The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

Mitochondrial RNAs are abundant in the poly(A)+RNA population of early frog embryos

Mitochondrial sequences have been identified within a set of cloned complementary DNAs that had been copied from poly(A)⁺RNA of two embryonic stages of Xenopus laevis (Dworkin and Dawid, 1980, Dworkin and Dawid, 1980, Develop. Biol.76, 435–448 and 449–464). Mitochondrial sequences were found to be highly abundant in gastrula stage poly(A)⁺RNA sequences; in tadpole RNA their relative abundance is reduced severalfold. Mitochondrial sequences account for the most abundant poly(A)⁺RNA molecules in the gastrula population. The high abundance of mitochondrial RNA in early stages may be the consequence of the accumulation of large numbers of mitochondria in the egg.     

5'-terminal structures of poly(A)+ cytoplasmic messenger RNA and of poly(A)+ and poly(A)- heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster.

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. ³²P-labeled RNA was digested with ribonucleases T₂, T₁ and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes.Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m⁷G^5′ppp^5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N⁶,2′-O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base.Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.     

Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos

To obtain information on the amounts and major classes of RNA stored in the mouse egg and accumulated during cleavage, we determined the contents of total RNA, total poly(A), and ribosomes from the 1-cell stage to blastocyst. Using purified RNA for assay, we obtained an RNA content of 0.35 ng in the unfertilized egg, 0.24 ng in 2-cell, 0.69 ng in 8- to 16-cell, and 1.47 ng in early bastocyst (32 cells). As derived from EM morphometry, the number of ribosomes accounts for 60–70% of the total RNA content at all these stages; the marked increase in ribosomal number during cleavage is attributable entirely to new synthesis. Hybridization with [³H]poly(U) in solution yielded a poly(A) content of 0.7 pg for the unfertilized egg and 0.83 pg for the 1-cell embryo. The poly(A) content dropped sharply, to 0.26 pg per embryo, by the late 2-cell stage and increased to 0.44 pg in 8- to 16-cell embryos and 1.42 pg in early blastocysts. Hybridization in situ gave a similar pattern and also revealed a heavy labeling of embryo nuclei from the 2-cell onward but very little, if any, labeling of the pronuclei of 1-cell embryos, suggesting an absence, or low level, of poly(A)+ RNA synthesis at the 1-cell but an active synthesis at the 2-cell and later stages. These findings and other available evidence(e.g., R. Bachvarova and V. De Leon, 1980, Develop. Biol.74, 1–8) suggest that the mouse embryo inherits a large supply of maternal mRNA but that the bulk of this RNA is eliminated in the 2-cell embryo. In situ hybridization was used to study the relative concentration of poly(A) in ovarian oocytes. In growing oocytes, the cytoplasmic concentration of poly(A) remains about the same, suggesting that the accumulation of poly(A)+ RNA is proportional to oocyte growth. The poly(A) content declines about twofold between the time of completion of oocyte growth and fertilization. The germinal vesicle continues to be labeled up to the time of ovulation, raising the possibility that poly(A)+ RNA synthesis (and presumably turnover) occurs in fully grown oocytes.     

A gradient of poly(A)+ RNA sequences in Xenopus laevis eggs and embryos

Xenopus laevis eggs and gastrula stage embryos were fractionated into three equal sections normal to the animal-vegetal axis, and poly(A)⁺ RNA was isolated from each section. Hybridization of these poly(A)⁺ RNAs with [³²P]cDNA synthesized using animal or vegetal poly(A)⁺ RNAs showed no detectable differences in the extents or rates of reaction. Thus, the vast majority of poly(A)⁺ RNAs are not segregated along the animal-vegetal axis. To increase the sensitivity of these experiments, [³²P]cDNAs were prepared which had reduced levels of RNA sequences from the animal region of the gastrula stage embryo or spawned unfertilized egg. Hybridization reactions with these probes showed that 3 to 5% of the input cDNA represents poly(A)⁺ RNA sequences enriched 2- to 20-fold in the vegetal region of the egg or gastrula stage embryo.     

