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At least 73 records · Page 4Linked to original sources
PubMed1998

The pre-mRNA 5' cap determines whether U6 small nuclear RNA succeeds U1 small nuclear ribonucleoprotein particle at 5' splice sites.

Efficient splicing of the 5'-most intron of pre-mRNA requires a 5' m7G(5')ppp(5')N cap, which has been implicated in U1 snRNP binding to 5' splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5' cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5' splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5' splice site and not with any loss of U1 snRNP binding.

Alternative Splicing↗
PubMed1981

Nuclear RNA is spliced in the absence of poly(A) addition.

Splicing of newly formed nuclear RNA transcripts has been demonstrated during adenovirus type 2 (Ad2) mRNA formation in HeLa cells in the presence of "cordycepin," 3' deoxyadenosine, a drug that stops poly(A) addition to nuclear RNA. Nuclear RNA prepared from Ad2-infected cells after a 30 min label time in the presence or absence of 3' deoxyadenosine was hybridized to and eluted from Ad2 DNA sequences in the transcription units of region E1b and region E2. The nuclear RNA from the 3' dA-treated cells did not contain poly(A) but did contain Ad2-specific molecules approximately 200 to 250 bases shorter than the spliced mRNAs of the control infected cells. In addition, the approximately 2 kb RNA from Ad2 region E2 was shown to have sequences that lie more than 3.5 kb apart on the DNA, suggesting that correct cutting and splicing of the primary transcript in the absence of poly(A) synthesis had occurred. Therefore, although poly(A) addition usually precedes splicing during mRNA formation, poly(A) is not required for splicing.

Adenoviruses, Human↗
PubMed1976

Message and non-message sequences adjacent to poly(A) in steady state heterogeneous nuclear RNA of HeLa cells.

Highly purified steady state heterogeneous nuclear RNA from HeLa cells has been prepared by a new procedure. Detergent-washed nuclei are disrupted in 0.4 M ammonium sulfate, which also disociated contamination polysomes. The hnRNA remains bound to chromatin, which can be pelleted by gentle centrifugation. Ribonuclease inhibitors permit the preparation of very high molecular weight nuclear RNA. The hnRNA was cleaved with alkali. The poly(A)-containing fragments were separated from those containing oligo(A), and a cDNA copy was prepared. Hybridization of this nuclear cDNA to cytoplasmic mRNA showed that the scarce (complex) message sequences make up a larger proportion of nuclear RNA than of cytoplasmic RNA. In addition, at least 30% of the poly(A)-adjacent sequences in nuclear RNA have no apparent counterparts in the cytoplasm. cDNA prepared from hnRNA sedimenting faster than 45S under denaturing conditions gives similar results, showing the presence of both message and non-message sequences in very large transcripts. cDNA complementary to mRNA was separated into the abundant and scarce sequences, and hybridized separately to the poly(A)-adjacent sequences in nuclear RNA. The hybridization of the abundant sequence cDNA was used to set an upper limit on possible cytoplasmic contamination. Hybridization of the scarce cytoplasmic sequences are represented in nuclear RNA approximately once per cell.

Cell Nucleus↗
PubMed1997

RNA-dependent phosphorylation of a nuclear RNA binding protein.

The human C1 heterogeneous nuclear ribonucleoprotein particle protein (hnRNP protein) undergoes a cycle of phosphorylation-dephosphorylation in HeLa cell nuclear extracts that modulates the binding of this protein to pre-mRNA. We now report that hyperphosphorylation of the C1 hnRNP protein is mediated by a kinase activity in nuclear extracts that is RNA-dependent. Although the basal phosphorylation of the C1 hnRNP protein in nuclear extracts reflects a casein kinase II-type activity, its RNA-dependent hyperphosphorylation appears to be mediated by a different kinase. This is indicated by the unresponsiveness of the RNA-stimulated hyperphosphorylation to casein kinase II inhibitors, and the distinct glycerol gradient sedimentation profiles of the basal versus RNA-stimulated C1 hnRNP protein phosphorylation activities from nuclear extracts. RNA-dependent phosphorylation was observed both for a histidine-tagged recombinant human C1 hnRNP protein added to nuclear extracts and also for the endogenous C1 hnRNP protein. Additional results rule out protein kinase A, protein kinase C, calmodulin-dependent protein kinase II, and double-stranded RNA-activated protein kinase as the enzymes responsible for the RNA-dependent hyperphosphorylation of the C1 hnRNP protein. These results reveal the existence in nuclear extracts of an RNA-dependent protein kinase activity that hyperphosphorylates a known pre-mRNA binding protein, and define an additional element to be integrated into the current picture of how nuclear proteins are regulated by phosphorylation.

