RNA synthesis during early embryogenesis of mass cultured highly synchronized coleopteran embryos

Summary Bruchidius embryos are shown to be well suited for biochemical studies during early embryogenesis. Mass cultivation is easy, and highly synchronized embryos can be obtained in large numbers (10⁴–10⁵ eggs). A method for in vivo incubation is described which allows the labelling of newly synthesized RNA. The kinetics of³H-ruidine uptake, phosphorylation and incorporation into RNA are presented. By autoradiography, the distribution of newly synthesized RNA is shown. Thereby, stage-specific differences were found in the labelling pattern of vitellophage nuclei, of blastoderm nuclei and of the nuclei of pole cells. The labelling of the cytoplasm remains weak until cellular blastoderm is formed. During late blastoderm and at gastrulation this label increases markedly. Gel electrophoresis of isolated RNA shows that at cellular blastoderm formation most of the label occurs in a region between 18 S and 7 S. Later on, at the onset of gastrulation, the³H-uridine incorporation found in isolated RNA is raised about 10 fold and rRNA synthesis becomes prominent. In a chase experiment, the processing of precursor RNA molecules into shorter RNA species, especially into mature rRNA and 5S RNA, is shown. The advantages of theBruchidius embryo for the biochemical analysis of early RNA synthesis and the regulation of rRNA synthesis in insect embryos are discussed.Dedicated to Professor Dr. Dr. h. c. Bernhard Rensch at the occasion of his 80th birthday     

Effect of cytokinins on ribosomal RNA gene expression in excised cotyledons of Cucurbita pepo L.

Excised pumpkin (Cucurbita pepo L.) cotyledons were used to investigate the effects of two different types of cytokinins: N⁶-benzyladenine and N₁-(2-chloro-4-pyridyl)-N₂-phenylurea on RNA synthesis in isolated nuclei. Treatment of cotyledons with both cytokinins resulted in a rapid enhancement of nuclear RNA-polymerase-I activity (EC 2.7.7.6). Maximum stimulation of RNA polymerase I, responsible for rRNA synthesis, was observed 4–6 h after the start of cytokinin action. The activity of RNA polymerase II was stimulated much more slowly and to a lesser extent. Uridine 5-monophosphate-uridine analysis of the alkalidigested nascent pre-rRNA chains showed that the stimulation of RNA-polymerase-I activity was the consequence of an increase of the polyribonucleotide-clongation rate. No significant change in the number of transcribing enzyme molecules was defected after hormone treatment (86·10³ RNA-polymerase-I molecules per diploid genome).Indications that de-novo protein synthesis is necessary for cytokinin-mediated RNA-polymerase stimulation were derived from experiments showing inhibition by cycloheximide.Abbreviations BA N⁶-benzyladenine - [PU]-30 N₁-(2-chloro-4-pyridyl)-N₂-phenylurea - UMP undine 5-monophosphate - UTP udine 5-triphosphate     

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.     

Characterization of ribonucleic acid synthesis by nuclei isolated from Zea mays

Nuclei were isolated from the shoots of Zea mays and assayed for endogenous RNA polymerase activity in vitro. Maximum incorporation from radioactive precursors (70 pmol [³H]uridine 5 monophosphate/100 g DNA) was reached after incubation for 1 h at 25°C. The RNA product, analysed by polyacrylamide gel electrophoresis, was polydisperse in size with an upper limit of 2x10⁶ daltons. Discrete peaks of rRNA were not detected, probably because of endogenous ribonuclease activity. The inclusion of -amanitin (4 g/ml) in the incubation reduced the total incorporation by approximately 40% but did not significantly alter the size of the RNA product. Although 40% of the total activity could be attributed to RNA polymerase II, [³H]RNA synthesised in vitro was found not to contain long sequences of poly (A).Abbreviations oligo (dT) oligo (deoxythymidylic acid) - poly (A) poly (adenylic acid) - GTP guanosine 5 triphosphate - ATP adenosine 5 triphosphate - CTP cytidine 5 triphosphate - UTP utidine 5 triphosphate - UMP uridine 5 monophosphate - PPO 2,5-diphenyloxazole - POPOP 1,4-di-2-(5-phenyloxazolyl) benzene     

Isolation of RNA polymerases from the water mold Achlya

The DNA-dependent RNA polymerases I, II, and III (ribonucleosidetriphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6) from Achlya ambisexualis E87 (male), have been isolated. The highly purified RNA polymerase I was found to be composed of polypeptides with the following molecular weights (·10^-4): 18.5, 14, 11.8, 7.3, 6.1, 4.9, 4.4, 2.8. RNA polymerase II showed a 400-fold higher resistance against -amanitin than mammalian or higher plant RNA polymerase II.     

