirely different fingerprint. We utilized Escherichia coli RNase III as a highly specific reagent for the recognition of RNA:RNA duplex structure. This enzyme cleaves within the six characteristic RNase T1-resistant oligonucleotides of HeLa- and KB-cell dAfRNA (Robertson et al., J. Mol. Biol. 115:571-589, 1977). In addition, the size of heterogeneous nuclear RNA from all three cell types is reduced from greater than 32S to about 15S after RNase III treatment. We conclude that this size shift is a result of cleavage within dAfRNA regions and that such regions are present in most or all of the large RNA transcripts of these cells.

Animals↗

PubMed1980

Developmental changes in the molecular weight of heterogeneous nuclear RNA.

The size distribution of heterogeneous nuclear RNA (hnRNA) in the sea urchin embryo changes markedly during early development. Measurement of cleavage (4.5 h) to late gastrula (40 h) hnRNA by sedimentation in aqueous and denaturing solvent indicates that in the stages tested cleavage (4.5 h) hnRNA is smallest and rotating blastula (13 h) hnRNA is largest. The molecular weight of cleavage hnRNA is calculated to be about one-third that of rotating blastula hnRNA. Sedimentation of early embryo hnRNA in denaturing solvent to disaggregate complexes demonstrated mean S values lower than those obtained in aqueous solvent.

Animals↗

PubMed1998

Molecular cloning of a cDNA encoding human SPH-binding factor, a conserved protein that binds to the enhancer-like region of the U6 small nuclear RNA gene promoter.

Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III. Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library. The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function. Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters. Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors. Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes. Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription.

Animals↗

PubMed1979

Effect of 5-bromodeoxyuridine on heterogeneous nuclear RNA in rat hepatoma cells.

Heterogeneous nuclear RNA HnRNA) was isolated from untreated and 5-bromodeoxyuridine (BrdUrd) treated hepatoma tissue culture (HTC) cells. analysis of this RNA by either electrophoresis on polyacrylamide-agarose gels or centrifugation in sucrose gradients demonstrated that BrdUrd caused a shift in the labeled HnRNA population toward a smaller size distribution. This effect was produced by concentrations of BrdUrd which specifically lower the level of the differentiated enzyme tyrosine aminotransferase, but do not greatly affect cell growth. Differential binding to oligo(dT) cellulose was used to fractionate HnRNA further into classes containing poly(A) (alpha), oligo(A) (beta) or neither category of A-rich sequences (gamma). BrdUrd did not alter the relative rates of uridine incorporation into the three classes. The shift in the labeled HnRNA population due to BrdUrd was observed in all three subclasses of HnRNA.

Animals↗