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PubMed1977

Synthesis of heterogeneous nuclear RNA in full-grown oocytes of Xenopus laevis (Daudin).

At various times following injection of either 3H-GTP or 32PO4 into full-grown (stage 6) Xenopus laevis oocytes, RNA has been extracted and fractionated on polyacrylamide gels. Based on size, base composition and incorporation data, we have defined the kinetics of synthesis and accumulation of ribosomal RNA (40S, 28S, 18S), heterogeneous RNA of high molecular weight (greater than 40S) and heterogeneous RNA migrating with molecular weights of from 4S to 40S. Nuclear isolations have been performed to determine the cellular distribution of these classes of RNA as a function of time. Evidence is presented which shows that stage 6 oocytes synthesize RNA which by virtue of its size, base composition, rapid turnover and nuclear location is equivalent to the heterogeneous nuclear RNA observed in somatic cells. In addition, the data suggest synthesis of a class of nuclear RNA with a half-life of several hours. A small fraction (5%) of the nuclear RNA is stable, enters the cytoplasm and may represent RNA added to the stockpile of maternal transcripts known to be present in stage 6 oocytes.

Animals↗
PubMed1978

Effects of ultraviolet irradiation and postirradiation incubation on heterogenous nuclear RNA size in murine cells.

We have analyzed the decrease in synthesis of individual size classes of heterogeneous nuclear RNA (hnRNA) in ultraviolet (UV)-irradiated Merwin plasmacytoma (MPC-11) cells at various times of postirradiation incubation. HnRNA from nonirradiated control cells is distributed over a wide range from approximately 60S to 5S, with 42S RNA carrying more label than any other size class. HnRNA from UV-irradiated cells shows a dose-dependent shift in size distribution toward lower molecular weight. The size distribution of hnRNA synthesized after prolonged times of postirradiation incubation is restored toward normal, i.e., synthesis of long RNA molecules increases relative to the synthesis of short ones. Analysis of the total number of hnRNA chains synthesized during a 20-min [(3)H]uridine pulse shows a considerable reduction in their number with increasing UV dose. Murine cell lines are excision-repair-deficient but capable of post replication repair inhibited by caffeine. HnRNA transcripts of cells incubated in its presence were studied. The caffeine, which has no effect on hnRNA size in control cells, inhibits to a considerable extent the restoration of full-length transcripts during postirradiation incubation. The lack of excision repair in MPC-11 was confirmed by the analysis of pyrimidine dimers in trichloracetic acid-insoluble and soluble fractions within 8 h of postirradiation incubation.The size of parental and daughter strand DNA in UV-irradiated cells was correlated with RNA transcript size. The parental DNA in these experiments does not change its size as a consequence of UV exposure and postirradiation incubation. In contrast, daughter DNA strands are short in UV-irradiated cells and they increase in size during postirradiation incubation to reach the size of parental strands after 8 h.

Animals↗
PubMed1979

Early termination of heterogeneous nuclear RNA transcripts in mammalian cells: accentuation by 5,6-dichloro 1-beta-D-ribofuranosylbenzimidazole.

Labeling of RNA in isolated HeLa cell nuclei in vitro reveals an abundance of short RNA chains made by RNA polymerase II. These short chains were initiated prior to isolation of the nuclei. The short abundant chains are increased in amount in nuclei isolated from cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Kinetic evidence indicates that the bulk of the putative heterogeneous nuclear RNA (hnRNA) precursor molecules that are terminated early in vivo are terminated approximately 100-300 nucleotides from sites of initiation. DRB increases the frequency of early termination, but there is a fraction of hnRNA precursor molecules whose elongation is not affected by DRB. Heparin is useful in studies of hnRNA transcription in isolated nuclei because it enhances chain elongation.

Amanitins↗
PubMed1976

Developmental shifts in the synthesis of heterogeneous nuclear RNA classes in the sea urchin embryo.

Three molecular classes with different properties can be distinguished in the heterogeneous nuclear RNA (HnRNA) of sea urchin embryos. The relative proportions of these classes, which we have previously designated alpha, beta and gamma HnRNA, undergo marked changes during development from morula to early gastrula. Such changes suggest the existence of more than a single HnRNA function, as well as specific roles for different HnRNA classes in embryonic development.

Age Factors↗
PubMed1977

Evidence from UV transcription mapping in HeLa cells that heterogeneous nuclear RNA is the messenger RNA precursor.

