Vasopressin mRNA in the suprachiasmatic nuclei: daily regulation of polyadenylate tail length.

Daily variation has been found in the length of the polyadenylate tail attached to vasopressin messenger RNA in the suprachiasmatic nuclei, which is the location of an endogenous circadian pacemaker in mammals. No such variation was found in the supraoptic or paraventricular nuclei. This variation in the length of the polyadenylate tail may underlie the circadian rhythm of vasopressin peptide levels in cerebrospinal fluid and is a unique example of a daily rhythm in messenger RNA structure.

Existence of high abundance antiproliferative mRNA's in senescent human diploid fibroblasts.

Polyadenylated RNA isolated from senescent human diploid fibroblasts (HDF) inhibited DNA synthesis in proliferation-competent cells after microinjection, whereas polyadenylated RNA from young HDF had no inhibitory effect. Polyadenylated RNA from young cells made quiescent by removal of serum growth factors had a slight inhibitory effect on DNA synthesis. The abundance level of inhibitor messenger RNA (mRNA) from senescent cells was estimated at 0.8 and that of quiescent cells at 0.005 percent. These results demonstrate the existence of one or more antiproliferative mRNA's in nonproliferating normal human cells; these RNA's code for factors that either work antagonistically to initiators of DNA synthesis or regulate the expression of the initiators in some way. The abundance level of the inhibitory mRNA in senescent cells indicates the feasibility of developing a complementary DNA probe that will be useful in studying cell cycle control mechanisms.

Genetic expression in the developing brain.

The adult mouse brain contains complex populations of polyadenylated [poly(A)+] and nonpolyadenylated [poly(A)-] messenger RNA's (mRNA's). These mRNA's are separate sequence populations, similar in complexity, and in combination are equivalent to approximately 150,000 different mRNA sequences, of average length. Essentially all of the "adult" poly(A)+ mRNA's are present in the brain at birth. In contrast, most of the poly(A)- mRNA's are absent. Brain poly(A)- mRNA's begin to appear soon after birth, but the full adult complement is not reached until young adulthood. This suggests that these poly(A)- mRNA's specify proteins required for the biological capabilities of the brain that emerge during the course of postnatal development.

Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTP-binding proteins in Xenopus oocyte after injection of rat brain messenger RNA.

The molecular mechanism underlying the signal transduction from muscarinic and serotonergic receptors to Cl- channels were investigated in Xenopus oocyte microinjected with rat brain poly(A)+ mRNA. Transient Cl- current responses of the mRNA-injected oocytes to acetylcholine (ACh) and serotonin (5-HT) were similar in amplitude and onset. Although pharmacological characterization indicated that distinct M1-like and S1-like receptors of rat brain are involved in the ACh and 5-HT responses, respectively, these responses cross-desensitized each other completely. A common involvement of the GTP-binding proteins coupled to phosphoinositide breakdown was suggested by the findings that intracellular application of guanosine 5'-O-(2-thio)bisphosphate (GDP beta S) or neomycin greatly suppressed both ACh and 5-HT responses. These responses were not affected by exposure of the mRNA-injected cells to cholera toxin, but they were inhibited by pertussis toxin. The increase in inositol trisphosphate (IP3) responsive both to ACh and 5-HT coincided with the expression of Cl- current responses. However, only 5-HT but not ACh slightly increased the cyclic AMP (cAMP) content of the mRNA-injected cells. Intracellular injection of either IP3 or Ca2+ produced a transient Cl- current in the mRNA-injected cells as well as in non-injected cells, while 1-oleoyl-2-acetylglycerol (OAG), cAMP or cyclic GMP (cGMP) never elicited chloride current responses. It was proposed that muscarinic and serotonergic receptors are commonly linked to phosphoinositide breakdown through the mediation of GTP-binding proteins Ni and/or No in mRNA-injected oocytes.

Could poly(A) align the splicing sites of messenger RNA precursors?

In general, poly(A)-mRNA appears to be derived from larger nuclear RNA precursors. The maturation of these precursors involves excision of sequences of variable length from within the molecule and splicing of the remaining structural and coding sequences. The mechanism by which this process occurs is not known. It does not appear to operate solely through the recognition of a defined primary sequence or through the formation of a consistent secondary structure. We propose an alternative model in which poly(A) facilitates the splicing event by promoting the formation of triple-stranded structures within the mRNA precursor.

Characterization of vaccinia virus transcripts involved in selective inhibition of host protein synthesis.

