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Double stranded RNA in heterogeneous nuclear RNA from normal and chronic lymphocytic leukemic lymphocytes.

Tritium labelled heterogeneous nuclear RNA (HnRNA) from normal and chronic lymphocytic leukemic (CLL) lymphocytes was investigated before and after fractionation into non-poly(A) containing (-HnRNA) and poly(A) containing (+HnRNA) HnRNA with respect to double stranded RNA (dsRNA). Statistically significant higher amounts of rapidly labelled RNA were recovered from CLL lymphocytes when compared to normal cases. Within the CLL cases a significant linear correlation (r = 0.95) was found between white blood cell counts and the amount of dsRNA in total HnRNA. After fractionation into (-) and (+) HnRNAs the ratios of dsRNAs, expressed as the dsRNA in (-) HnRNA divided by the dsRNA in (+) HnRNA, was lower than the corresponding values in normal cases for all the CLL cases except one. The relationship between (+) HnRNA and the total dsRNA level was different when comparing CLL and normal lymphocytes indicating a RNA processing abnormality.

Humans

Hybridization properties of DNA sequences directing the synthesis of messenger RNA and heterogeneous nuclear RNA.

The relationship of the DNA sequences from which polyribosomal messenger RNA (mRNA) and heterogeneous nuclear RNA (NRNA) of mouse L cells are transcribed was investigated by means of hybridization kinetics and thermal denaturation of the hybrids. Hybridization was performed in formamide solutions at DNA excess. Under these conditions most of the hybridizing mRNA and NRNA react at values of D(o)t (DNA concentration multiplied by time) expected for RNA transcribed from the nonrepeated or rarely repeated fraction of the genome. However, a fraction of both mRNA and NRNA hybridize at values of D(o)t about 10,000 times lower, and therefore must be transcribed from highly redundant DNA sequences. The fraction of NRNA hybridizing to highly repeated sequences is about 1.7 times greater than the corresponding fraction of mRNA. The hybrids formed by the rapidly reacting fractions of both NRNA and mRNA melt over a narrow temperature range with a midpoint about 11 degrees C below that of native L cell DNA. This indicates that these hybrids consist of partially complementary sequences with approximately 11% mismatching of bases. Hybrids formed by the slowly reacting fraction of NRNA melt within 4 degrees -6 degrees C of native DNA, indicating very little, if any, mismatching of bases. Hybrids of the slowly reacting components of mRNA, formed under conditions of sufficiently low RNA input, have a high thermal stability, similar to that observed for hybrids of the slowly reacting NRNA component. However, when higher inputs of mRNA are used, hybrids are formed which have a strikingly lower thermal stability. This observation can be explained by assuming that there is sufficient similarity among the relatively rare DNA sequences coding for mRNA so that under hybridization conditions, in which these DNA sequences are not truly in excess, reversible hybrids exhibiting a considerable amount of mispairing are formed. The fact that a comparable phenomenon has not been observed for NRNA may mean that there is less similarity among the relatively rare DNA sequences coding for NRNA than there is among the rare sequences coding for mRNA.

Animals

Messenger and heterogeneous nuclear RNA in HeLa cells: differential inhibition by cordycepin.

Cordycepin (3'-deoxyadenosine) suppresses the labeling of messenger RNA in HeLa cells. The drug has no effect on either the labeling of nuclear heterogeneous RNA or on the transport of messenger RNA into the cytoplasm. The results suggest that messenger RNA and nuclear heterogeneous RNA are synthesized separately, and that the transcription of messenger RNA is inhibited by the drug.

Carbon Isotopes

Identification of ceruloplasmin messenger RNA sequences in heterogeneous nuclear RNA from rat liver.

The distribution of the sequences of ceruloplasmin mRNA in different fractions of heterogeneous nuclear RNA from rat liver was studied using cDNA transcripts of highly purified mRNA as hybridization probe. The content of ceruloplasmin mRNA sequences in poly(A)-containing and poly(A)-free subfractions of heterogeneous nuclear RNA is respectively 1 and 27 molecules per a hepatocyte. Heterogeneous nuclear RNA carrying the sequences of ceruloplasmin mRNA sedimented in sucrose gradients containing formamide, as a broad zone around the 56S peak. Denaturing electrophoresis followed by the transfer of RNA onto diabenzyloxymethyl paper and hybridization with [32P]-cDNA revealed multiple high molecular weight fractions of ceruloplasmin pre = mRNA (9.0, 6.6, 2.2 and 1.6 megadaltons) in the non-adenylated fraction of nuclear RNA and a single 1.1-1.2 megadalton zone in poly(A)-containing nuclear RNA, the latter being equal in size to the mature ceruloplasmin mRNA from liver polysomes.

