In vitro synthesis of RNA by Xenopus spermatogenic cells I. Evidence for polyadenylated and non-polyadenylated RNA synthesis in different cell populations.

Premeiotic and postmeiotic (haploid) gene expression during spermatogenesis in the anuran, Xenopus laevis, was studied by analyzing the accumulation of radioactively labelled cytoplasmic polyadenylated [poly (A +)] and non-polyadenylated [poly (A -)] RNAs. Dissociated spermatogenic cells were labelled and maintained in an in vitro system capable of supporting cell differentiation. Labelled cells were separated by density gradient centrifugation into subpopulations enriched for individual spermatogenic stages. RNA was extracted and purified from each cell fraction, and separated into poly (A +) and poly (A -) species. Comparison of poly (A +) to non-poly (A) radioactivity in cells labelled with tritiated uridine or adenosine demonstrated that (1) all cell fractions produced significant quantities of polyadenylated RNA relative to total RNA synthesis; and (2) that a cell fraction enriched for pachytene spermatocyte RNA contained up to 15% of total cytoplasmic and 35% of total polysomal RNA labelled as poly (A +) containing species. RNA was also characterized by sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. All cell types showed typical poly (A -) peaks of 4S, 18S and 28S, corresponding to tRNA (4S) and rRNAs (18, 28S) respectively. Spermatids and spermatozoa had additional absorbance peaks at 13 and 21S which cosedimented with Xenopus oocyte mitochondrial rRNA. Patterns of incorporation of uridine and adenosine into poly (A +) RNA in all germ cell fractions tested were complex. In all cases, major areas of radioactivity were found in a broad band sedimenting between 6-17S. Spermatid fractions showed a prominent peak of incorporation at 6-8S, while pachytene cells also showed heavier poly (A +) peaks in the 17-25S region. A non-polyadenylated RNA species sedimenting at 6-8S with a relatively rapid rate of turnover was also observed in spermatids. From these results it is concluded that synthesis of transfer, ribosomal, and putative messenger RNA species continues in spermatogenic cells throughout all but the very last stages of spermatogenesis in Xenopus.     

Plant Desiccation and Protein Synthesis : V. Stability of Poly (A) and Poly (A) RNA during Desiccation and Their Synthesis upon Rehydration in the Desiccation-Tolerant Moss Tortula ruralis and the Intolerant Moss Cratoneuron filicinum

Upon desiccation of gametophytes of the desiccation-tolerant moss Tortula ruralis preexisting pools of poly(A)⁻ RNA (rRNA) remain inact, regardless of the speed at which desiccation is achieved. Preexisting poly(A)⁺ RNA pools (mRNA) are unaffected by slow desiccation but are substantially reduced during rapid desiccation. Poly(A)⁻ RNA involved in protein synthesis is also unaffected by desiccation, whereas the levels of polysomal poly(A)⁺ RNA in rapid- and slow-dried moss closely reflect the state of the protein synthetic complex in these dried samples.

Poly(A)⁻ RNA pools, both total and polysomal, are also stable during the rehydration of both rapid- and slow-dried moss. The total poly(A)⁺ RNA pool decreases upon rehydration, but this reduction is simply an expression of the normal turnover of poly(A)⁺ RNA in this moss. Analysis of polysomal fractions during rehydration reveals the continued use of conserved poly(A)⁺ RNA for protein synthesis. The rate of synthesis of poly(A)⁺ RNA upon rehydration appears to depend upon the speed at which prior desiccation is administered. Rapidly dried moss synthesizes poly(A)⁺ RNA at a faster rate, 60 to 120 minutes after the addition of water, than does rehydrated slowly dried moss. Recruitment of this RNA into the protein synthetic complex also follows this pattern. Comparative studies involving the aquatic moss Cratoneuron filicinum are used to gain an insight into the relevance of these findings with respect to the cellular mechanisms associated with desiccation tolerance.

     

The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke.

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 X 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 X 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 X 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 X 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.     