Vaccinia virus transcripts synthesized in vitro inhibit protein synthesis directed by globin mRNA, EMC RNA, and total cytoplasmic RNA isolated from HeLa and CHO cells in micrococcal nuclease-treated reticulocyte lysates. In contrast, RNA isolated from vaccinia virus-infected cells is efficiently translated in the same system in the presence of the in vitro transcripts (G. Coppola and R. Bablanian (1983), Proc. Natl. Acad. Sci. USA 80, 75-79). In this study, we have fractionated the in vitro transcripts by acid-urea-agarose gel electrophoresis and demonstrated that the smallest RNA fraction inhibited translation of HeLa cell mRNA in vitro but had no effect on vaccinia virus mRNA translation. On the other hand, the larger RNA species inhibited nonselectively. These results indicate that the selective inhibitory activity of vaccinia virus transcripts resides only in the small poly(A)-containing in vitro RNA fraction.

Patterns of maternal messenger RNA accumulation and adenylation during oogenesis in Urechis caupo.

We have investigated the accumulation and adenylation of the maternal mRNA during oogenesis in the oocytes of the marine worm Urechis caupo. The analysis, using in vitro translation and cDNA probes to assay for specific mRNAs, demonstrates that different maternal mRNAs accumulate with different patterns during oogenesis. One class of maternal mRNAs accumulates throughout oogenesis and remains at a steady level in the full-grown oocyte. These mRNAs do not have a poly(A) tail long enough to mediate binding to oligo(dT)-cellulose in oocytes, but are rapidly adenylated immediately following fertilization. The other maternal mRNAs accumulate in growing oocytes as poly(A)+ RNA and undergo some deadenylation in full-grown oocytes and embryos. Some of these mRNAs attain their highest concentration fairly early in oogenesis, while others continue to accumulate during later stages. Many of the mRNAs that accumulate as poly(A)+ RNA in growing oocytes diminish dramatically in concentration in full-grown oocytes.

DNA sequence required for efficient transcription termination in yeast.

The cyc1-512 mutation is a 38 base pair deletion in the 3' nontranslated region of the CYC1 locus in the yeast Saccharomyces cerevisiae. The deletion occurred between two 7 bp directly repeated sequences. The cyc1-512 mutant produces approximately 10% of the normal amount of the CYC1 gene product, iso-1-cytochrome c, and produces 5%--10% of the normal steady-state amount of CYC1 mRNA. Most of the mRNAs in cyc1-512 are longer at their 3' ends by up to 1000 nucleotides, suggesting that the 38 bp deletion in cyc1-512 prevents proper transcription termination. The improper transcription termination is shown to cause converging transcription between CYC1 and an adjacent gene. The fact that all of the aberrantly sized mRNAs in cyc1-512 are polyadenylated leads us to suggest that polyadenylation may be coupled to transcription termination in yeast. We have uncovered a consensus sequence between the region deleted in cyc1-512 and the 3' nontranslated regions of some but not all yeast genes, and discuss the possible role of this sequence in transcription termination.

Polyadenylate-deficient analogues of poly(A)-containing mRNA sequences in cultured AKR mouse embryo cells.

Five to six percent (by mass) of AKR-2B mouse embryo cell polysomal RNA consists of messenger RNA sequences which may exist in polyadenylated form. In the steady state, however, only 30--40% of these molecules are retained by extensive passage over oligo(dT)-cellulose, the remainder being present in the form of poly(A)-deficient analogues. Within experimental limits, these poly(A)-deficient analogues contain representatives of all poly(A)-containing mRNA sequences in these cells. An analysis of the kinetics of hybridization of cDNA probes enriched for either abundant or rare poly(A)-containing mRNA sequences suggests that the frequency distributions of poly(A)-containing and poly(A)-deficient analogues are dissimilar, and that a relationship exists between the intracellular frequency of a given mRNA sequence and the number of poly(A)-deficient analogues of that sequence. High frequency sequences appear to be enriched in the poly(A)-containing fraction, while low frequency sequences are predominately associated with the poly(A)-deficient fraction, thus, poly(A) may play a role in the regulation of mRNA frequency in the cytoplasm.

Translation of messenger RNA of pig kidney D-amino acid oxidase in a cell-free system.

In vitro synthesis of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3], one of the peroxisomal flavin enzymes, was performed using a rabbit reticulocyte lysate system in order to elucidate the biosynthetic pathway of the enzyme. The apparent molecular weight of the synthesized enzyme protein was the same as that of D-amino acid oxidase purified from pig kidney. On the other hand, the enzyme protein was not detectable when a wheat germ lysate system was used for the translation. Denaturation of pig kidney poly(A)+ RNA with methylmercury hydroxide prior to the translation was found to enhance the synthesis of the enzyme protein. These results suggest a tight conformational structure of the mRNA used.

Functions of maternal mRNA as a cytoplasmic factor responsible for pole cell formation in Drosophila embryos.

Injection of mRNA extracted from Drosophila cleavage embryos or mature oocytes restored pole cell-forming ability to embryos that had been deprived of this ability by uv irradiation. However, mRNA extracted from blastoderms did not show the restoration activity. Pole cells thus formed in uv-irradiated embryos bear similarities to normal pole cells both in their morphology and their ability to migrate to the gonadal rudiments. But this mRNA does not appear to be capable of rescuing uv-induced sterility, or inducing pole cells in the anterior polar region.