Poly(A)+ RNA synthesis in germinating pollen ofNicotiana tabacum L

Poly(A)⁺RNA is synthesized during the first hours of pollen germination and is rapidly incorporated into polysomal structures. After a 2-h pulse with uracil-¹⁴C, 42% of the transcribed fraction of polysomal RNA is polyadenylated. Following 4 h of germination the amount of the newly-made poly(A)⁺RNA decreases steadily at the rate of about 14% per h, whereas that of rapidly-labelled poly(A)⁻RNA continues to grow. Beginning 1 h of cultivation the ratio of poly(A)⁻/poly(A)⁺RNA increases exponentially. Similarly as in non-polyadenylated mRNA the main portion of the synthesized polysomal poly(A)⁺RNA sediments at a rate of 4 to 14 S and its mean size decreases slightly with the time of labelling. RNA isolated from nuclei and cell wall containing pollen tube fraction differed from the polysomal one in higher apeoific radioactivity and the polyadenylated RNA exhibited higher size distribution. The comparison of the results with earlier observations suggests the involvement of poly(A)in mRNA translation in pollen tubes.     

Regulation of poly(A) polymerase activity and poly(A)+ RNA in germinated wheat embryos under conditions of pathogenesis

The induction of poly(A) polymerase was accompanied by a rise in the level of poly(A)⁺ RNA during early germination of excised wheat embryos (48 h). Fractionation of this RNA-processing enzyme by acrylamide gel electrophoresis and also by molecular sieving on Sephadex G-200 revealed a single molecular form of poly(A) polymerase with a molecular weight of 125 000. Wheat poly(A) polymerase specifically catalyzed the incorporation of [³H]AMP from [³H]ATP into the polyadenylate product only in the presence of primer RNA. Substitution of [³H]ATP by other labelled nucleoside triphosphates, such as [³H]GTP, [³H]UTP or [α-³²P]CTP in the assay mixture did not yield any labelled polynucleotide reaction product. The ³H-labelled reaction product was retained on poly(U)-cellulose affinity column and was not degraded by RNAase A and RNAase T₁ treatment. In addition, the nearest-neighbour frequency analysis of the ³²P-labelled reaction product predominantly yielded [³²P]AMP. Thus, characterization of the reaction product clearly indicated its polyadenylate nature. The average chain length of the [³H]poly(A) product was 26 nucleotides. Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) brought about a severe inhibition (62–79%) of poly(A) polymerase activity. Concurrently, there was a parallel decrease (73%) in the level of poly(A)⁺ RNA. Inhibition of poly(A) polymerase activity in infected embryos could be due to enzyme inactivation, which in turn brought about a downward shift in the level of poly(A)⁺ RNA. The crude extract of the cultured pathogen contains a non-dialysable, heat-labile factor, which, along with a ligand, inactivates (65–74%) poly(A) polymerase in vitro. The fungal extracts also contained a dialysable, heat-stable stimulatory effector which activated wheat poly(A) polymerase (3.6–4.0-fold stimulation) in vitro. However, the stimulatory fungal effector was not expressed in vivo, but was detectable after the inhibitory fungal factor had been destroyed by heat-treatment in our in vitro experiments.     

Major transcripts containing B1 and B2 repetitive sequences in cytoplasmic poly(A)+RNA from mouse tissues

The cytoplasmic poly(A)+RNAs containing ubiquitous B1 and B2 repeats of the mouse genome in normal tissues and tumors have been studied. Only one strand of the repeats is represented in cytoplasmic RNA in all the cases. Some tumor cells were found to be enriched in 1.4 kb B1+mRNA, 1.6 kb B2+mRNA and small (0.2-04 kb) B1+ and B2+ poly(A)+RNAs. On the other hand, mouse liver and kidney contained high amounts of 2 kb B2+mRNA. Its content increased noticeably in the regenerating liver, but in hepatoma it dropped to a zero level. Thus, the switching on (or off) of B1- and B2-containing mRNAs occurred noncoordinately. At the same time, the activation of the synthesis of small B2+RNA and small B1+RNA was simultaneous.     

Modulation of poly(A)+ RNA levels in fungal-infected wheat embryos through the selective inactivation of RNA polymerase II and poly(A) polymerase

Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) drastically lowered (70–73%) the relative abundance of poly(A)⁺ RNA. This was paralleled by a significant loss in the activities of RNA polymerase II (60–70%) and poly(A) polymerase (80–85%) enzymes. The inhibition of RNA polymerase II (60–65%) and poly(A) polymerase (70–85%) activities was also witnessed by the in vitro addition of the fungal extract to the enzyme preparations isolated from healthy embryos. The fungal extract showed negligible phosphatase and nuclease activities. This ruled out the possibility of rapid degradation of the labelled substrate [³H]ATP, primer RNA, or even the labelled reaction products under our assay conditions. The inhibitory effect of the fungal extract could be alleviated by fractionating the treated enzyme preparation by phosphocellulose chromatography. This indicated that the fungal extract was directly responsible for the inactivation of the polymerases in a reversible manner. The inhibitory function of the fungal extract was destroyed by treatment with pronase, but not with RNAase A and RNAase Ti. Poly(A) ‘tails’ were enzymatically excised from ³²P-labelled poly(A)⁺ RNA and fractionated on acrylamide gels for autoradiographic analysis. The lengths of the ³²P-labelled poly(A) ‘tails’ in control and infected embryos turned out to be identical (64 nucleotides). Our results suggest that the relative abundance of poly(A)⁺ RNA is diminished in fungal-infected wheat embryos through the selective inactivation of RNA polymerase II and poly(A) polymerase enzymes.     

Small cytoplasmic poly(A)+RNA, homologous to the B2 repetitive sequence of the mouse genome, is present in ribonucleoproteins

Small cytoplasmic poly(A) + RNA homologous to a highly repeated sequence B2 of the mouse genome (scB2 RNA) was not found as free RNA within a cell. This RNA is bound to small (12-18S) ribonucleoprotein particles as well as to heavy RNP particles, apparently informosomes. After deproteinization of the heavy RNP the major part of scB2 RNA molecules cosedimented with heavy RNAs. It seems that scB2 RNA is associated with mRNA in informosomes via short complementary regions. About half of the scB2 RNA molecules was revealed in the cytoskeleton fraction. The possibility that scB2 RNAs are involved in mRNA transport or in the regulation of mRNA translation is discussed.     

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.     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.     

The effect of epinephrine and serotonin on hepatic poly(A)+ RNA synthesis

In vivo administration of epinephrine or serotonin has been shown to stimulate the incorporation of 14C-orotic acid into Poly(A)+ RNA. However, only epinephrine and not serotonin could stimulate DNA dependent RNA polymerase activity of isolated hepatic nuclei in in vitro experiments.     

Changes in hepatic RNA, poly(A)+RNA, and poly(A)-RNA during the acute phase response to inflammation

The steady state changes in total rat hepatic cytoplasmic RNA, poly(A)+ RNA and poly(A)-RNA were assessed in response to turpentine induced inflammation. From 18 to 24 h after injury, cytoplasmic RNA doubled, while poly(A)+ RNA peaked at 24 h, 3.5 times over control animals. Cell-free translation showed significant increases in messenger RNA levels beginning at 18 h. Gel electrophoresis of translation products revealed significant increases in several polypeptides and a decrease in others. Poly(A)-RNA from control and injured rats translated to an insignificant level and the electrophoretic gel patterns of their proteins were similar. Furthermore, no change had occurred in the 3' poly(A)-sequences during the course of inflammation.     

Poly(A)+ RNA from Tetrahymena: stimulation of protein synthesis in vitro.

Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at approximately 17S and 25S. After heat denaturation all the 25S RNA was converted to 17S, indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A) +/- RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15-fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28,000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.     

Mitochondrial poly(A) RNA synthesis during early sea urchin development

The synthesis of mitochondrial messenger RNA during early sea urchin development was examined. Oligo(dT) chromatography and electrophoresis on aqueous or formamide gels of mitochondrial RNA from pulse-labeled embryos showed the presence of eight distinct poly(A)-containing RNA species, ranging in size from 9 to 22 S. Nuclease digestion of these RNAs revealed poly(A) sequences of 4 S size. Using sea urchin anucleate fragments, we were able to demonstrate that all eight messenger RNAs are transcribed from mitochondrial DNA, rather than being transcribed from nuclear DNA and imported into the mitochondria.There was no change in the electrophoretic profile of the eight poly(A) RNAs when embryos were pulsed with [³H]uridine at various times after fertilization. Neither was there any change in the incorporation of [³H]uridine into these species or in the percentage of total newly synthesized mitochondrial RNA that contains poly(A) sequences as development progresses. Even though these RNAs appear to be transcribed at a constant rate throughout early development, they were not detected in mitochondrial polysomes until 18 hr after fertilization.     

Poly(A)-containing RNA from germinating wheat embryos

Rapidly labelled mRNAs were isolated from informosomes and polyribosomes of imbibed wheat embryos. The distribution of poly(A) sequences in these fractions were studied by poly(U) Sepharose chromatography. It was shown that informosomes contain 11% polyadenylated mRNA while polyribosomes--38%. This fact suggests the important role of poly(A) sequences for translation of mRNA.