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 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) length,cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos

The poly(A) content of early mouse embryos fluctuates widely: after a transient increase in the one-cell embryo, there is a 70% drop in the two-cell and an approximately fivefold increase between the two-cell and early blastocyst stages (L. Pikó and K. B. Clegg, 1982, Dev. Biol.89, 362–378). To shed light on the significance of these changes, we analyzed the size distribution of total poly(A) from embryos at different stages of development by gel electrophoresis and hybridization with [³H]poly(U). The number-average size of poly(A) tracts varies only slightly, from 61 to 77 nucleotides, indicating that the changes in poly(A) content are due primarily to changes in the number of poly(A) sequences, i.e., the number of poly(A)+ mRNA. From these data, the number of poly(A)+ mRNA can be estimated as follows: ovulated egg, 1.7 × 10⁷; one-cell embryo, 2.4 × 10⁷; late two-cell, 0.7 × 10⁷; late eight-cell, 1.3 × 10⁷; and early blastocyst, 3.4 × 10⁷. These results suggest the elimination of the bulk of maternal poly(A)+ mRNA at the two-cell stage, to be replaced by newly synthesized mRNA derived from the embryonic genome. To study the synthesis of poly(A)+ mRNA, we cultured mouse embryos in vitro with [³H]adenosine and analyzed the labeled poly(A)+ RNA as to molecular size, length of the poly(A) tail, and relative distribution of label in poly(A) vs internal locations. We observed an active incorporation of label into large-molecular-weight (average size about 2 kb) poly(A)+ RNA at all stages from the one-cell to the blastocyst. However, in the one-cell embryo, about 70% of the label was localized in the poly(A) tail, suggesting cytoplasmic polyadenylation, and only about 30% was localized in the remainder of the molecule, suggesting the complete new synthesis of a small amount of poly(A)+ RNA. Differences in the size distribution of the labeled poly(A) as compared with the total poly(A) in the one-cell embryo indicate that the labeling is not due to a general turnover of poly(A) tails, but rather to the polyadenylation of previously nonpolyadenylated, stored RNA. Significant new synthesis of poly(A)+ RNA is evident from the two-cell stage onward and most likely accounts for the sharp rise in the number of poly(A)+ RNA molecules by the early blastocyst stage.     

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.     

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.     

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.     

Circulating human blood platelets retain appreciable amounts of poly (A)+ RNA

Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A)+ RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A)+ RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A)+ RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A)+ RNA and that this RNA can be harvested by the described approach. The poly (A)+ RNA provides templates for the synthesis of cDNA's that code for platelet proteins.     

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.     

Role of calcium in the localization of maternal poly(A)+RNA and tubulin mRNA in Xenopus oocytes

Summary Poly(A)⁺RNA and tubulin mRNA are localized in the periphery of Xenopus oocytes and become delocalized during meiotic maturation. Delocalization of this RNA can be triggered by incubation in agents which reduce entry of calcium ions into the cell (e.g. lanthanum chloride and verapamil). Although these agents ordinarily promote meiotic maturation, addition of theophylline to the medium will inhibit maturation but not delocalization. Manipulations which prevent calcium entry without inducing meiotic maturation (e.g. calcium-free buffer) are also shown to trigger disruption of the RNA localization. In addition, manipulations which reduce chloride efflux from the cell (e.g. increasing the external chloride ion concentration with choline chloride) result in disruption of the localization of poly (A)⁺ RNA and tubulin mRNA without inducing meiotic maturation. The calcium-dependent chloride efflux present in Xenopus oocytes disappears after the oocyte has been stimulated to proceed through meiotic maturation. We show that reduction of the influx of calcium ions or efflux of chloride ions induces the delocalization of poly (A)⁺RNA and tubulin mRNA without inducing meiotic maturation. We suggest, therefore, that reducing the transmembrane movement of these ions is likely to be the natural trigger for the delocalization of poly(A)⁺RNA and tubulin mRNA.     