Protein biosynthetic capacity in the endosperm tissue of ripening castor bean seeds

Endosperm tissue was excised from Ricinus communis plants at different stages during seed maturation. The various stages were characterized on the basis of total RNA, protein and lipid content. Polyadenylated RNA was recovered from the total RNA by affinity chromatography on oligo(dT)cellulose. With the exception of that isolated from dry seeds, this poly(A⁺) RNA actively programmed protein synthesis in cell-free systems containing either wheat germ S30 extracts or nuclease treated rabbit reticulocyte lysates at each developmental stage examined. Translational products were separated electrophoretically and were visualized by fluorography. The capacity to synthesize protein was also estimated during in vivo labelling studies. Developmental changes in the capacity of maturing endosperm tissue to synthesize a characteristic protein, R. communis agglutinin, were followed by immunoprecipitating this protein from the total in vitro products synthesized at various stages. Endoplasmic reticulum membranes were isolated from maturing endosperm tissue by sucrose density gradient centrifugation. The role of the endoplasmic reticulum (ER) in protein glycosylation was indicated by (a) localizing the enzymes catalysing the incorporation of N-acetylglucosamine and mannose into mono- and oligosaccharide lipid and into glycoprotein, (b) localizing particulate ³H-labelled glycoprotein amongst cellular fractions prepared from endosperm tissue which had been incubated with [³H]N-acetylglucosamine.Abbreviations polyCA⁺)RNA polyadenylated RNA - SDS sodiumdodecylsulphate - TCA trichloroacetic acid - RCA₁₂₀ Ricinus communis agglutinin, type I - NP40 Nonidet P40 - PMSF phenylmethylsulphonyl fluoride     

In vitro inhibition of HeLa cell nuclear ribonucleases by ADP-ribosylation

Regeneration of cilia in Tetrahymena cells treated with 1 µg/ml of 4,6-diamidino-2-phenylindole (DAPI) takes place about 30 min later then in untreated controls. Analysis of RNA isolated at 50-th min of reciliation, that is when the control cells – but not DAPI-treated ones — have just restored their motility, revealed that; –1. Two peaks of polyadenylated RNA were present in DAPI-treated cells as well as in the control ones; –2. The mobility of the polyadenylated RNA was the same in the two compared cell samples; –3. In wheat germ cell-free translational system these poly (A⁺)-RNA probably directed the synthesis of -and -tubulin; –4. The amount of polyadenylated RNA in compared cell samples was different, in DAPI-treated Tetrahymena its amount was estimated at 1.43% vs. 1.89% in control ones.     

Spezifische Aktivitätssteigerung eines Balbianiringes durch Mg2+ in isolierten Zellkernen von Chironomus

Mg²⁺ induces in isolated salivary gland nuclei of Chironomus thummi and Ch. tentans a specific enlargement of one Balbiani ring, whereas Na⁺ or K⁺ do not. Incorporation of ³H-uridin suggests that this specific enlargement is a sign of increased RNA synthesis of this chromosome region. Based on recent studies on the structure and function of Balbiani rings a model is proposed to explain the mechanism by which Mg²⁺ differentially influences chromosomal RNA synthesis.     

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.     

Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene

Summary A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5 end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.     

Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments

Summary The advent of methods for preparing ¹⁵N- and ¹³C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the ¹³C label. It may therefore sometimes be desirable to prepare a relatively inexpensive ¹⁵N-labeled sample only. Here we show that the two-bond ¹H-¹⁵N HSQC experiment can be used on ¹⁵N-labeled RNA to correlate the intranucleotide H1 and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected ¹H-¹⁵N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1 and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.     

Measurement and application of 1H-19F dipolar couplings in the structure determination of 2′-fluorolabeled RNA

Residual dipolar couplings can provide powerful restraints for determination and refinement of the solution structure of macromolecules. The application of these couplings in nucleic acid structure elucidation can have an especially dramatic impact, since they provide long-range restraints, typically absent in NOE and J-coupling measurements. Here we describe sensitive X-filtered-E.COSY-type methods designed to measure both the sign and magnitude of long-range ¹H-¹⁹F dipolar couplings in selectively fluorine labeled RNA oligonucleotides oriented in solution by a liquid crystalline medium. The techniques for measuring ¹H-¹⁹F dipolar couplings are demonstrated on a 21-mer RNA hairpin, which has been specifically labeled with fluorine at the 2-hydroxyl position of three ribose sugars. Experimentally measured ¹H-¹⁹F dipolar couplings for the 2-deoxy-2-fluoro-sugars located in the helical region of the RNA hairpin were found to be in excellent agreement with values predicted using canonical A-form helical geometry, demonstrating that these couplings can provide accurate restraints for the refinement of RNA structures determined by NMR.     