RNA Synthesis in Division Synchronized Tetrahymena: Macronuclear and Cytoplasmic RNA*

SYNOPSIS. Synthesis of RNA in the macronucleus and appearance of RNA in the cytoplasm were studied in heat synchronized Tetrahymena pyriformis GL and compared to those found under conditions of logarithmic growth (28 C) and during heat shocks (34 C). In macronuclei of logarithmically growing cells precursors were processed to 2 rRNA species (25S and 17S). In addition, another RNA (15S), more homogeneous than the RNA (8-15S) in the cytoplasm, was observed in the macronucleus. Both 17S and 25S rRNA species were found in the cytoplasm, 17S rRNA appearing more rapidly than 25S rRNA. Synthesis of rRNA was suppressed at 34 C in cells subjected to heat synchronization; 8-15S RNA synthesis appeared to be inhibited to a lesser extent. During the time preceding the first synchronized division, the synthesis of rRNAs in the macronucleus slowly recovered. Early in the cycle, almost no newly synthesized rRNAs were extracted. By 30 min after the last heat shock (EH), most of the RNA synthesized was not identified as rRNA. By 60 min after EH, the pattern of RNA synthesis had not returned to that observed in logarithmically growing cells.     

Poly(ADP-ribose) Polymerase inhibitors preserve nicotinamide adenine dinucleotide and adenosine 5'-triphosphate pools in DNA-damaged cells: mechanism of stimulation of unscheduled DNA synthesis

Inhibitors of poly(ADP-ribose) polymerase stimulated the level of DNA, RNA, and protein synthesis in DNA-damaged L1210 cells but had negligible effects in undamaged L1210 cells. The poly(ADP-ribose) polymerase inhibitors stimulated DNA repair synthesis after cells were exposed to high concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (68 and 136 microM) but not after exposure to low concentrations (13.6 and 34 microM). When the L1210 cells were exposed to 136 microM N-methyl-N'-nitro-N-nitrosoguanidine, the activation of poly(ADP-ribose) polymerase resulted in the rapid depletion of oxidized nicotinamide adenine dinucleotide (NAD+) levels and subsequent depletion of adenosine 5'-triphosphate (ATP) pools. After low doses of N-methyl-N'-nitro-N-nitrosoguanidine (13.6 microM), there were only small decreases in NAD+ and ATP. Poly(ADP-ribose) polymerase inhibitors prevented the rapid fall in NAD+ and ATP pools. This preservation of the ATP pool has a permissive effect on energy-dependent functions and accounts for the apparent stimulation of DNA, RNA, and protein synthesis. Thus, the mechanism by which poly(ADP-ribose) polymerase inhibitors stimulate DNA, RNA, and protein synthesis in DNA-damaged cells appears to be mediated by their ability to prevent the drastic depletion of NAD+ pools that occurs in heavily damaged cells, thereby preserving the cells' ability to generate ATP and maintain energy-dependent processes.     

Ribonucleic acid synthesis in rabbit erythroid cells.

Kinetic studies on the synthesis of RNA in mature bone-marrow erythroid cells from rabbits were made by measuring the incorporation of [2-3H]adenosine into the ATP pool and RNA over periods up to 8h. By use of equations to fit the pool specific radioactivity and an equation using the same type of pool to generate the rate of linear DNA synthesis, good agreement between the pool parameters is found, provided that the ATP pool is measured in whole cell extracts, and assuming that the dATP and ATP pools equilibrate rapidly. RNA-synthesis rates were measured by using curve fits to equations developed by using the pool specific-radioactivity curves. The rate of synthesis of poly(A)-containing RNA varied in three experiments from 90 to 220mol/min per cell, with half-life of nuclear processing of 12-22 min with a mean of 16 min. Ribosomal RNA is synthesized at a rate of 70-200 mol/min per cell with an average half-life of nuclear processing of 37 min for the 18S RNA and 214 min for the 28S RNA. When the stable rRNA components are subtracted from the nRNA synthesis, the rate of nRNA synthesis is between 2 and 6fg/min per cell with an average half-life of degradation of 27 min. The rate of synthesis of poly(A)-containing RNA is 1.5-3.5% of the RNA-synthesis rates. These rates are compared with the RNA-synthesis rates found in L cells and concentrations of globin mRNA found in various erythroid-cell preparations.     

Onset of ribosome degradation during cessation of growth in BHK-21/C13 cells.

