Intracellular distribution of low molecular weight RNA species in HeLa cells

Intracellular distributions of the low molecular weight RNA species of HeLa cells were determined by a nonaqueous method of cell fractionation, in which lyophilized cells were homogenized and centrifuged in anhydrous glycerol. The nonaqueous method was used to avoid artifactual extraction of weakly bound nuclear RNA during cell fractionation. We found that the mature small RNA species K, A, C, and D were almost entirely (greater than 95%) nuclear, and that mature 4S tRNA was partially (5-10%) nuclear. Our results gave higher nuclear content of the mature species K, A, C, and 4S than was shown previously with conventional aqueous cell fractionation. The nonaqueous method also gave higher nuclear proportions of some short-lived precursors to mature small RNAs. We found that approximately one-half of recently synthesized pre-4S RNA and more than one-half of recently synthesized 5S RNA were nuclear, whereas these species had been thought to be cytoplasmic from previous work. The species C' and D', precursors to the stable nuclear species C and D respectively, were found to be partially nuclear, also in contrast to earlier work. The stable cytoplasmic species L (oncornavirus 7S RNA) was found to be mostly nuclear shortly after synthesis.     

Intracellular distribution of low molecular weight RNA in Chironomus tentans

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4–5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10⁵ and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10⁵ and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10⁴ and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (β-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4–5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

Subnuclear particles containing a small nuclear RNA and heterogeneous nuclear RNA

Five of the stable low molecular weight RNA species in the HeLa cell nucleus have been localized in RNP complexes in the cell nucleus. The two abundant species C and D and the three minor species F, G′ and H are found in RNP particles following two different methods of preparation. Sonication of nuclei releases the five small RNAs and also the hnRNA in RNPs that sediment in a range from 10 to 150 S. Alternatively, incubation of intact nuclei at elevated temperature and pH releases four of the small RNAs and degraded hnRNA in more slowly sedimenting structures.When nuclear RNPs obtained by sonication are digested with RNAase in the presence of EDTA, the hnRNA is degraded and the hnRNPs sediment at 30 S. The structures containing the small RNA species D are similarly shifted to 30 S particles by RNAase and EDTA but not by either agent alone. In contrast, the sedimentation of complexes containing species G′ and H are not altered by exposure to RNAase/EDTA and small RNA species C and F are unstable under these conditions.In isopycnic metrizamide/²H₂O gradients species D and hnRNA accumulate at a density characteristic of RNP particles. They have a similar but not identical distribution.Species D is released from large RNPs by salt concentrations of 0.1 m-NaCl or greater, while the hnRNA remains in large RNP particles. In contrast, the structures containing species G′ and H are stable in 0.3 m-NaCl. All five of the small nuclear RNA species and the hnRNAs are released from rapidly sedimenting complexes by the ionic detergent sodium deoxycholate.It is suggested that the low molecular weight RNA species play a structural role in RNP particles in the cell nucleus and that a subpopulation of species D may be associated with the particles that package the hnRNA.     

Characteristics of ribonucleic acids isolated from Botryodiplodia theobromae pycnidiospores

Nucleic acids isolated from dormant and germinated Botryodiplodia theobromae pycnidiospores contain five distinct species of RNA. They include two ribosomal species, two ribosomal-associated species and transfer RNAs. Sedimentation coefficients of 25.1S and 18S were obtained for the two ribosomal RNA species and 5.8S and 5S for the two ribosomal-associated RNA components. Molecular weights of 1.20, 0.67, 0.054 and 0.035x10⁶ daltons were obtained after formaldehyde treatment and electrophoresis on polyacrylamide gels for these same four RNAs. Methylated nucleotides were present in the transfer RNAs and large and small ribosomal RNAs; in contrast 5.8S and 5S RNAs contained few methylated nucleotides. In addition to the 5 distinct RNA species, polyadenylate-containing RNA was isolated from both dormant and germinated spores.Published with the approval of the Director as paper no. 5006, Journal Series, Nebraska Agricultural Experiment Station. The work was conducted under Nebraska Agricultural Experiment Station Project no. 21-17.     

