Low Molecular Weight RNA Research Articles

Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes.

Several cDNA clones coding for the high molecular weight (alpha) subunit of the voltage-sensitive Na channel have been selected by immunoscreening a rat brain cDNA library constructed in the expression vector lambda gt11. As will be reported elsewhere, the amino acid sequence translated from the DNA sequence shows considerable homology to that reported for the Electrophorus electricus electroplax Na channel. Several of the cDNA inserts hybridized with a low-abundance 9-kilobase RNA species from rat brain, muscle, and heart. Sucrose-gradient fractionation of rat brain poly(A) RNA yielded a high molecular weight fraction containing this mRNA, which resulted in functional Na channels when injected into oocytes. This fraction contained undetectable amounts of low molecular weight RNA. The high molecular weight Na channel RNA was selected from rat brain poly(A) RNA by hybridization to a single-strand antisense cDNA clone. Translation of this RNA in Xenopus oocytes resulted in the appearance of tetrodotoxin-sensitive voltage-sensitive Na channels in the oocyte membrane. These results demonstrate that mRNA encoding the alpha subunit of the rat brain Na channel, in the absence of any beta-subunit mRNA, is sufficient for translation to give functional channels in oocytes.

High-speed preparative-scale separation and purification of ribosomal 5S and 5.8S RNA's via Sephacryl S-300 gel filtration chromatography.

In this work we present a rapid and economical alternative to Sephadex and high performance size exclusion chromatography (HPSEC) for preparative-scale separation and purification of low molecular weight RNA's: 5.8S RNA, 5S RNA, and tRNA's. These three RNA species can be well resolved from each other and from higher molecular weight RNA species via Sephacryl S-300 gel filtration chromatography under mild eluting conditions: 10 mM Tris-HCl buffer, pH 7.5, containing 1.0 M NaCl. For a sample load of about 250 mg, the resolving power of a Sephacryl S-300 column (78 X 3.2 cm) is comparable to that of a 4.5 times larger Sephadex G-75 column (144 X 5 cm). Moreover, the total separation period is 2.5 times shorter for the Sephacryl method. Up to 500 mg or more of crude ribosomal RNA mixtures could be separated via two Sephacryl S-300 columns operated in tandem.

A discontinuous small subunit ribosomal RNA in Tetrahymena pyriformis mitochondria.

We show here that in the mitochondria of Tetrahymena pyriformis, the small subunit (SSU) rRNA is discontinuous, being comprised of two separate components which we term "alpha" (a novel low molecular weight RNA, approximately equal to 200 nucleotides long) and "beta" (a previously described 14 S RNA). The SSU alpha rRNA has been sequenced in its entirety; it represents the immediate 5'-terminal domain of conventional SSU rRNA. The sequences at the ends of the SSU beta rRNA have also been determined; they show that this molecule corresponds to the 3'-terminal 7/8 of conventional SSU rRNA. A 2.5-kilobase pair XbaI restriction fragment of T. pyriformis mitochondrial DNA which contains the SSU alpha and SSU beta rRNA genes was cloned and its complete nucleotide sequence was determined. This revealed that the genes encoding the two segments of SSU rRNA are separated by a 54-base pair (A + T)-rich spacer. The alpha and beta sequences can be fitted to a generalized secondary structure model for eubacterial 16 S rRNA, with the two RNA species associating through long range interactions to form base-paired regions characteristic of SSU rRNA. In this model, the spacer is situated in a region of pronounced primary and secondary structural variation among SSU rRNAs. The significance of these findings with respect to rRNA biosynthesis and processing and the possible evolutionary relationship between spacers and variable regions in rRNA genes is discussed.

Complementarity of sequences in low molecular weight RNAs to regions of messenger and ribosomal RNAs.

