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PubMed1988

[Biosynthesis and processing of heterogeneous nuclear RNA in rat thymocytes].

Using [14C]adenine as a labeled precursor, the biosynthesis and processing of heterogeneous nuclear RNA (hnRNA) in chromatin and nucleoplasm were studied. It was shown that intraperitoneally injected actinomycin D (50-500 micrograms/100 g of body weight) did not inhibit the biosynthesis of hnRNA and rRNA in rat thymocytes. Besides biosynthesis, the polyadenylation of the bulk of primary transcripts was also localized in the chromatin. rRNA precursors constituted only a small part of the newly synthesized hnRNA molecules. During processing, highly polymeric (28S-55S) poly(A+)-hnRNA fractions passed from chromatin to the nucleoplasm, as a result of which a large number of high molecular weight polyadenylated hnRNA molecular were pooled in the nucleoplasm. Thymocyte hnRNA was tightly bound to nuclear structures and its isolation tributed stringent conditions. The bulk of poly(A+)-hnRNA molecules were degraded without formation of stable intermediates. The pattern of poly(A-)-hnRNA maturation in chromatin and nucleoplasm was identical, i.e., in both compartments the decrease in the size and accumulation of processed 16S-20S RNA molecules was observed. About 6% of newly synthesized poly(A+)-hnRNA was converted into polysomal mRNA. Cytoplasmic poly(A+)-RNA consists of two fractions, i.e., metabolically stable high molecular weight (greater than or equal to 40S) RNA molecules and a less stable 10S-20S RNA.

Animals↗
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↗
PubMed1983

Delayed processing of dihydrofolate reductase heterogeneous nuclear RNA in amino acid-starved mouse fibroblasts.

We have studied the metabolism of dihydrofolate reductase (DHFR) RNA in cells synchronized in the G1 phase of the cell cycle by starvation for isoleucine and glutamine. The relative content and stability of DHFR mRNA and the relative rate of transcription of the DHFR gene are similar in starved and exponentially growing cells. However, the relative rate of labeling of DHFR mRNA is about three times lower in starved cells than in exponentially growing cells. When the starved cells are stimulated to reenter the cell cycle by feeding them with complete medium, the relative rate of labeling of DHFR mRNA increases about fourfold within 6 h. However, the relative rate of transcription of the DHFR gene changes very little during this period. Continuous labeling experiments show that starved cells convert DHFR heterogeneous nuclear RNA into cytoplasmic DHFR mRNA much more slowly than serum-limited or exponentially growing cells. Pulse-chase experiments show that DHFR mRNA sequences contained in DHFR heterogeneous nuclear RNA appear to be conserved in starved cells. In addition, the content of DHFR RNA sequences in the nuclei of starved cells is about three times greater than that in exponentially growing cells. Delayed processing of DHFR heterogeneous nuclear RNA is also observed when exponentially growing cells are treated with inhibitors of protein synthesis. Our results suggest that, although delayed processing leads to a decrease in the initial labeling rate of DHFR mRNA, it does not result in a decrease in the actual rate of production of the message.

Amino Acids↗
PubMed1977

Complexity of the structure of particles containing heterogeneous nuclear RNA as demonstrated by ribonuclease treatment.

Brain ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) and a large number of polypeptides ranging from 23000 to 150000 molecular weight were treated with pancreatic and T1 ribonucleases at low (0.1 or 0.5 microng plus 5 or 25 units/ml), high (2 microng plus 100 units/ml) and very high (20 microng plus 1000 units/ml) concentrations. At low enzyme concentration, a large fraction of the particle material accumulated at 35-45 S. The accumulation was more marked for proteins in the 30000-38000 molecular weight range. Heterogeneous complexes containing a large spectrum of proteins with a characteristic distribution were disclosed between 60 and 200 S. At high ribonuclease concentration the total quantity of proteins at 35-45 S decreased, but the proteins of 30000-38000 molecular weight predominated. Heterogeneous complexes were also present. Finally at very high enzyme concentration, the particles were almost entirely hydrolyzed. Only a small amount of heterogeneous complexes subsisted at 30-190 S. The RNA content of the remaining ribonucleoprotein complexes as well as its size decreased upon ribonuclease treatment as shown by CsCl density determination and electrophoretic analysis. The results suggested that the stability of the complexes depended upon protein-protein as well as RNA-protein interactions. They also showed that sequences up to 200-300 nucleotides were protected by proteins against ribonuclease. The results were compatible with the existence of at least 3 constituents in the particles. The previously defined monoparticle population accumulating at 35-45 S was heterogeneous in respect to ribonuclease sensitivity and protein composition. The 30000-38000 molecular weight proteins accumulating at low ribonuclease and predominating afterwards were assumed to belong to monoparticles alpha. Monoparticles beta contained a larger range of proteins more easily released by the enzymes. The heterogeneous complexes were a third constituent whose relationship with the monoparticles war not established yet. A large fraction of the particle phosphoproteins was associated to these complexes. On the basis of our experiments in vitro it is assumed that monoparticle alpha can be preferentially isolated from the nuclei under conditions where endogeneous ribonuclease is high and (or) not inhibited. This might explain why, in certain cases, only one or a few proteins were described in nuclear particles instead of the complete set present in the native particles.