Transfer RNAs as genotypic fingerprints of eubacteria

A new method was developed for rapid genotypic identification and classification of bacteria. The method is based on high resolution gel electrophoresis of the stable, low molecular weight (LMW) RNA fraction of single bacterial strains. This fraction comprises the total transfer RNA pool and the 5S ribosomal RNA. On a one-dimensional gel, every eubacterial strain exhibited a distinct LMW RNA profile, a set of bands belonging to three different size classes: 5S rRNAs (110–131 nt), class 2 tRNAs (82–96 nt) and class 1 tRNAs (72–79 nt). LMW RNA profiles of members of five of the ten major eubacterial groups, previously defined by 16S rRNA sequence analysis, were highly diverse. For some major groups, like flavobacteria and planctomyces, the distinctive sizes of their 5S rRNAs allowed the assignment of strains to these groups. More specific taxonomic information was gained from analysis of the tRNA part of the profile. Strains could be grouped as species and genera due to species- and genus-specific tRNA bands. From an evolutionary point of view, this order found in the total tRNA pool of eubacteria could indicate that cytoplasmic tRNA evolution reflects ribosomal RNA evolution. Given the universality of tRNAs, it is to be expected that their electrophoretic mobility profiles may serve as a convenient RNA fingerprint for defining bacterial species operationally and for identifying new genotypes by differing patterns.     

Species of RNA synthesized during early germination in the radicle of the lentil embryo

RNA synthesis is activated in cells of the plant embryo very soon after the start of imbibition by the seed. This study was undertaken to determine whether synthesis of one particular RNA or all the major RNA species was initially activated in the radicle of lentil embryos ( Vicia lens ). Two different methods were used after incorporation of radioactive precursor to identify the newly synthesized RNA species. First, RNA was extracted and analyzed using gel electrophoresis, gel filtration, affinity chromatography and base composition analysis. The second method was to localize the labelled RNA molecules within cells using autoradiography of sections of embryonic radicles. The data indicate that newly synthesized heterogeneous nuclear RNA and possibly messenger RNA, transfer RNA, 5S ribosomal RNA and precursor of ribosomal RNA are detectable 3 h after the start of imbibition of the decoated embryo and before completion of initial water uptake. It is concluded that synthesis of all major species of RNA is simultaneously initiated in the radicle of the germinating embryo.     

Stable low molecular weight RNA analyzed by staircase electrophoresis, a molecular signature for both prokaryotic and eukaryotic microorganisms

Low-molecular weight RNA (LMW RNA) analysis using staircase electrophoresis was performed for several species of eukaryotic and prokaryotic microorganisms. According to our results, the LMW RNA profiles of archaea and bacteria contain three zones: 5S RNA, class 1 tRNA and class 2 tRNA. In fungi an additional band is included in the LMW RNA profiles, which correspond to the 5.8S RNA. In archaea and bacteria we found that the 5S rRNA zone is characteristic for each genus and the tRNA profile is characteristic for each species. In eukaryotes the combined 5.8S and 5S rRNA zones are characteristic for each genus and, as in prokaryotes, tRNA profiles are characteristic for each species. Therefore, stable low molecular weight RNA, separated by staircase electrophoresis, can be considered a molecular signature for both prokaryotic and eukaryotic microorganisms. Analysis of the data obtained and construction of the corresponding dendrograms afforded relationships between genera and species; these were essentially the same as those obtained with 16S rRNA sequencing (in prokaryotes) and 18S rRNA sequencing (in eukaryotes).     

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.     

Changes in the metabolism of nucleic acids during the growth and senescence of tobacco callus

Changes in DNA and RNA metabolism, DNA composition and RNA species in callus of tobacco ( Nicotiana rustica L. cv. Gansu Yellow Flower) were investigated during the growth and senescence. DNA and RNA contents remained almost unchanged during the callus growth period, but started to decrease synchronously at the time that callus senescence was initiated. Synthesis of DNA and RNA, as measured by incorporation of [³H]-labelled precursor, increased during the growth period and did not decrease until late in senescence. The activities of DNase and RNase (pH 4.5) increased during the early senescence period in accordance with the decrease in the levels of DNA and RNA, but appeared to decrease during late senescence. These results suggest that the decrease in the levels of DNA and RNA in senescing tobacco callus may stem from the increase in the hydrolytic activities of DNase and RNase (pH 4.5) in the early stage of senescence, and that the slowdown of synthesis in the late senescence period may also be a cause. DNA and RNA electrophoresis showed that a low-molecular-weight satellite DNA band disappeared after the onset of senescence and that the nuclear main band DNA gradually decreased, whereas the high-molecular-weight satellite DNA seemed to undergo no significant changes during the senescence period tested. Of the RNA species, 4–5S RNA was far more susceptible to damage during senescence than 25S and 18S rRNA. This suggests different susceptibilities of different DNA and RNA components to damage during the senescence of tobacco callus or alternatively a highly sequenced degradation of DNA and RNA molecules.     