Total low molecular weight nuclear RNAs of mouse ascites cells have been labeled in vitro and used as probes to search for complementary sequences contained in nuclear or cytoplasmic RNA. From a subset of hybridizing lmw RNAs, two major species of 58,000 and 35,000 mol. wt. have been identified as mouse 5 and 5.8S ribosomal RNA. Mouse 5 and 5.8S rRNA hybridize not only to 18 and 28S rRNA, respectively, but also to nuclear and cytoplasmic poly(A+) RNA. Northern blot analysis and oligo-dT cellulose chromatography have confirmed the intermolecular base-pairing of these two small rRNA sequences to total poly(A+) RNA as well as to purified rabbit globin mRNA. 5 and 5.8S rRNA also hybridize with positive (coding) but not negative (noncoding) strands of viral RNA. Temperature melting experiments have demonstrated that their hybrid stability with mRNA sequences is comparable to that observed for the 5S:18S and 5.8S:28S hybrids. The functional significance of 5 and 5.8S rRNA base-pairing with mRNAs and larger rRNAs is unknown, but these interactions could play important coordinating roles in ribosome structure, subunit interaction, and mRNA binding during translation.

Ribonucleic acid is a component of the oligomeric, transformed mouse AtT-20 cell glucocorticoid receptor.

The glucocorticoid receptor from mouse AtT-20 pituitary tumor cells exists in three forms. The largest form is an untransformed (non-DNA-binding), oligomeric species (9.1 S, 8.3 nm, Mr 319 000). Two transformed (DNA-binding) forms can be generated. One is an oligomeric protein (5.2 S, 6-8.3 nm, Mr 132 000-182 000), while the other is the monomeric, hormone-binding subunit (3.8 S, 6 nm, Mr 96 000). The composition of the oligomeric, transformed receptor and its relationship to the monomeric protein were examined. The 3.8S monomer can be isolated from DEAE-cellulose (0.12 M step elution) in a form that continues to sediment at about 3.8 S on molybdate-containing sucrose gradients and at about 4.2 S on molybdate-free gradients. Addition of a non-hormone-binding component isolated from the same DEAE-cellulose column (0.5 M KCl step) can apparently interact with the 3.8-4.2 S monomer, increasing its sedimentation coefficient to 5.2 S (on molybdate-containing gradients) or 6.6 S (on low-salt, molybdate-free gradients). This factor is a macromolecule (nondialyzable) and is heat-stable (100 degrees C, 20 min). A dose-dependent shift to the higher sedimentation coefficient is observed when increasing quantities of the 0.5 M step material are added to the receptor monomer. This activity is abolished when the 0.5 M step material is treated with ribonuclease A. Further, when RNA is purified from the 0.5 M step by phenol/chloroform extraction, its ability to increase the S value of the monomer is retained. Ribonuclease treatment of the untransformed, 9.1S, oligomeric complex does not cause a significant decrease in sedimentation rate, while the same treatment of the 5.2S, oligomeric, transformed receptor (obtained after Sephadex G-25 transformation) causes a decrease in sedimentation rate to about 3.8 S. The addition of bovine liver mRNA and rRNA does not cause a shift in sedimentation rate of the receptor monomer to a discrete, higher sedimenting receptor form. However, the addition of total rabbit liver tRNA or three distinct tRNA species causes a shift in sedimentation to a similar, but not identical, form as that with the 0.5 M step material. We propose that the 5.2S, oligomeric transformed glucocorticoid receptor is composed of one monomeric hormone-binding, protein subunit (Mr 96 000) and a low molecular weight RNA (Mr 36 000). This interaction may be important for the role of the receptor in regulating gene expression.

Interaction of M protein and RNP of fowl plague virus in vitro

The ability of the fowl plague virus (FPV) M protein ∗∗ ∗∗ M protein refers to the m 1 protein throughout this paper. to form a complex with FPV RNP and to inhibit the RNP transcriptase activity in vitro depended on NaCl concentration and did not depend on the concentration of nonionic detergents. The results obtained indicate tha the M protein-RNP links formed were of an electrostatic rather than a hydrophobic nature. As demonstrated using individual RNP components, vRNA and RNA-free protein structures, M protein formed complexes only with vRNA, and the complex formation was salt-dependent. Analysis of products formed in the in vitro system containing RNP of FPV in the presence of the M protein showed impairment in the transcription of all RNA segments. The degree of inhibition correlated with the size of a segment, transcription of high molecular weight RNA segments being inhibited significantly more than that of low molecular weight RNA segments.