Animals↗
PubMed1975

Ribonucleoprotein particles containing heterogeneous nuclear RNA in the cellular slime mold Dictyostelium discoideum.

As in higher eukaryotes, heterogeneous nuclear RNA (HNRNA) in the cellular slime mold Dictyostelium discoideum is associated with proteins in the form of ribonucleoprotein particles. Mixing experiments with deproteinized hnRNA establish that the nuclear ribonucleoprotein particles are not formed artificially during isolation. In contrast to comparable material from mammalian cells (polydisperse, 40-25- S), Dictyostelium heterogeneous nuclear ribonucleoprotein particles sediment at only 55 S in sucrose gradients, possibly reflecting the smaller size of slime mold hnRNA relative to the large hnRNA found in higher eukaryotes. The RNA of the nuclear 55S ribonucleoprotein particles is shown to be hnRNA by virtue of its size (15S), content of polyadenylate sequences, and hybridization kinetics at DNA excess. The hnRNA-associated porteins are electrophoretically complex and have molecular weights between 20,000 and 150,000. In 0.35 M NaCl most of the proteins are released from the hnRNA. However, a single protein of 72,000-74,000 molecular weight remains bound, as indicated by its co-chromatography with the RNA on poly(U)-Sepharose and banding in Cs2SO4. The same protein is recovered when heterogeneous nuclear ribonucleoprotein is digested with T1 ribonuclease under conditions where the poly(A) is nuclease-resistant. The 73,000 molecular weight protein appears to be specifically bound to polyadenylate sequences in Dictyostelium hrRNA.

Adenine↗
PubMed1979

Heterogeneous nuclear RNA double-stranded regions probed in living HeLa cells by crosslinking with the psoralen derivative aminomethyltrioxsalen.

The psoralen derivative aminomethyltrioxsalen (AMT, 4'-aminomethyl-4,5',8-trimethylpsoralen) has been employed as a probe for heterogeneous nuclear RNA (hnRNA) double-stranded regions in experiments with living HeLa cells. hnRNA ribonucleoprotein (hnRNP) particles were purified from untreated or AMT-treated cells after irradiation with 365-nm light, and double-stranded hnRNA regions (dsRNA) were isolated by RNase A + T1 digestion of hnRNP, followed by preparative Cs2SO4 isopycnic centrifugation. The purified, hnRNP-derived dsRNA was then assayed for interstrand crosslinks by measurement of its "snapback" to RNase-resistant form after thermal denaturation. By this procedure, the amount of crosslinked dsRNA was found to be increased 3- to 7-fold in cells exposed to AMT in vivo. The levels of crosslinking in vivo compared favorably with those observed in model experiments with pure dsRNA in vitro. These results establish that double-stranded hnRNA regions exist in the living cell, and they further demonstrate that these base-paired regions are organized as rather accessible sites within the nucleus.

Furocoumarins↗
PubMed1979

The 5' terminal capping of heterogeneous nuclear RNA at different embryonic stages of the sea urchin.