A polymerase activity forming 5S and pre-4S RNA in isolated HeLa cell nuclei

Isolated HeLa cell nuclei are capable of synthesizing 5S and pre-4S RNA. The labeling of these low molecular weight species has been compared with the labeling of nucleolar RNA and nuclear heterogeneous RNA. The 5S and pre-4S RNA molecules made in vitro were identified by their mobility on SDS acrylamide gels and by the sensitivity of pre-4S RNA to enzymes which cleave it in vitro to 4S RNA. Their mobilities and cleavage properties are similar to the RNA made in vivo. Unlike the nuclear heterogeneous RNA, the synthesis of the two small molecular weight RNAs is resistant to α-amanitin.A large proportion of 4S RNA labeled in vitro appears to be formed de novo. The ratio of the terminal uridine to the internal uridine 3′-monophosphate remains constant with time, even though there is linear incorporation into the pre-4S RNA in the isolated nuclei.Production of the nucleolar RNA and pre-4S RNA has been compared in the presence of various ions. The pre-4S RNA synthesis has a sharper maximum for (NH₄)SO₄ and MgCl₂ than does the synthesis of nucleolar RNA. The in vitro synthesis of pre-4S is more sensitive to ellipticine and pCMB than the production of nucleolar RNA. These differences between the production of pre-4S RNA and nucleolar RNA are discussed with respect to in vitro reinitiation and the possibility that different polymerases are involved in their synthesis.     

Reinitiation of synthesis of small cytoplasmic RNA species K and L in isolated HeLa cell nuclei in vitro.

Isolated HeLa cell nuclei were used to synthesize low molecular weight RNA species in-vitro. The labelled RNA released from the nuclei during the incubation mainly consists of 5S RNA, pre-tRNA and small cytoplasmic RNA species K and L. All these low molecular weight RNA species are synthesized by RNA polymerase C (or III). The polyanion heparin was applied to study the reinitiation of these RNA molecules in-vitro. A comparison of the kinetics of RNA synthesis in the absence and in the presence of this inhibitor demonstrates a highly efficient in-vitro reinitiation of scRNA species K and L as well as 5S and pre-tRNA by RNA polymerase C. These results indicate a general competence of this enzyme to catalyze the de-novo formation of specific gene products in-vitro.     

THE TURNOVER AND SUBCELLULAR LOCALIZATION OF THE CHICK FOREBRAIN SMALL NUCLEAR RNAs

Analysis of 2 day old chick forebrain RNA on polyacrylamide gels revealed the presence of the 29S, 18S, 5.5s and 5s rRNAs, 4S tRNA and the small mol wt nuclear RNA (snRNA) species K, L, C, DD and G. The turnover of these molecules was studied following the intracerebral injection of [³H]uridine into 2 day old animals. The specific activities of these molecules declined over a 23 day time course and all displayed first-order decay kinetics. Apparent half-lives (in days) for 29S (7). 18S (S), L (24), C (6.5), DD′ (8.5), 5.5S (7), 5S (13), G (6) and 4S (7) were obtained. The subcellular localization of the snRNAs was studied in 5 day old chick forebrain. The snRNAs accounted for almost 17% of nuclear RNA, in contrast to their low levels in whole forebrain (1.2%). This was due to an approx 20-fold nuclear enrichment of C, DD′, G′ and H. Relatively low levels of K and L were present within the nuclear fraction. However, K and L, and to a lesser extent C and DD, were readily detected within the post-microsomal supernatant. Production of a pH 5 enzyme fraction from this supernatant resulted in a 2-3-fold enrichment of K, L, C and DD′ with respect to tRNA. The polysome containing fractions also displayed significant levels of L with about 1 mol of L per 30 of 5S rRNA. A new method for the determination of the subcellular distribution of the snRNAs is described. It depends upon the purity of the nuclear, microsomal and cell sap subfractions rather than upon total recoveries from such fractions. Utilizing the relative amounts of these RNA species within whole forebrain and the major subfractions, distributions (as a percentage within each fraction) were obtained. The observation of significant levels of K, L, C and DD within the cytoplasmic fractions raises some doubts about previous suggestions of an exclusively nuclear role for these molecules. It is proposed that the snRNAs may be involved in a multistage interaction within the process of eukaryotic gene expression.     