Vira RNA synthesis by a particulate fraction from cucumber seedlings infected with cucumber mosaic virus

Particulate preparations from cucumber mosaic virus (CMV)-infected cucumber cotyledons incorporated [α- 32P]GTP into products which, after phenol extraction, appeared to be double-stranded forms of the four CMV RNAs. In preparations isolated 3 to 5 days after inoculation, the label was incorporated mostly into plus RNA as shown by dot-blot hybridization using single-strand recombinant DNA clones of CMV RNA. After a short pulse the labeled material consisted mostly of small heterogeneous products, but part of the label could be chased into full-length RNAs, demonstrating that the synthetic process was continuous strand elongation rather than terminal addition. CMV RNAs added to the particulate fraction did not give rise to high molecular weight transcripts. In addition to the high molecular weight defined products which remained in the particulate fraction, small heterogeneous products, complementary to plant RNA and to viral RNA of both plus and minus polarity, were released into the incubation medium. They appeared to be products of the virus-induced M r 100,000 (100K) protein which is solubilized from the particulate fraction during incubation. However, when the particulate fraction was first subjected to an extensive solubilization and washing treatment in order to remove the M r 100K protein [ D. S. Gill, R. Kumarasamy, and R. H. Symons (1981) Virology 113, 1–8], and then used for product synthesis, a large amount of the product was still of small size, suggesting that the synthesis of high and low molecular weight RNA was intrinsically connected, and that the M r 100K protein was not merely contaminating the particulate fraction.

A search for nuclear RNA lost in azo dye-induced transplantable hepatoma.

Studies were conducted to characterize the nuclear RNA (nRNA) species that were present in rat liver but absent in the hepatoma. Nuclear RNA was compared between Donryu rat liver and AH136B hepatoma, an azo dye-induced transplantable cell line, by DNA-RNA competitive hybridization. The hepatoma lacked 13-14% of nRNA according to measurements of radioactivity of the hybridized 32P-labeled liver nRNA, and this loss was shown to be due to the failure to transcribe such RNA rather than to the deletion of the relevant DNA in the genome. Characterization of the lost RNA was first attempted by fractionating liver nRNA by density gradient sedimentation and polyacrylamide gel electrophoresis. A comparison of the additive effects of the fractionated RNA's in the competitive hybridization indicated that the pertinent RNA was present in the large RNA molecules (greater than 14S), not in the low molecular weight RNA's. Then poly(A) nRNA was found to show a strong additive effect in the competitive hybridization while nucleolar RNA showed little additive effect, indicating that the pertinent RNA was present in the heterogeneous nRNA, not in the ribosomal precursor. Further characterization was done by fractionating DNA with regard to the repetition in the genome. A comparison of the competitive hybridizations on the fractionated DNA's showed that the loss occurred mostly in RNA transcribed from highly repetitive DNA. In conclusion, the RNA species lost in the hepatoma were components of heterogeneous nRNA transcribed from highly repetitive DNA.

Transmission to apple seedlings of a low molecular weight RNA extracted from apple scar skin diseased trees.

Two species of low molecular weight RNA have been associated with apple scar skin disease. One of the RNAs, ASSARNA 1 was detected in the bark tissue as well as in the fruit tissue of diseased apple trees. Inoculation of young seedlings with extracted RNA was performed by rubbing with carborundum or by slashing with a razor. Nucleic acid was extracted from the bark of inoculated seedlings and analysed by two cycles of polyacrylamide gel electrophoresis. ASSARNA 1 was detected in one of 13 seedlings after 28 months following inoculation by the rubbing method with phenol-extracted nucleic acid and in 8 of 14 seedlings after 13 months following inoculation by the razor-slash method with purified ASSARNA 1. The results indicate that ASSARNA 1 was transmitted to apple seedlings and multiplied in them. These facts provided additional evidence for the viroid etiology of the causal agent of apple scar skin disease. However, the pathogenecity of ASSARNA 1 and the role of another disease-associated RNA, ASSARNA 2, are still unknown.