5' Terminal cap structures of hnRNA have been characterized and the extent of capping determined as a function of embryonic development. Sea urchin embryo hnRNA contains only the type-1 cap, m7GpppNmpNp, with the type-2 cap, which has a 2'-0-methylated subpenultimate nucleotide, being associated only with stable small nuclear RNAs. These cap 2-containing RNAs are synthesized at a rate of approximately 70 molecules min-1 nucleus-1 compared to approximately 1000 molecules for hnRNA cap 1. Approximately 70% of nuclear cap 1 is associated with greater than 15S RNA in denaturing solvent, but under non-denaturing conditions the percentage is much higher. Cap 1 in low and high molecular weight nuclear RNA have the same kinetics of methyl labeling. Thus all cap 1 structures may belong to a single class either covalent or H-bonded to high molecular weight RNA. hnRNA greater than 15S is 35% capped; however, adding caps in less than 15S RNA gives an estimate of 50% capping for total hnRNA. In development from early blastula to late gastrula, there is little if any change in the extent of capping of hnRNA. These results coupled with others indicate that the fraction of hnRNA molecules serving as precursor to mRNA does not change quantitatively during embryonic development.

Animals↗
PubMed1978

Oligonucleotides in heterogeneous nuclear RNA: similarity of inverted repeats and RNA from repetitious DNA sites.

A comparison has been made by oligonucleotide analysis of three fractions of HeLa cell hnRNA: (1) the "snap-back" fraction (ds-hnRNA, 5% of the total); (2) the fraction that self-anneals during prolonged incubation (25% of total); and (3) the fraction that hybridizes most rapidly to an excess of HeLa cell DNA (rep-hnRNA, 10% of the total). T1 fingerprints of each of these hnRNA fractions were similar to one another and featured the largest T1 oligonucleotides of known sequence previously isolated from ds-hnRNA (Robertson, H.D., et al. (1977) J. Mol. Biol. 115, 571--590; Jelinek, W. (1977 J. Mol. Biol. 115, 591--602). When hybridized to DNA either in solution or immobilized on filters, the isolated ds-hnRNA and the rep-hnRNA fractions showed similar hybridization kinetics in the COt range of "intermediate" repetitive DNA sequences; the ds-hnRNA and the rep-hnRNA also self-annealed to equal extents in the absence of any DNA. DNA of all buoyant density classes contained the T1 oligonucleotides diagnostic of the ds-hnRNA and the rep-hnRNA. While hnRNA is rich in inverted repeated sequences, cytoplasmic mRNA contains far fewer such sequences.

Base Sequence↗
PubMed1976

Adenylate-rich sequences of heterogeneous nuclear RNA from normal and chronic lymphocytic leukaemia lymphocytes.

Rapidly labelled high molecular weight heterogeneous nuclear RNA (Hn RNA) from normal and chronic lymphocytic leukaemia (CLL) lymphocytes has been fractionated into two classes by chromatography on poly (U) sephasose. The (+) Hn RNA is bound to poly (U) sepharose and contains both a long poly (A) segment (100-200 nucleotides) and shorter (A) rich sequences (approximately 20-30 nucleotides. (-) Hn RNA is not bound to poly (U) sepharose and lacks the longer poly (A) segment but does contain shorter (A) rich sequences. Partial association of the short (A) rich segments with double-stranded regions was found for both classes of Hn RNA but was most pronounced in (-) Hn RNA from high WBC cases of CLL. Both classes of Hn RNA from CLL lymphocytes contain varying but generally higher amounts of the short (A) rich fraction than those in normals. Similar amounts of the longer poly (A) segments were found in (+) Hn RNA from both CLL and normal lymphocytes.

Base Sequence↗
PubMed1996

Quantitative RT-PCR on CYP1A1 heterogeneous nuclear RNA: a surrogate for the in vitro transcription run-on assay.

A quantitative reverse transcription polymerase chain reaction (RT-PCR) assay was developed to amplify a region of the CYP1A1 heterogeneous nuclear RNA (hnRNA) transcript encompassing the first intron-exon boundary. The RT-PCR protocol uses a CYP1A1 recombinant RNA internal standard identical to the target hnRNA except for an engineered unique internal restriction site. Its inclusion enables normalization between reactions and a measurement of the absolute number of target hnRNA transcripts. Specificity for the hnRNA was achieved by using intron-directed primers in both the RT and the PCR. Nuclear run-on assays and the hnRNA RT-PCR assay detected an equivalent increase in transcription of Cyp1a-1 in cultured murine Hepa 1c1c7 cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The RT-PCR assay also revealed TCDD-dependent transcriptional activation of the Cyp1a-1 gene in murine skin, a tissue unsuited to the nuclear run-on assay because of inherent difficulties associated with the isolation of nuclei. These examples demonstrate that the hnRNA RT-PCR assay is a facile surrogate for the nuclear run-on assay. Moreover, the sensitivity and design characteristics of the RT-PCR assay suggest the potential for its broad application in general transcriptional research.