The effects of UV irradiation on the incorporation of [3H]uridine in HeLa (human) cell mRNA, rRNA, heterogeneous nuclear RNA (hnRNA) and early mRNA from adenovirus type 2 have been compared. The UV target size of cell mRNA is at least 3 times larger than the average size of the mRNA itself and larger than the adenovirus-2 early mRNA, which is known to derive from transcription units of about 1.5-5.0 kilobases. The UV target size of hnRNA, in contrast, is about the same as its size determined by sedimentation and overlaps with the target size of mRNA. It is concluded that most mRNA derives from a higher molecular weight hnRNA molecule.

Adenoviridae↗
PubMed1984

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs.

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1975

Characteristics of heterogeneous nuclear RNA in normal small lymphocytes and in acute leukemia blast cells. An outline.

In this short review of the characteristics of heterogeneous nuclear RNA of normal small lymphocytes and of leukemic blast cells the author points out that the average life time of the molecules of this RNA class in both types of cell is much longer than that so far reported for rapidly proliferating cells. In both types of cell the newly synthesized RNA includes rapidly hybridizing sequences. There is evidence, although circumstantial, that in leukemic cells these sequences are markedly different from that of normal lymphocytes. In both normal lymphocytes and leukemic blast cells the rapidly hybridizing RNA sequences are in part involved in secondary structure. In fact, a significant fraction of the hnRNA appears in RNase-resistant form with double-stranded properties. About one fifth of the hnRNA molecules of normal lymphocytes carries poly(A) segments, whereas this proportion is markedly higher in leukemic cells.

Base Sequence↗
PubMed1975

Units of transcription and translation: sequence components of heterogeneous nuclear RNA and messenger RNA.

Defining the units in which the eucaryotic genome is transcribed and translated is central to any analysis of eucaryotic gene expression. The relationship between heterogeneous nuclear RNA and messenger RNA raises the question of whether the primary transcript may be more complex than the sequence which is translated; as I concluded last month in the first part of this review, kinetic analyses of these two RNA populations provide some suggestive indications but cannot prove whether the nuclear population includes messenger precursors that are much longer than mature cytoplasmic messengers (Lewin, 1975). Here I discuss recent analyses of the sequence components present in hnRNA and mRNA and how they may be related to each other and to the organization of the genome.

Animals↗
PubMed1971

Polyadenylic acid sequences in the heterogeneous nuclear RNA and rapidly-labeled polyribosomal RNA of HeLa cells: possible evidence for a precursor relationship.

Polyadenylate sequences have been found covalently linked in heterogeneous DNA-like nuclear RNA of HeLa cells. This poly(A) material seems homogeneous in size and accounts for about 0.5% of such RNA. Similar poly(A) sequences were found in rapidly-labeled polyribosomal RNA, thought to be messenger RNA. A possible model for mRNA synthesis from large heterogeneous nuclear RNA precursor molecules is discussed.

Adenine Nucleotides↗
PubMed1976

5,6-Dichloro-1-Beta-D-ribofuranosylbenzimidazole inhibits initiation of nuclear heterogeneous RNA chains in HeLa cells.

The nucleoside analog 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) at 75 to 150 micromolar concentrations inhibits the synthesis of nuclear heterogeneous RNA (hnRNA) in HeLa cells by 60 to 70 percent. The sedimentation profile of hnRNA labeled with (3H)uridine for 45 seconds after brief treatment (45, 90, or 180 seconds) with DRB showed a progressive decrease in the labeling of shorter hnRNA molecules relative to longer molecules. Prior exposure of the cells to actinomycin D, an inhibitor of RNA chain elongation, did not alter the sedimentation profile of hnRNA. These results suggest that DRB preferentially inhibits the initiation of hnRNA chains so that after exposure to DRB for a brief period the longer nascent chains still remain to be finished and thus incorporate a greater share of the pulse label. By progressively increasing the time of exposure to DRB, and measuring the rate of increase in the average size of the labeled, nascent RNA, it was estimated that the chains were growing at rates between 50 and 100 nucleotides per second.

Benzimidazoles↗
PubMed1991

Levels of ornithine decarboxylase genomic sequences, heterogeneous nuclear RNA and mRNA in human myeloma cells resistant to alpha-difluoromethylornithine.