When BHK-21/C13 cells growing exponentially in 10% serum are transferred to a medium containing only 0.25% serum, cell growth is decreased. After initial changes in RNA synthesis and degradation, protein content of the cultures reaches a plateau and eventually DNA synthesis is arrested. rRNA is relatively stable in exponentially growing cells. Immediately after 'step-down' rRNA degradation commences, but poly(A)-containing RNA does not appear to be degraded any faster than in control cells. Reutilization of RNA precursors has been independently measured and amounts to less than 1%/h for rRNA, insufficient to influence the conclusion that rRNA degradation begins almost immediately after 'step-down'. The degree of reutilization of uridine is much greater for poly(A)-containing RNA than for poly(A)-free RNA.     

Repression of rRNA synthesis during slug reconstruction in Dictyostelium discoideum

When cells dissociated from "slugs" are allowed to reaggregate, they reconstruct slugs. During this process, the cells showed a marked decrease in the ratio of labeled RNA lacking poly(A) to labeled total RNA as compared with that of cells in normal slugs. Irrespective of the change in total labeled RNA, the ratio remained low even after 6 h of reconstruction. Sucrose density gradient analysis of RNA showed that the synthesis of high molecular weight rRNA (26S and 17S) was considerably repressed in reconstructed slugs as compared with normal slugs. Polyacrylamide gel electrophoresis of low molecular weight rRNA revealed that the synthesis of 5S rRNA, but not 4S tRNA, was repressed.     

Changes in amount and synthesis of poly(A)- and poly(A)+ RNA in growing and resting cultures of Tetrahymena

This investigation deals both qualitatively and quantitatively with the changes of RNA content and synthesis during the culture growth cycle of Tetrahymena. Affinity chromatography with an oligo(dT) column was used to separate poly(A)+ RNA from total RNA. The rates of synthesis of poly(A)- and poly(A)+ RNA were determined in terms of the incorporation of [5-3H]uridine. During the log phase, the cellular RNA and protein contents decreased steadily, whereas during the resting stage, both were constant. The extents of decrease of both fractions of RNA were essentially the same (54.4% and 50.6% for total and poly(A)+ RNA, respectively). Therefore, the relative contents of poly(A)+ RNA was constant from the beginning of the log to the resting stage (4.58%). The decrease in protein content, however, amounted to only 24.8%. Theoretically, a change in the age distribution during culture growth would cause a lower content of both fractions of RNA. The extents of the decrease in the rate of synthesis of both fractions were the same (75% and 79% for poly(A)- and poly(A)+ RNA, respectively). However, this reduction is so large that it cannot be solely the result of a shift of the age distribution of the cell population.     

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.     

Isolation and fractionation of total nucleic acids from tissues and cells

A simple and efficient method was devised to permit the isolation of total nucleic acids (poly(A+) and poly(A-) RNAs and DNA) from cells and tissues (e.g. testis) enriched in DNA. Chaotropic reagents (guanidine thiocyanate and LiBr) were utilized to inactivate nucleases rapidly, minimize the viscosity of the homogenization solution and to precipitate RNA selectively (LiBr). Both total and poly(A+)-enriched mRNAs were recovered in a biologically active form as demonstrated by their ability to programme an in vitro translation reaction. High molecular weight DNA (greater than 22 kilobases) was recovered from the LiBr-soluble supernatants by selective ethanol precipitation, subsequently purified and was in a form suitable for further biochemical analysis.     

Poly(A) RNA metabolism during change of physiological state of Tetrahymena pyriformis cells

In the present work the metabolism of poly(A)+ RNA was investigated in cells of Tetrahymena pyriformis derived either from stationary cultures or from starved suspensions that were initiating growth. Under these circumstances the organisms derived from stationary cultures synthesize ribosomal and poly(A)+ RNA and form polysomes. In the presence of actinomycin D (actD) the observed expansion of the polysomal population is arrested. Pre-starved cells, on the other hand, start making polysomes in the virtual absence of ribosomal and poly(A)+ RNA synthesis soon after being transferred to peptone medium. In this case polysome formation is only partially sensitive to actD. These results have been interpreted as indicating that, in the beginning of growth, cells derived from stationary cultures are dependent on RNA synthesis for polysome formation, whereas pre-starved cells use pre-synthesized RNA for the same purpose.     