Immunoprecipitation of a male-specific polypeptide after in vitro translation of testicular poly(A)+ RNA

Summary Poly(A)⁺ RNAs from male and female gonads of 20-day-old rats were translated in vitro using rabbit reticulocyte lysates. The resulting polypeptides were incubated with specific anti-male (anti-H-Y) antisera raisedin female rats by intrasplenic immunization with syngeneic male skin. Using a second antibody, a male-specific polypeptide of molecular weight approximately 18,000 was immunoprecipitated. This male-specific polypeptide was not cotranslationally modified in vitro and appeared to be identical to serological H-Y antigen.     

Half-lives and relative amounts of stored and polysomal ribosomes and poly(A)+ RNA in mouse oocytes

Growing mouse oocytes were labeled in vitro with [³H]uridine and chased for 2 or for 7 days to estimate the relative amounts of RNA appearing in different fractions and to follow their turnover. Oocytes were lysed and thoroughly dispersed in the presence of 1% DOC, and centrifuged on sucrose gradients to separate polysomes from smaller components not engaged in translation. After the short chase, one-third of the labeled ribosomes appeared in EDTA-sensitive polysomes. The proportion of ribosomes in both fractions remained stable during the long chase, demonstrating no net flow from one fraction to the other. When gradient fractions were analyzed by poly(U) Sepharose chromatography, it was found that about 20% of the labeled poly(A)+ RNA appeared in polysomes after the short chase. The half-lives of stored and translated mRNA were followed relative to stable rRNA during the long chase. Stored mRNA was completely stable, but translated mRNA turned over with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 6 days. Other methods for separating stored from translated components were not successful, including sedimentation of putative large complexes (fibrillar lattices) containing stored components, or chromatography of lysates on oligo(dT)-cellulose. Results presented here combined with our previous results demonstrate that, during meiotic maturation, the percent of labeled stable RNA which is polyadenylated declines from 19 to 10%, suggesting deadenylation or degradation of half of the accumulated maternal mRNA.     

A study on the steady-state population of poly(A)+RNA during early development of Xenopus laevis

Autoantisera against rabbit testes and rabbit ejaculated spermatozoa have been used to study the appearance of surface autoantigens during spermatogenesis. Two distinct subclasses of autoantigens have been identified: an early subclass which first appears on pachytene spermatocytes and a late subclass which first appears on differentiating spermatids. These spermatids are just beginning to demonstrate migration of the nucleus and overlying acrosomal cap to the cell periphery and changes in nuclear shape. Some autoantigens of the early subclass do not appear on spermatozoa, but those that do are predominantly found over the acrosomal region. Autoantigens of the late subclass are predominantly found over the postacrosomal and middle-piece regions of the spermatozoon. It is suggested that morphological constraints during spermiogenesis may be responsible for the regional localization of the two subclasses.     

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.     

Changes in ribosomal RNA, poly(A)+ RNA, and collagen alpha 2(I) mRNA synthesis and processing during hypertonic treatment of cultured chick embryo cells

The effect of hypertonic conditions on RNA synthesis in cultured chick embryo cells was examined. The appearance of newly synthesized 28 S, 18 S, and 4 S and 5 S RNA into the cytoplasm was found to be decreased by hypertonic conditions. The appearance of newly synthesized poly(A)+ RNA into the cytoplasm was also found to be depressed. To examine the behavior of a specific mRNA, nuclear and cytoplasmic levels of procollagen alpha 2(I) mRNA were measured during high salt treatment. While nuclear levels of this mRNA were found to increase, those of the cytoplasm fell markedly. S1 nuclease digestion studies of an intron flanked by two exons revealed that the pro alpha 2(I) collagen nuclear RNA that accumulated under hypertonic conditions was spliced. The nuclear accumulation of mRNA appears therefore to be due to a hypertonic block of nuclear-cytoplasmic transport, and not to an inhibition of RNA splicing.     

Biosynthesis of the neurofilament heavy subunit in Xenopus oocytes microinjected with rat brain poly(A)+ RNA

In order to study the expression of the major subunit of neurofilaments (NFs), rat brain poly(A)⁺ RNA was purified by three different procedures and was injected in Xenopus laevis oocytes. This system was able to translate efficiently the 200 kDa NF subunit as shown by a dot-blot immunoassay and by immunoprecipitation of labeled NF polypeptides.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethanesulphonyl fluoride - PBS phosphate buffered saline - BSA bovine serum albumin