Casein Kinase II↗
PubMed1978

Proteins bound to heterogeneous nuclear RNA of simian-virus-40-infected cells.

Heterogeneous nuclear RNA . protein (hnRNA . protein) complexes from simian-virus-40 (SV40)-infected cells late in infection contain 7--10% RNA sequences specific to SV40 DNA. The SV40 nuclear RNA . protein complexes sediment at 60--70 S. The reality and specificity of the RNA-protein association is shown in metrizamide gradients. Protein and RNA lebels of hnRNA . protein-particles in SV40-infected cells follow a parallel pattern with a peak at 1.28 g/cm2 whereas a mixture of ribosomal RNA and soluble cytoplasmic proteins is separated according to the different densities in metrizamide. Analysis of hnRNA . protein from infected cells by two-dimensional gel electrophoresis shows the presence of a number of new proteins. It is demonstrated that three of these proteins are cellular ones induced by the virus infection and hence constitute good candidates to be specific RNA . protein particles for virus nuclear RNA. The presence of actin in hnRNA . protein particles from normal and SV40-infected cells and the presence of the major capsid protein VP1 in hnRNA . protein particles from SV40-infected cells is discussed.

Base Sequence↗
PubMed1992

Cyclic 2',3'-phosphates and nontemplated nucleotides at the 3' end of spliceosomal U6 small nuclear RNA's.

Spliceosomal U6 small nuclear RNA (U6 RNA) in species as diverse as man, frog, fruitfly, and soybean have at their 3' ends a cyclic 2',3'-phosphate (greater than p) apparently derived from uridylic acid residues that were added post-transcriptionally. The 3' ends of U6 RNA's from various sources may be processed in different ways, or to different extents, depending on the organism or stage of development. The presence of a greater than p terminus on U6 RNA may influence the activity of U6 RNA either directly during splicing or indirectly by ensuring that the RNA has a defined length or proper conformation (or both).

Animals↗
PubMed1990

Flow cytometry measurement of nuclear RNA content in uveal melanoma.

We retrospectively studied the nuclear RNA content in uveal melanomas with known, long-term follow-up. Thirty patients had spindle cell melanomas, 25 had mixed cell tumors, and 9 had epithelioid cell neoplasms. Epithelioid cell types had a higher nuclear RNA content compared to mixed or spindle cell types (p less than 0.05). Using the Cox proportional hazards model, the nuclear RNA content appeared to be an independent prognostic indicator in uveal melanomas, and an increased nuclear RNA content was associated with a worse prognosis (p = 0.001).

DNA, Neoplasm↗
PubMed2003

ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA.

Stable addition of U2 small nuclear ribonucleoprotein (snRNP) to form the prespliceosome is the first ATP-dependent step in splicing, and it requires the DEXD/H box ATPase Prp5p. However, prespliceosome formation occurs without ATP in extracts lacking the U2 snRNP protein Cus2p. Here we show that Prp5p is required for the ATP-independent prespliceosome assembly that occurs in the absence of Cus2p. Addition of recombinant Cus2p can restore the ATP dependence of prespliceosome assembly, but only if it is added before Prp5p. Prp5p with an altered ATP-binding domain (Prp5-GNTp) can support growth in vivo, but only in a cus2 deletion strain, mirroring the in vitro results. Other Prp5 ATP-binding domain substitutions are lethal, even in the cus2 deletion strain, but can be suppressed by U2 small nuclear RNA mutations that hyperstabilize U2 stem IIa. We infer that the presence of Cus2p and stem IIa-destabilized forms of U2 small nuclear RNA places high demands on the ATP-driven function of Prp5p. Because Prp5p is not dispensable in vitro even in the absence of ATP, we propose that the core Prp5p function in bringing U2 to the branchpoint is not directly ATP-dependent. The positive role of Cus2p in rescuing mutant U2 can be reconciled with its antagonistic effect on Prp5 function in a model whereby Cus2p first helps Prp5p to activate the U2 snRNP for prespliceosome formation but then is displaced by Prp5p before or during the stabilization of U2 at the branchpoint.