The increases in the rates of synthesis of ribosomal proteins and ribosomal RNA during refeeding of starved Tetrahymena cells are not dependent on DNA replication

We have analysed the effects of an inhibition of DNA replication by hydroxyurea on the synthesis of ribosomal proteins (r-proteins) and ribosomal RNA (rRNA) in Tetrahymena cells resuming growth after long-term starvation. The coordinate regulation of the synthesis of individual r-proteins and their increased rate of synthesis during refeeding are not impaired by inhibition of DNA replication. Moreover, the presence of hydroxyurea does not prevent an increase in the rate of synthesis of rRNA around 70-80 min after refeeding. Previously, this increase was claimed to be gene dose-dependent. Up to 180 min after refeeding, the synthesis of r-proteins appears to be closely coupled with that of rRNA and proceed in stoichiometric balance, irrespective of whether hydroxyurea is present or not. After 180 min of refeeding in the presence of hydroxyurea, this stoichiometric balance breaks down, and the rate of synthesis of r-proteins clearly exceeds that of the rRNA synthesis.     

A temporal analysis of the synthesis of the mRNA sequestered in zoospores of Blastocladiella emersonii

To determine when the dormant mRNA of Blastocladiella emersonii zoospores is synthesized, the metabolism of poly(A) RNA and rRNA was studied during growth and sporulation using pulse-chase techniques. Zoospore poly(A) RNA is synthesized at all stages of the growth cycle investigated in cultures grown either on a normal 15-hr growth cycle or in minicyclic cultures induced to sporulate after only 6.5 hr growth. For cells labeled during the growth phase the specific activity of the pulse-labeled poly(A) RNA and rRNA was identical at the beginning and end of sporulation for any of the 2-hr labeling times investigated. From this it was concluded there is neither a preferential conservation nor degradation during sporulation of the poly(A) RNA and rRNA synthesized at various times during growth. Poly(A) RNA synthesized during early sporulation is preferentially degraded; in contrast, poly(A) RNA synthesized during late sporulation is conserved in the zoospore. Approximately one-third of the total zoospore poly(A) RNA accumulates during the final 15–20 min of sporulation. The accumulation rate for both poly(A) RNA and rRNA decreases as sporulation proceeds. In addition, the rate of degradation for both types of RNA decreases at later stages of sporulation.     

Ribonucleic acid labelling and nucleotide pools during compensatory renal hypertrophy

During the first 48h of compensatory renal hypertrophy induced by unilateral nephrectomy, RNA content per cell increased by 20-40%. During this period, rates of RNA synthesis derived from the rates of labelling of UTP and RNA after a single injection of [5-(3)H]uridine showed no change in the rate of RNA synthesis (3.1nmol of UTP incorporated into RNA/min per mg of RNA). ATP and ADP pools were not changed. The rate of RNA synthesis was considerably in excess of the increment of total RNA appearing in the kidneys. With [5-(3)H]uridine as label, only continuous infusion for 24h could produce an increase (60%) in the specific radioactivity of renal rRNA in mice with contralateral nephrectomies. With a single injection of [methyl-(3)H]methionine used to identify methyl groups inserted into newly synthesized rRNA, the specific radioactivity of this rRNA was unchanged 5h after contralateral nephrectomy, increased by 60% at 9-48h, and returned to normal values at 120h. Most RNA synthesized in both nephrectomized and sham-nephrectomized mice has a short half-life. Since total cellular RNA content increases in compensatory hypertrophy despite unchanged rates of rRNA synthesis, the accretion of RNA might involve conservation of ribosomal precursor RNA or a change in rate of degradation of mature rRNA.     

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis.

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.     

DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1

RNA synthesis and DNA replication cease after DNA damage. We studied RNA synthesis using an in situ run-on assay and found ribosomal RNA (rRNA) synthesis was inhibited 24 h after UV light, gamma radiation or DNA cross-linking by cisplatin in human cells. Cisplatin led to accumulation of cells in S phase. Inhibition of the DNA repair proteins DNA-dependent protein kinase (DNA-PK) or poly(ADP-ribose) polymerase 1 (PARP-1) prevented the DNA damage-induced block of rRNA synthesis. However, DNA-PK and PARP-1 inhibition did not prevent the cisplatin-induced arrest of cell cycle in S phase, nor did it induce de novo BrdU incorporation. Loss of DNA-PK function prevented activation of PARP-1 and its recruitment to chromatin in damaged cells, suggesting regulation of PARP-1 by DNA-PK within a pathway of DNA repair. From these results, we propose a sequential activation of DNA-PK and PARP-1 in cells arrested in S phase by DNA damage causes the interruption of rRNA synthesis after DNA damage.