We previously isolated and characterized a human myeloma cell line overproducing ornithine decarboxylase (ODC) due to gene amplification [Leinonen, Alhonen-Hongisto, Laine, Jänne & Jänne (1987) Biochem. J. 242, 199-203]. We have now employed the PCR combined with reverse transcription to determine semiquantitatively ODC gene dosage and the amounts of heterogeneous nuclear (hn) RNA and of mature mRNA of the enzyme in parental and alpha-difluoromethylornithine-resistant human myeloma cells. Experiments with dilution series revealed that the ODC gene copy number and the amount of both hnRNA and mRNA were increased to the same extent (about 100-fold) in the resistant cells. Similar dot-blot analyses of ODC-specific genomic DNA and total RNA indicated that the ODC gene copy number was increased by a factor of 380 and the amount of ODC mRNA by a factor of 700. Our results indicate that the PCR combined with reverse transcription is at least as useful as blot analyses to give semiquantitative assessment of the amounts of specific DNA or RNA sequences. In addition, the use of the PCR enables the analysis of minute sample amounts in extremely short time.

Base Sequence↗
PubMed1982

[Dependence of cordycepin inhibition of heterogeneous nuclear RNA biosynthesis in rat brain on the size of the poly(A) segments].

A study was made of the effect of cordicepin on the biosynthesis of rat brain heterogeneous nuclear RNA (hnRNA) fractions, such as poly(A-)hnRNA, oligo(A+)hnRNA and poly(A+)hnRNA differing in the size of poly(A)-segments. Cordicepin was shown to inhibit the biosynthesis of poly(A+)hnRNA alone. However, small doses of the antibiotic do not virtually inhibit the biosynthesis of hnRNA with shorter segments of poly(A). At the same time they inhibit the biosynthesis of hnRNA containing long chains of poly(A) in the 3'-end. A possible molecular mechanism of the phenomenon reviewed is under discussion.

Animals↗
PubMed1978

Message sequences are not adjacent to poly(A) in heterogeneous nuclear RNA of Friend leukemic cells.

Hybridization of labeled RNA with excess amounts of unlabeled complementary DNA (cDNA) was used to investigate the location of cytoplasmic mRNA sequences in heterogenous nuclear RNA (hnRNA) of noninduced Friend leukemic cells. A heterologous hybrid between hnRNA and cytoplasmic cDNA was formed. Two homologous hybrids were also formed, one between poly(A)-containing mRNA and cytoplasmic cDNA, and the other between poly-(A)-containing hnRNA and nuclear cDNA. All hybrids were selected on hydroxyapatite columns after RNase treatment. The hybrids were further investigated for the presence of poly(A). No poly(A) was found in the heterologous hybrid, while both homologous hybrids contained poly(A). From these results we conclude that there exists a spacer nucleotide sequence between the poly(A) and the message sequences in hnRNA.

Cell Line↗
PubMed1974

Differential metabolism of large and small poly(A) sequences in the heterogeneous nuclear RNA of HeLa cells.

The heterogeneous RNA of the HeLa cell nucleus contains short internal poly(A) sequences which, in contrast to the longer poly(A) sequences at the 3' ends, are not found in messenger RNA of the cytoplasm. A distinct origin for each of these homologous sequences is evident when the effects of actinomycin D and 3'-deoxyadenosine on their biosynthesis are compared. The shorter poly(A) appears to be transcribed, while the longer one does not. A kinetic analysis of the metabolism of each type of poly(A) sequence within different size classes of HnRNA after treatment of the cells with 3'-deoxyadenosine reveals distinctive distribution and metabolism of these sequences and also provides insight into mechanisms proposed for the generation of messenger RNA from these large RNA molecules in the nucleus.

Adenine Nucleotides↗
PubMed1987

Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins.

In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins.

Amino Acid Sequence↗
PubMed1982

ADP-ribosylation of proteins associated with heterogeneous nuclear RNA in rat liver nuclei.

Purified rat liver nuclei were incubated in vitro in the presence of [adenylate-32P]nicotinamide adenine dinucleotide. The label was rapidly incorporated into trichloroacetic acid-insoluble material and also detected in particles carrying heterogeneous nuclear RNA. The particles were isolated by density gradient centrifugation, and their size determined to be 30-40 S from parallel experiments using nuclei labelled with [3H]uridine 5'-triphosphate under similar conditions. Treatment of the 30-40 S-particles with enzymes of different specificities showed that the label was tightly bound to proteins, not incorporated into nuclei acids and not utilized in phosphorylation of proteins. The label was detached by phosphodiesterase I from snake venom and identified as ADP-ribose and adenosine 5'-phosphate present at a ratio of 7.5 to 1 using thin layer chromatography on poly(ethyleneimine)-cellulose. Radioactively labelled (ADP-ribosylated) proteins were visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. They included several major species of the ribonucleoprotein with molecular weights of 36000, 39000 and 42000, and a limited number of high molecular weight polypeptides.

Adenosine Diphosphate↗
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↗