Incorporation of mevalonic Acid into ribosylzeatin in tobacco callus ribonucleic Acid preparations

The incorporation of ¹⁴C-2-mevalonic acid into transfer RNA and ribosomal RNA (high molecular weight RNA) in rapidly growing, cytokinin-dependent tobacco (Nicotiana tabacum var. Wisconsin No. 38) callus cultures has been investigated. Approximately 40% of the label incorporated into transfer RNA was present in a ribonucleoside with chromatographic properties identical to those of cis-ribosylzeatin. The remainder of the label in the transfer RNA appears to be nonspecific incorporation resulting from degradation and metabolism of ¹⁴C-2-mevalonic acid by the tobacco callus tissue. Although the total radioactivity incorporated into ribosomal RNA was roughly the same as in transfer RNA, the specific radioactivity of the transfer RNA was about four times higher than that of the ribosomal RNA, and the ribosomal RNA labeling could be distinguished from the cytokinin labeling observed in transfer RNA. The distributions of the ¹⁴C-2-mevalonic acid label and cytokinin activity in tobacco callus transfer RNA fractionated by benzoylated diethylaminoethylcellulose chromatography indicate that at least two cytokinin-containing transfer RNA species are present in this tissue.     

Sugarbeet leaf disc culture: an improved procedure for inducing morphogenesis

In preparation for gene transfer experiments we investigated factors that might affect the production of shoots and somatic embryos from the wound callus of cultured sugarbeet leaf discs. A complex interaction was found between the leaf disc plating density, the disc culture medium, the source-shoot culture medium and the frequency of disc transfer to fresh medium. The most productive protocol utilized: source shoots maintained on MS medium containing 0.25 mg 1^-1 BA; multiple leaf discs (ten 4-mm discs/plate) plated onto an enriched modification of MS medium (RV) containing 1.0 mg 1^-1 BA and solidified with 0.3% Gelrite (not permitted to dry during hardening); and transfer of the discs to fresh medium every two weeks during the first month. This standard protocol produced more callus per plate and higher rates of morphogenesis per unit dry weight of callus than did the one-step method of Saunders and Doley. Water availability considerations were found to be critical to obtaining high morphogenic rates. Root induction frequency and quality was superior on shoots transplanted to MS medium containing 1 mg 1^-1 NAA as the sole growth regulator compared to IAA at the same concentration.Abbreviations BA N⁶-benzyladenine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Low molecular weight RNAs hydrogen-bonded to nuclear and cytoplasmic poly(a)-terminated RNA from cultured chinese hamster ovary cells

A group of RNAs 90–100 nucleotides long were isolated by melting them from poly(A)-terminated nuclear or cytoplasmic RNA from cultured Chinese hamster ovary cells. Conditions that favor hydrogen bond formation allowed the reassociation of these low molecular weight RNAs with poly(A)-terminated RNA. The nuclear poly(A)-terminated molecules contained 1.3 moles of the low molecular weight RNAs per mole of poly(A), while the cytoplasmic poly(A)-terminated RNA contained only one seventh as much. These low molecular weight RNAs were also isolated from the total 4S RNA of either the nucleus or cytoplasm by polyacrylamide gel electrophoresis. They formed a prominantly labeled band of RNA in the gels after cells had been labeled with H₃³²PO₄ for 4 hr. The low molecular weight RNAs melted from the nuclear poly(A)-terminated RNA were slightly different (although not necessarily in primary nucleotide sequence) from those melted from the cytoplasmic poly(A)-terminated RNA.