Animals

Identification of human RNA transcripts among heterogeneous nuclear RNA from man-mouse somatic cell hybrids.

In man-mouse hybrid line from our cell library, the only cytological detectable portion of the human genome is the X chromosome, and the only genetic markers regularly expressed are coded by genes known to be ?X-linked. A component of the heterogeneous nuclear RNA of these cells was found to be complementary to repetitive human DNA sequences by means of RNA-DNA hybridization on nitrocellulose filters. The same procedure also permitted the identification of hybrid cell DNA sequences that are complementary to human heterogeneous nuclear RNA. This experimental approach, coupled with hybridization studies in situ, is expected to yield critical data on the distribution and the specificity of the repetitive DNA sequences present in the human genome and to provide a new tool for cytological mapping of human chromosomes.

Animals

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA.

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.

Base Composition

Double-stranded regions in heterogeneous nuclear RNA from Hela cells.

Heterogeneous nuclear RNA from HeLa cells contains double-stranded regions that arise by base pairing of complementary sequences that exist as parts of the same molecule (intramolecular base pairing). When denatured, the RNA sequences that form the double-stranded regions hybridize rapidly to HeLa cell DNA, suggesting that they are transcribed from reiterated sites in the genome. The messenger RNA does not contain the same class or amount of double-stranded RNA regions found in heterogeneous nuclear RNA.

Cell Nucleus

[Metabolically stable classes of messenger-like nuclear RNA. II. Existence of homology between the 28S fraction of nuclear RNA, giant non-polyadenylated molecules of heterogeneous nuclear RNA and polyadenylated molecules of cytoplasmic RNA].

The existence and extent of homology between the 28S fraction of messenger-like nuclear RNA on one hand, and giant (greater than 45S) molecules of heterogeneous nuclear RNA and various fractions of cytoplasmic RNA on the other hand were studied by the method of competitive hybridization. The results obtained lead to the conclusion that the poly(A)-subfraction of hnRNA molecules with the size of greater than 15 000 nucleotides contains molecules subjected to rapid but incomplete processing with the formation of chains approximately 5000 nucleotides long (approximately 28S), which are then stored in the nucleus for up to 30 hours. Later polyadenylated and non-polyadenylated molecules of cytoplasmic RNA with the size of less than or equal to 2000 nucleotides are formed from the stored precursors.

Animals

The status of small nuclear RNA in the ribonucleoprotein fibrils containing heterogeneous nuclear RNA.

hnRNP are made of two classes of unit, monoparticles and heterogeneous complexes. The monoparticles are much more easily dissociated by salt than the heterogeneous complexes. We made use of this differential salt sensitivity to determine the localization of snRNA in hnRNP. 1, About 50% of the snRNA were released by NaCl under the conditions of dissociation of monoparticles, U1 RNA which was enriched in monoparticles was preferentially released. 2, When the proteins resistant to salt dissociation were digested with proteinase K, an additional small proportion of snRNA was released, in particular a species designated 5 Sa RNA. Therefore, 5 Sa RNA seems to be preferentially associated with the proteins of heterogeneous complexes. 3, 40% of the snRNA remained associated with the hnRNA in the absence of any detectable protein. U1 and U2 RNA were the major RNAs in this fraction. The same RNA pattern was obtained for phenol-extracted RNA. The results indicate that all snRNA species are associated with the proteins of monoparticles, with those of heterogeneous complexes and with hnRNA. The existence of these pools of snRNA may reflect different functional states.

Animals

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

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

Oligouridylate stretches in heterogeneous nuclear RNA.

Three classes of heterogeneous nuclear RNA (HnRNA) alpha, beta, and gamma, with different chemical and physical properties, can be identified in the early sea urchin embryo by hybridization with poly(U). The relative amounts of these classes vary as a function of embryonic development. It is demonstrated here that the adenyl-containing classes of HnRNA, alpha and beta, can be subfractionated by hybridization with poly(A)-agarose into species containing and lacking oligo(U)-enriched segments. These oligo(U) segments could not be detected in gamma HnRNA, which was previously shown to also lack adenylate segments. The relative proportions of these species undergo marked changes during development from early blastula (7 hr) to mesenchyme blastula (20 hr). I propose models to explain the possible effects of complementary sequences of adenylate and uridylate on the secondary structure of HnRNA, and speculate on the functional significance of such complexes.

Animals