Evidence that the Low Molecular Weight RNA Associated with Chicory Yellow Mottle Virus is a Satellite

AbstractChicory yellow mottle virus, ringspot strain (CYMV‐RS), supports the replication of a low molecular weight RNA (0.17 × 106 daltons) associated with CYMV‐T (type stain).Competition hybridization experiments revealed lack or nucleotide sequence homology between 0.17 × 106 mol. wt. RNA (Sat RNA) and CYMV‐RS genomic RNAs, and partial homology (33 %) with CYMV‐T genomic RNAs. However, such apparent partial homology can be due to contamination of CYMV‐T genomic RNAs with a multimeric form of Sat RNA having a similar molecular weight. On this account the hypothesis that CYMV‐T Sat RNA is a true satellite RNA becomes tenable.

Co-ordinate control of gene expression: Muscle-specific 7 S RNA contains sequences homologous to 3′-untranslated regions of myosin genes and repetitive DNA

We have cloned and sequenced a complementary DNA copy (pSS48) of a novel muscle-specific, low molecular weight RNA, 7 S RNA, isolated from embryonic chick cardiac muscle cells. The hybridization pattern of plasmid pSS48 DNA to chick genomic DNA suggests that 7 S RNA is derived from the repetitive chick DNA with a repetition frequency of about 300 copies per haploid genome. Under low stringency, pSS48 DNA also hybridizes with high specificity to the single copy gene for chick myosin light chain (MLC) and to myosin heavy chain (MHC), and possibly to other co-ordinately expressed genes for chick muscle proteins. The sequence analysis of recombinant plasmids pSS48, pML10 and pMHC8, for 7 S RNA, MLC mRNA and MHC RNA, respectively, indicated that short nucleotide stretches homologous to 7 S RNA reside in the 3′ untranslated regions of the respective genes. The 7 S RNA sequence appears to be highly specific for the chick muscle tissue, since RNA and DNA from several sources did not hybridize to pSS48 DNA. Furthermore, the 7 S RNA-like sequence(s) appears in chick blastodermal cells preferentially earlier than the onset of transcription of genes for major muscle proteins. These results, taken together, suggest a possible function for 7 S RNA in expression of muscle-specific genes during chick development.

Low molecular weight RNAs with homologies to cellular DNA at sites of adenovirus DNA insertion in hamster or mouse cells.

The adenovirus type 2 (Ad2)-transformed hamster cell line HE5 contains one or very few integrated copies of Ad2 DNA. At the site of insertion of Ad2 DNA, the cellular DNA sequence has been completely preserved and has homologies to small unpolyadenylated, cytoplasmic RNAs of 300 nucleotides in length and to minority populations of smaller RNAs present in HE5 cells and in normal hamster cells. The 300-nucleotide RNA is present on average in approximately 20 copies per cell. This RNA, and shorter RNAs, reveal homologies to the hamster DNA sequence of approximately 400 nucleotides to the right of the site of insertion of Ad2 DNA, which is present in one or very few copies per genome. The nucleotide sequence of the DNA segment homologous to this RNA does not contain open reading frames in excess of a sequence encoding 18 amino acids. Thus, it is unlikely that the small RNAs are actually translated and their function is unknown. The nucleotide sequence does not exhibit similarities to known low mol. wt. RNAs of eukaryotic origin. The low mol. wt. cellular RNA has been found in HE5 cells, in other hamster cell lines and organs, and also in mouse cells. There are differences with respect to size and abundance in the RNAs smaller than 300 nucleotides between HE5 cells and LSH hamster embryo cells. The adenovirus type 12 (Ad12)-induced mouse tumor CBA-12-1-T carries greater than 30 copies of integrated Ad12 DNA. The cellular DNA sequence at the site of Ad12 DNA insertion exhibits homologies to small RNAs (approximately 300 nucleotides long) from mouse cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the template activity of chromatin and the transcriptive specificity of RNA polymerase II of wheat leaves during the early stages of rust infection