Change in the Template Specificity of RNA Phage SP-Replicase by the <Emphasis Type="Italic">E. coli</Emphasis> Cell Component

Qβ-REPLICASE was isolated from E. coli infected with the RNA bacteriophage Qβ as RNA-dependent RNA polymerase which had template specificity¹. RNA phage SP², which is distinct from RNA phages isolated previously^3,4, has been isolated in our laboratory and SP-replicase⁵ was purified from E. coli infected with SP-phage. SP-replicase has a template specificity different from that of Qβ-replicase. By using this new RNA-replicase, comparison between two distinct replicases has become possible.     

Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.     

Thermodynamics of stabilization of RNA pseudoknots by cobalt(III) hexaammine

Equilibrium unfolding (folding) studies reveal that the autoregulatory RNA pseudoknots derived from the bacteriophage T2 and T4 gene 32 mRNAs exhibit significant stabilization by increasing concentrations of divalent metal ions in solution. In this report, the apparent affinities of exchange inert trivalent Co(NH₃) have been determined, relative to divalent Mg²⁺, for the folded, partially folded (K_f), and fully unfolded (K_u) conformations of these molecules. A general nonspecific, delocalized ion binding model was developed and applied to the analysis of the metal ion concentration dependence of individual two‐state unfolding transitions. Trivalent Co(NH₃) was found to associate with the fully folded and partially unfolded pseudoknotted forms of these RNAs with a K_f of 5–8 × 10⁴ M⁻¹ in a background of 0.10 M K⁺, or 3‐ to 5‐fold larger than the K_f obtained for two model RNA hairpins and hairpin unfolding intermediates, and ≈ 40–50‐fold larger than K_f for Mg²⁺. The magnitude of K_f was found to be strongly dependent on the monovalent salt concentration in a manner qualitatively consistent with polyelectrolyte theory, with K_f reaching 1.2 × 10⁵ M⁻¹ in 50 mM K⁺. Two RNA hairpins were found to have affinities for Co(NH₃) and Ru(NH₃) of 1–2 ×10⁴ M⁻¹, or ≈ 15‐fold larger than the K_f of ∼ 1000 M⁻¹ observed for Mg²⁺. Additionally, the K_u of 4,800 M⁻¹ for the trivalent ligands is ≈ 8‐fold larger than the K_u of 600 M⁻¹ observed for Mg²⁺. These findings suggest that the T2 and T4 gene 32 mRNA pseudoknots possess a site(s) for Mg²⁺ and Co(NH₃) binding of significantly higher affinity than a “duplexlike” delocalized ion binding site that is strongly linked to the thermodynamic stability of these molecules. Imino proton perturbation nmr spectroscopy suggests that this site(s) lies near the base of the pseudoknot stem S2, near a patch of high negative electrostatic potential associated with the region where the single loop L1 adenosine crosses the major groove of stem S2. © 1999 John Wiley & Sons, Inc. Biopoly 50: 443–458, 1999     

Local helix content and RNA-binding activity of the N-terminal leucine-repeat region of hepatitis delta antigen

Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV) which provides the surface antigen for the viral coat. Our results show that the N-terminal leucine-repeat region of hepatitis delta antigen (HDAg), encompassing residues 24–50, binds to the autolytic domain of HDV genomic RNA and attenuates its autolytic activity. The solution conformation of a synthetic peptide corresponding to residues 24–50 of HDAg as determined by two-dimensional ¹H NMR and circular dichroism techniques is found to be an -helix. The local helix content of this peptide was analyzed by NOEs and coupling constants. Mutagenesis studies indicate that Lys³⁸, Lys³⁹, and Lys⁴⁰ within this -helical peptide may be directly involved in RNA binding. A structural knowledge of the N-terminal leucine-repeat region of HDAg thus provides a molecular basis for understanding its role in the interaction with RNA.     

Nucleic acid and protein synthesis and loss of vigour in germinating wheat embryos

A study has been made of the RNA and protein synthesising systems of wheat embryos isolated from seed lots having high viability but differing in vigour. The rate of RNA and protein synthesis in wheat embryos during the early hours of germination is related to the vigour of the seed lot. The imposition of a stress factor, in the nature of a sub-optimal germination temperature, during germination of isolated wheat embryos magnifies the differences in rates of protein and RNA synthesis between high and low vigour seed. Using cell-free protein synthesising systems it has been demonstrated that an important difference between high and low vigour embryos lies in the relative levels of messenger RNA in the embryo. High vigour embryos contain relatively higher levels of poly A⁺-RNA (i.e. potential mRNA species) than lower vigour embryos and furthermore the level of poly A⁺-RNA in high vigour embryos increases during early germination whilst in lower vigour embryos the level decreases. The difference in poly A⁺-RNA levels accounts, at least partially, for the differences in rates of protein synthesis observed between embryos from high and low vigour wheat seed during early germination at both optimal and sub-optimal germination temperatures.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - poly A⁺-RNA polyadenylated RNA - GM germination medium - PMS post-mitochondrial supernatant fraction