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system.

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.

Developmental regulation of cytochrome P-450 genes in the rat.

Synthetic oligomer probes were used in hybridization experiments to investigate the developmental regulation of cytochrome P-450 (P-450) genes in rat liver. Transplacental induction by phenobarbital of P-450b and P-450e mRNAs was not detectable in fetal rat livers prior to day 21 of gestation. The levels of these mRNAs increased approximately 2-fold from gestational day 21 to day 22 in phenobarbital-induced liver. P-450b and P-450e mRNAs were shown to be adenylated and the fractions associated with polysomes were similar in both fetal and adult livers. No P-450b or P-450e mRNAs were detected in fetal lung and kidney RNA preparations regardless of pretreatment. Southern blot data utilizing fetal liver DNA suggests that responsiveness to xenobiotic induction during development is not attained by rearrangement of P-450b or P-450e genes. Experiments with probes specific for P-450c and P-450d failed to detect their respective mRNAs in fetal livers from 3-methylcholanthrene (3-MC)-treated or untreated rats. Both species were detectable in 3-MC-treated rats 1 week after birth. The levels of 3-MC-inducible P-450c and P-450d mRNAs increased with age and peaked approximately 3 weeks after birth. Hepatic P-450d mRNA levels in 3-MC-treated or control rats was consistently higher than P-450c mRNA levels at all ages studied.

Separation, amino-terminal sequence and cell-free synthesis of the smallest subunit of sweet potato cytochrome c oxidase.

The smallest subunit (V) of sweet potato cytochrome c oxidase was separated into three polypeptides, Va, Vb and Vc with different molecular masses (7.4 kDa, 6.8 kDa and 6.2 kDa respectively) by highly resolving sodium dodecylsulfate polyacrylamide gel electrophoresis. Antibody against subunit V reacted specifically with the polypeptide Vc. When polyadenylated mRNA from sweet potato root tissue was translated in a wheat germ cell-free system, the smallest subunit (Vc) of the polypeptides was synthesized to the same size as the mature form, which suggests that the mature subunit retains the signal for import into mitochondria. Within the N-terminal first 25 amino acids there is a stretch of 16 non-polar residues, periodically linked by basic residues, which might form an amphiphilic helix as the targeting signal.

Synthesis of delta-aminolaevulinate synthase in vitro using hepatic mRNA from chick embryos with induced porphyria.

Polyadenylated mRNA was isolated from chick embryo liver following induction of hepatic porphyria. The RNA was translated in vitro using a wheat germ cell-free system and delta-aminolaevulinate synthase was identified in the translation products by indirect immunoprecipitation. The enzyme was not apparent in the translation products of polyadenylated RNA from non-induced livers. The molecular weight of delta-aminolaevulinate synthase synthesized in vitro was 70000 and the protein was estimated to represent up to 5% of total products synthesised in vitro. These data demonstrate for the first time that induction of chick embryo liver delta-aminolaevulinate synthase activity in hepatic porphyria correlates with a large increase in the translational capacity of isolated polyadenylated RNA for this enzyme and, together with preliminary cDNA . RNA hybridization studies, indicate that an increase in the level of delta-aminolaevulinic synthase mRNA is responsible.

The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes.

Synthetic RNAs coding for chicken lysozyme, calf preprochymosin and Xenopus globin were transcribed in vitro using Sp6 RNA polymerase. The effects of capping and adding a poly(A) tail on the stability, movement and translation of these RNAs in Xenopus oocytes was examined. Capping and polyadenylation increased stability of the transcripts, with at least 40% remaining intact 48 h after injection into oocytes. Capped poly(A)- transcripts moved more rapidly in oocytes than either capped poly(A)+ transcripts or naturally occurring mRNAs. The translational efficiency of most of the synthetic RNAs in oocytes increased with both capping and polyadenylation. The exception was one Xenopus globin transcript which had an unusual 3' end of 20As and 30Cs, where further polyadenylation decreased translational efficiency. Polyadenylation was essential for detectable expression of the synthetic RNAs in cultured cells, but decreased translation of the synthetic RNAs in vitro.

The role of RNA molecules in transduction of the proto-oncogene c-fps.

Transduction of cellular genes by retroviruses requires two recombinations: one to form the left-hand junction between cellular gene and viral genome, the other to form the right-hand junction. Previous findings raised the possibility that the right-hand recombination might use RNA molecules as intermediates. We now provide direct experimental support for this view by showing that the right-hand end of v-fps in the avian sarcoma virus PRCII was formed by recombination within the poly(A) tract at the 3' end of the mRNA for the proto-oncogene c-fps. Recombination of this sort may be mediated by "copy-choice" during reverse transcription, acting on either homologous or non-homologous nucleotide sequences.