The template potential of chromatin from healthy wheat leaves ( Triticum vulgare L. cv. W2691) and from those at 3 days after inoculation with wheat rust fungus ( Puccinia graminis f. sp. tritici, race 126-ANZ-6,7) was assessed by (i) actinomycin D binding and (ii) in vitro transcription with RNA polymerise from different sources. There was no quantitative difference in the actinomycin D binding to chromatin from either healthy or inoculated leaves. Commercial preparations of Escherichia coli RNA polymerase holoenzyme and wheat germ RNA polymerase II synthesized only low molecular weight RNA with chromatin as template and no difference was observed in the template activity of chromatin from healthy or inoculated leaves. By contrast, RNA polymerase II isolated from wheat leaves synthesized high, intermediate and low molecular weight RNA. A comparison of different size classes of RNA radiolabelled in vitro by wheat leaf RNA polymerase II in homologous and heterologous enzyme-chromatin combinations, revealed significant changes in the template activity of chromatin and in the transcriptive specificity of RNA polymerase II of wheat leaves during the early stages of rust infection.

Purification of Cucumber Pale Fruit Viroid

Cucumber pale fruit viroid was purified from infected cucumber leaves and stems. Nucleic acids were extracted from the frozen tissue by phenol-CHCl3-SDS, and precipitated with ethanol. Polysaccharides were removed by ethylene glycol monomethyl ether treatment and phenolic substrances by cetyltrimethyl ammonium bromide. After 2M LiCl soluble fraction was treated with DNase, low molecular weight RNAs were obtained. The viroid was further purified by CF-11 cellulose chromatography and 15% polyacrylamide gel electrophoresis. Yield of the purified viroid was about 3-6μg/200g of tissue. Five percent polyacrylamide gel electrophoresis of the purified viroid in the presence of urea revealed two bands both associated with infectivity.

The effect of 5-fluoroorotic acid on the early labelling of nucleotides and RNA in whole liver and of RNA in subnuclear fractions in rats given 3H-orotic acid.

Male white rats were given either orotic acid or 5-fluoroorotic acid 60 min, and 3H-orotic acid 3 1/2 min before sacrifice. Liver nucleotides were analyzed by isotachophoresis. The (F)UTP pool increased to the same extent in both groups and showed the same specific labelling. The RNA synthesis, as measured by nmol of UTP entering RNA-UMP/g wet liver tissue was significantly lower in fluoroorotic acid treated rats. In the nucleolar fraction there was an increase in the RNA/DNA ratio, a decrease in the specific RNA labelling and an essentially unaltered specific labelling of RNA/microgram/DNA. The effect on the specific labelling of RNA/microgram DNA was the same in the nucleolar fraction and the nucleolar-free pellet, suggesting that the transcription of RNA in these two fractions proceeded in a similar way. There was a slight increase in the specific RNA labelling of the supernatant subnuclear fraction, containing low-molecular weight RNA. Incorporation into UDP-hexoses and UDP-N-acetylhexosamine was unchanged.

An infectious low molecular weight RNA was detected in grapevines by molecular hybridization with hop stunt viroid cDNA.

An infectious low molecular weight RNA was detected in grapevines by molecular hybridization with hop stunt viroid cDNA.