Animals↗
PubMed1979

Photochemical cross-linking of secondary structure in HeLa cell heterogeneous nuclear RNA in situ 1.

The psoralen derivative 4'-hydroxymethyl-4, 5', 8-trimethylpsoralen (hydroxymethyltrioxsalen) has been used in experiments with isolated HeLa cell nuclei to photochemically cross-link double helical regions in heterogeneous nuclear RNA in situ. Although there are other self-complementary sequences in hnRNA that can form base-paired structures upon phenol deproteinization of annealing, the present in situ cross-linking results demonstrate that some double-stranded regions are an authentic component of native hnRNA structure. Moreover, these special regions of secondary structure are apparently highly accessible to chemical probes within the intact cell nucleus, despite the fact that hnRNA possesses a ribonucleoprotein organization.

Base Sequence↗
PubMed1982

Improved preparation of poly(A) Sepharose and the isolation of oligo(U) containing heterogeneous nuclear RNA from Friend erythroleukemic cells.

When poly(A) sepharose (prepared according to previously published procedures) was stored in aqueous buffer at 4 degrees C for 5 days or longer, it bound nonspecifically a high percentage of the input RNA which could not be eluted with formamide. We have found that treatment with ethanolamine, followed by dehydration with ethanol yielded poly(A) sepharose which was stable for many months and possessed a low degree of nonspecific binding. Chromatography on poly(A) sepharose permitted the specific isolation of that fraction of Friend erythroleukemic cell heterogeneous nuclear RNA (hnRNA) which contained oligo(U) sequences. Approximately 10% of the hnRNA which contained a poly(A) sequence [poly(A+)] also contained an oligo(U) sequence. Interestingly, prior HCHO denaturation of the hnRNA enhanced binding of the poly(A+) oligo(U+) hnRNA to poly(A) sepharose by tenfold. This suggested that the oligo(U) sequence may be in a region with secondary structure, possibly an intramolecular duplex with the 3' poly(A). Friend cell oligo(U) sequences ranged from 20 to 50 nucleotides in length and, thus, were similar to the oligo(U) sequences which heretofore had only been shown to be present in HeLa cell hnRNA. These results established that rodent cell hnRNA contain oligo(U) sequences and demonstrate, for the first time, that hnRNA containing both a poly(A) and an oligo(U) sequence can be separated from other classes of hnRNA. In addition, conditions are presented for the removal of HCHO from nucleic acid.

Animals↗
PubMed1985

Evidence for age-dependent impairment of ovalbumin heterogeneous nuclear RNA (HnRNA) processing in hen oviduct.

The expression of the ovalbumin gene in hen oviduct decreases during ageing. Northern transfer of electrophoretically fractionated total RNA from oviducts of mature egg-laying and old non-egg-laying hens, and hybridization to nick-translated plasmid pOV230 (ovalbumin complementary DNA cloned into pMB9) revealed with age increasing concentrations of distinct high-molecular-weight ovalbumin pre-mRNA species as compared with that of functional 18S ovalbumin mRNA. The accumulation of ovalbumin sequence-containing processing intermediates in old hen oviducts indicates lowered rates of RNA splicing and/or altered steady state concentrations of splicing intermediates during post-transcriptional maturation of heterogeneous nuclear RNA in old animals.

Age Factors↗
PubMed1970

A comparison of the heterogeneous nuclear RNA of HeLa cells in different periods of the cell growth cycle.

HeLa cells synthesize heterogeneous nuclear RNA (HnRNA) in the G(1), S, and G(2) portions of the cell cycle. HnRNA prepared from these various periods was compared by RNA-DNA hybridization experiments. The results indicated that some of the HnRNA molecules were equivalent at all times in the cell cycle, but limitations in the sensitivity of the hydridization reactions, as well as in the spectrum of hybridizing molecules, restrict the conclusions that can be drawn from these comparisons.

Carbon Isotopes↗