Adenosine Triphosphate↗
PubMed1977

The heterogeneous nuclear RNA of chicken erythroblasts.

Heterogeneous nuclear RNA (hnRNA) from chicken erythroblasts has a modal molecular weight of 1.6 -10(6) in 99% dimethylsulfoxide. When erythroblasts are labeled continuously with [14C]uridine, nuclear RNA is labeled as a single kinetic component with a half-life of 18 min. After a 10--20 min lag, label appears in cytoplasmic RNA at about 1% of the initial rate of total RNA synthesis. Of the hnRNA sedimenting faster than 28 S ribosomal RNA in both an aqueous sucrose gradient and a subsequent fructose gradient in 99% dimethylsulfoxide, about one-third is polyadenylated, although only about one in 2000 (i.e. about four molecules per cell) contain a globin messenger sequence. The hnRNA of erythroblasts isolated from 5.7- and 11-day chick embryos have the same content of globin messenger sequences as erythroblaasts from anemic adults.

Anemia↗
PubMed1972

Occurrence of uridylate-rich oligonucleotide regions in heterogeneous nuclear RNA of HeLa cells.

Heterogeneous nuclear RNA molecules from HeLa cells contain a specific segment of about 30 nucleotides length that is largely (about 80%) uridylic acid. This oligo(U) segment is located predominantly in the larger (70S-90S) heterogeneous nuclear RNA molecules, and is essentially absent in messenger RNA and 45S ribosomal precursor RNA molecules. The oligo(U) hybridizes rapidly to cellular DNA, suggesting that it is transcribed from the repeated regions of the DNA.

Alkaline Phosphatase↗
PubMed1983

Poly(A) RNA of the egg cytoplasm: structural resemblance to the nuclear RNA of somatic cells.

This paper concerns the structural characteristics of the poly(A) RNA stored in unfertilized amphibian and echinoderm eggs. Though located in the egg cytoplasm, at least two-thirds of these maternal transcripts display an interspersed sequence organization similar to that of nuclear RNA. In Xenopus laevis interspersed poly(A) RNA molecules are synthesized and deposited in the oocyte cytoplasm throughout the main growth phase of oogenesis. Regions of the sea urchin genome that are represented by interspersed maternal transcripts have been recovered from recombinant clone libraries. In one case the same single-copy sequence is found both in an abundant message-sized 1.6 kilobase (kb) maternal transcript and in a 7.5 kb maternal transcript that structurally resembles a precursor form and is not found in embryonic polysomes. In a second example considered, a 9.5 kb transcript was identified in embryo nuclear RNA that may be identical in structure with an interspersed maternal poly(A) RNA derived from the same transcription unit. Transcription of this sequence appears to be constitutive in somatic cell nuclei, though no homologous cytoplasmic RNAs are found after early cleavage. This may be a widespread form of regulation for transcription units expressed in female germ cells, and represented in the maternal poly(A) RNA pools of unfertilized eggs.

Animals↗
PubMed1997

Identification and characterization of a nuclease specific for the 3' end of the U6 small nuclear RNA.

The U6 small nuclear RNA is post-transcriptionally processed by the addition and removal of nontemplate uridylates (Us) at its 3' end prior to incorporation into the U4 x U6 small nuclear ribonucleoprotein complex. An enzyme responsible for removing Us from the U6 3' end has not been previously identified. Here we biochemically isolate and characterize an exonuclease activity from HeLa cells that removes template and nontemplate 3'-nucleotides specifically from U6 RNA. We also report the isolation of an inhibitor of this U6 nuclease. U6 nuclease rapidly removes the four terminal 3' Us found in the human U6 coding sequence in a magnesium-dependent manner. Mutagenesis studies on the U6 RNA define regions essential for processing. U6 nuclease recognizes specific sequences on both strands near the base of the major intramolecular stem loop of the U6 RNA. The preprocessed 3' Us form part of the base of the stem loop, but neither specific sequences nor secondary structure at the four terminal nucleotides are required to achieve processing.

Base Sequence↗
PubMed1984

Nuclear RNA metabolism in rat hippocampal slices incubated in vitro.

Rat hippocampal slices were incubated with [3H]uridine in vitro to analyze the metabolism of nuclear RNA and the RNA precursor fractions. Labeling of total nuclear RNA was linear for 4 h of incubation and proportional to the concentration of labeled uridine in the incubation medium. Addition of 3.5 X 10(-8) M corticosterone to the incubation medium produced an enhancement of nuclear RNA labeling with no significant effect on the labeling of the RNA precursor fraction. Progesterone and dexamethasone, at the same concentration, had no effect on either variable. Labeling of RNA by cerebellar slices under the same conditions was approximately one-half the value obtained using hippocampal slices and the cerebellar RNA precursor fraction accumulated only 65% of the radioactivity from [3H]uridine found in the hippocampal pool. Corticosterone had no effect on the labeling of total nuclear RNA in cerebellar slices. Nuclear poly(A)-containing RNA constituted 19% of the total labeled nuclear RNA in these incubations, as estimated by oligo (dT)-cellulose chromatography. Cordycepin (3'-deoxyadenosine) at a concentration of 25 micrograms/ml inhibited to some extent the labeling of total nuclear RNA and the RNA precursor fraction, but preferentially diminished the amount of labeled RNA bound to oligo (dT)-cellulose. Corticosterone increased the amount of [3H]RNA which bound to oligo (dT)-cellulose, while progesterone had no effect. These results show that hippocampal slices maintained in vitro, can be used to analyze nuclear RNA metabolism, one positive regulator of which in the rat hippocampus is the adrenal steroid, corticosterone.

Adrenalectomy↗
PubMed1979

Sequential Sepharose chromatographic isolation of polysomes and polysomal RNAs depleted in nuclear RNA from Xenopus.

Analyses of sequence complexities, stage specific gene expression, and mRNA sequence divergence require polysomal RNA preparations relatively free of nuclear RNA contamination. Conventional procedures for the isolation of uncontaminated polysomal RNAs which rely on sucrose density centrifugations are laborious and unsuitable for large scale isolations. We describe here a method using sequential Sepharose chromatography for isolating polysomes and polysomal RNAs depleted for nuclear RNA. Polysomes and polysomal RNAs isolated from livers of Xenopus stimulated to produce vitellogenin were capable of directing protein synthesis in vitro and showed little evidence of degradation. The polysomal RNAs contained less than 0.5% of nuclear RNA.

Animals↗
PubMed1975

Sequence complexity of heterogeneous nuclear RNA in sea urchin embryos.

The sequence complexity of heterogeneous nuclear RNA is sea urchin gastrulas was measured by RNA-driven hybridization reactions with nonrepetitive sea urchin DNA. 28.5% of the sequence complexity of the genome is represented in the nuclear RNA. This amounts to 1.74 X 10(8) nucleotides of diverse sequence, more than 10 times the nucleotide complexity of the polysomal messenger RNA extracted from sea urchin embryos at the same stage. The complex set of nuclear RNA sequences driving this hybridization reaction was shown to be the same as the rapidly labeled hnRNA, using pulse-labeled nuclear RNA as driver.

Animals↗
PubMed1981

Purification and characterization of a simple ribonucleoprotein particle containing small nucleoplasmic RNAs (snRNP) as a subset of RNP containing heterogenous nuclear RNA (hnRNP) from HeLa cells.

A ribonucleoprotein complex whose RNA complement consists exclusively of small nuclear RNA species (snRNA) has been purified from particles containing heterogenous nuclear RNA (hnRNP) from HeLa cells. This was accomplished by taking advantage of their ability to band at a density of about 1.43 g/cm3 in plain cesium chloride as well as in cesium chloride gradients containing 0.5% sarkosyl without prior aldehyde fixation. After these two steps of equilibrium density centrifugation, these snRNPs were still largely contaminated by free proteins (and especially phosphoproteins). A final step of purification by velocity sedimentation in a sucrose gradient containing 0.5 M cesium chloride and 0.5% sarkosyl was efficient in completely eliminating all free proteins. U1, U2, U4, U5 and U6 species according to the nomenclature of Lerner et al. (Nature, (1980) 283, 220-224) were found in these purified snRNPs, while a significant part of U6 and a small amount of U2 were found in the bottom fraction. 5S species behaved entirely as free RNA and is presumably a contaminant of cytoplasmic origin. Electrophoresis of proteins from snRNP labeled in vivo with (35S) methionine, revealed four bands with migrations corresponding to molecular weights ranging between 10,000 and 14,000 daltons.

Base Sequence↗