Dimers of 5 S RNA and 5.8 S RNA in low molecular weight RNA preparations

When purified 5 S RNA is exposed to concentrated solutions of sodium chloride or sodium phosphate, it is partly converted to the 5′ S conformer but also partly to dimers and oligomers of 5 S RNA which are stable during gel filtration and electrophoresis. Presence of 5 S dimers can not be demonstrated in cellular RNA preparations extracted with phenol. 5.8 S RNA dimers are observed in RNA from cells extracted with phenol at 40–60°C. No 5.8 S dimer is observed when RNA is extracted at 0°C. The amount of dimer increases with the temperature of extraction and may account for about 10% of the total 5.8 S RNA. When RNA extracted at 40–60°C is electrophoresed on polyacrylamide gels under nondenaturing conditions the 5.8 S RNA dimer co-migrates with the low molecular weight RNA component L.

Biosynthesis of J-chain in human lymphoid cells producing immunoglobulins of various isotypes

The relationship between synthesis, secretion, and subcellular localization of J-chain, IgM, IgA, and IgG was investigated in cultures of PWM-stimulated human PBL and in lymphoblastoid cell lines. Cells were examined for surface, cytoplasmic, and secreted immunoglobulins (Igs) and J-chain by immunofluorescence and radioimmunoassay (RIA). By these techniques, J-chain was detected in cells that produce polymeric or monomeric Igs. In PWM-stimulated PBL the synthesis of J-chain paralleled the production of Igs. In both PWM-stimulated (for 2 days) and unstimulated PBL, equal proportions of free and disulfide-linked J-chain were found. Increased amounts of intracellular J-chain were produced at later stages in PWM-stimulated PBL and J-chain occurred mostly in a free form. In tissue culture fluids, J-chain was not secreted in a free form but was always disulfide-linked to polymeric Igs. In lymphoblastoid cell lines, J-chain was present in a disulfide-linked form in IgM and IgA producers, but in IgG cells and in an IgM cell line (DAUDI) that did not secrete IgM but expressed it on the cell membrane, intracellular J-chain was present in free form. Although various proportions of polymeric and monomeric IgA were seen in culture fluids from IgA-secreting cell lines, intracellular IgA occurred mostly in a monomeric form. Further studies revealed that the ability to produce polymers was not equally distributed among all cells and might vary according to their content of J-chain and stage of maturation. Subcellular fractionation and subsequent analyses for J-chain and Ig in PWM-stimulated PBL and in IgM or IgG-producing cell lines revealed that these proteins were associated with fractions that contained ribosomes, cell sap, and low molecular weight RNA. In lysates of IgG and J-chain producing cells grown in the presence of 3H-labeled amino acids, intracellular J-chain was not disulfide-linked to IgG.

Two small RNAs encoded by Epstein-Barr virus can functionally substitute for the virus-associated RNAs in the lytic growth of adenovirus 5.

Adenovirus (Ad) serotypes 2 and 5 synthesize large amounts of two low molecular weight RNAs designated virus-associated (VA) 1 and 2. Recently, genetic and biochemical approaches have been used to show that Ad2 VA1 RNA is required for efficient translation of viral mRNAs produced at late times after infection. Primate cells harboring the Epstein-Barr virus genome (EBV) synthesize large amounts of two low molecular weight RNAs: EBER1 and EBER2. Striking similarities of gene organization have been noted between the genes coding for Ad5 VA RNAs and the EBV EBERs [Rosa, M. D., Gottlieb, E., Lerner, M. R. & Steitz, J. A. (1981) Mol. Cell. Biol. 1, 785-796]. To examine whether the EBRs can functionally substitute for the VA RNAs for the lytic growth of Ad5, we have constructed an Ad5 substitution mutant in which the two VA RNA genes have been deleted and replaced by an EBV DNA segment coding for the two EBERs. The resulting Ad5 mutant synthesizes large amounts of the EBERs and is viable. Thus, two small RNAs of EBV origin, having primary and secondary structures different from those of the VA RNAs, can functionally substitute for the VA RNAs in the lytic growth of Ad5. These results are discussed in the context of mechanism of function of the VA RNAs.

Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis.

This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [alpha-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, alpha-difluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [32P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 microM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32P]guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis.