Temperature-sensitive Yeast Mutant Defective in Ribonucleic Acid Production

A single, recessive mutation in a nuclear gene confers a temperature-sensitive growth response in a mutant of Saccharomyces cerevisiae, ts(-) 136. The mutant grows normally at 23 C, but exhibits a rapid and preferential inhibition of ribonucleic acid (RNA) accumulation after a shift to 36 C, demonstrating a defect in stable RNA production. Cultures of the mutant which were shifted from 23 to 36 C display the following phenomena which indicate that messenger RNA (mRNA), as well as stable RNA production, is defective. The entrance of pulse-labeled RNA into cytoplasmic polyribosomes is even more strongly inhibited than is net RNA accumulation. The rate of protein synthesis, at first unaffected, decreases slowly; this decrease is paralleled by the decay of polyribosomes to monoribosomes with a half-time of 23 min. The polyribosomes which remain after a 30-min preincubation of the mutant at 36 C are active in polypeptide synthesis in vivo, whereas the monoribosomes which accumulate are not. Furthermore, ribosomes isolated from a culture of the mutant preincubated for 1 hr at 36 C are inactive in polypeptide synthesis in vitro, but can be restored to full activity by the addition of polyuridylic acid as mRNA. We conclude that mutant ts(-) 136 is defective either in the synthesis of all types of cytoplasmic RNA, or in the transport of newly synthesized RNA from the nucleus to the cytoplasm, and that the mRNA of a eucaryotic organism (yeast) is metabolically unstable, having a half-life of approximately 23 min at 36 C.     

Coupling of transcription and translation in Dictyostelium discoideum nuclei

The nuclei of Dictyostelium discoideum cells have been found to contain polyribosomes active in protein synthesis. mRNA molecules enter nuclear polyribosomes while they are still being synthesized. "Non sense mediated mRNA decay" occurs in the nucleus, through the interaction of the mRNAs containing a nonsense codon with newly formed nuclear ribosomes, rather than with cytoplasmic ribosomes, as previously generally supposed.     

Transport of messenger RNA into different classes of membrane-associated polyribosomes in Ehrlich-ascites-tumor cells.

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.     

Differences in the cytoplasmic distribution of newly synthesized poly (A) in serum-stimulated and resting cultures of BALB/c 3T3 cells.

Density-inhibited, serum-stimulated, and SV40 virus-transformed BALB/c 3T3 cultures were compared with respect to the rates of accumulation of cytoplasmic RNA molecules and with respect to the distribution of newly synthesized messenger RNA (mRNA) between polyribosomes and the post-ribosomal cell fraction. mRNA was isolated and quantitated by virtue of its association with radioactive polyadenylate (poly(A))-synthesized during a 90 min exposure of the cultures to ³H-adenosine. The rate of accumulation of cytoplasmic poly(A) rose slowly after serum stimulation and reached a value of 1.8 times that of resting cultures at 12 h after serum stimulation, which was also the time of onset of DNA synthesis. A change in the cytoplasmic distribution of newly synthesized poly(A) occurred more rapidly than the change in the rate of its synthesis, however. Resting cultures contained 37% of newly synthesized cytoplasmic poly(A)-containing RNA large enough to be mRNA in the post-ribosomal cell fraction, whereas virtually all of this material was found in polyribosomes at 3, 6 and 12 h after stimulation and in transformed cultures. The relatively infrequently translated mRNA of resting cultures was shown to be functional by cycloheximide treatment. (All BALB/c 3T3 cultures, resting or stimulated, contained about 20% of newly synthesized cytoplasmic poly(A) as nearly pure poly(A) in molecules of 4–6 Svedbergs in size, presumably too small to be mRNA.) We conclude that serum stimulation of density-inhibited cultures resulted in a more efficient use of the protein-synthesizing ability of the cell, and that the change in efficiency preceded increases in numbers of ribosomes and mRNA molecules.     

The effect of cycloheximide upon polyribosome stability in two yeast mutants defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis

Summary The effect of cycloheximide upon protein synthesis, RNA metabolism, and polyribosome stability was investigated in the parent and in two temperature-sensitive mutant yeast strains defective respectively in the initiation of polypeptide chains and in messenger RNA synthesis. Cycloheximide at high concentrations (100 g/ml) severely inhibits but does not completely stop protein synthesis (Fig. 1); the incorporation of ¹⁴C-amino acids into polyribosome-associated nascent polypeptide chains continues at a slow but measurable rate (Figs. 2 and 3). Polyribosome structures are stable in the parent strain at 36° whether or not cycloheximide is present (Fig. 5). However, in Mutant ts^- 136, a mutant defective in messenger as well as in stable RNA production, polyribosomes decay at the restrictive temperature (36° C) at the same rate whether or not cycloheximide is present (Fig. 5). Thus the maintenance of polyribosome structures is dependent upon the continued synthesis of messenger RNA even under conditions of extremely slow polypeptide chain elongation. In mutant ts^- 187, a mutant defective in the initiation of polypeptide chains, all of the polyribosomes decay to monoribosomes within 2 minutes after a shift to the restrictive temperature; cycloheximide completely prevents this decay demonstrating that this mutant is capable of continued messenger RNA synthesis at 36° C. Consistent with these observations is the fact that a newly synthesized heterogeneously sedimenting RNA fraction continues to enter polyribosomes in the presence of cycloheximide whereas the entrance of newly synthesized ribosomal RNA is severely inhibited (Figs. 7, 8, 9). The decay or lack of decay of polyribosomes at the restrictive temperature is, therefore, a rapid and discriminating test for the analysis of mutants defective in macromolecule synthesis. Mutants which exhibit a decay of polyribosomes in the presence of cycloheximide are likely to be defective directly or indirectly in the synthesis of messenger RNA whereas mutants in which decay is prevented or slowed by cycloheximide are likely to be defective in some factor required for the association of ribosomes and messenger RNA.     

Analysis of the control of citrulline synthesis in isolated rat-liver mitochondria

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross-link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA-protein complexes (mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28 000, 32 000, 46 000, 65 000 and 150 000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130 000 and 43 000 was higher in free mRNPs than in polysomal mRNP complexes. To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat-shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre-existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA-binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes. Heat-shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr = 81 000, 65 000 and 25 000 (heat-shock polypeptides). Whether this altered pattern of protein synthesis following heat-shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat-shock treatment. A [35S]methionine-labeled polypeptide of Mr = 80 000 was detected in mRNPs of heat-shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80 000-Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat-shock mRNAs. The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effects of iron and inflammation on ferritin synthesis on free and membrane-bound polyribosomes of rat liver.

We have examined the distribution of ferritin mRNA to free and endoplasmic reticulum (ER)-bound liver polyribosomes during inflammation and iron treatment of rats. Postnuclear tissue supernatants were fractionated on a discontinuous sucrose gradient developed to separate free and bound polyribosomes. Total RNA recovered averaged 3.2 mg/g tissue, 40% of which was with ER and 30% with the free polyribosomes, about 25% being with the postribosomal/RNP fraction. Slot-blot hybridization of equal portions of RNA revealed that 12 h after injection of turpentine to induce inflammation, ferritin mRNA was concentrated on the ER-bound polyribosomes, while it was concentrated on the free polyribosomes 2 h after injection of ferric ammonium citrate. Differences were highly significant, based on multiple determinations and densitometry. Profiles of ferritin mRNA distribution on linear sucrose gradients corroborated the differential findings. Concentrations of total ferritin mRNA per gram liver doubled with iron treatment but were not significantly different 12 h after turpentine treatment. At the same time point after turpentine, ferritin protein synthesis was increased twofold, as measured by the 1 h incorporation of [14C]leucine. We conclude that a significant portion of ferritin mRNA always associates with the ER-bound polyribosomes, and that inflammation and iron differentially alter the polysomal distribution of ferritin mRNA, suggesting that two different kinds of mRNA may be involved.     

The effect of cycloheximide on incorporation of newly formed mRNA into membrane-bound polyribosomes in rat liver cells]

Incorporation kinetics of new synthesized mRNA into free and endoplasmic membrane-bound polyribosomes in the absence of normal translation (when protein synthesis in inhibited by 98% with cycloheximide) is studied. mRNA is found to incorporate into both free and bound polyribosomes. Relative content of new synthesized membrane-bound polyribosomes in the presence of cycloheximide within 2.5-4.5 hours is by 30-40% lower as compared with the control. This fact can be explained either by the absence of a growing peptide of a sufficient length, which is necessary for the formation of a part of membrane-bound polyribosomes, or by a restricted number of attachment sites on membranes as a result of delayed translation of mRNA in pre-existed polyribosomes. It is suggested that 1) the growing peptide in liver cells is responsible for the recognition of a membrane only under the formation of only one type of membrane-bound polyribosomes, or 2) the formation of all bound polyribosomes has a single mechanism and the growing peptide does not participates in the membrane recognition.     

Polyribosome formation and poly(A)-containing RNA in embryos of the sand dollar,Dendraster excentricus

Summary The pattern of appearance of ribosomes, newly synthesized mRNA, and poly(A)-containing mRNA in polyribosomes has been examined in sand dollar embryos. From early blastula until shortly before hatching small polyribosomes engaged in histone synthesis predominate. At the time of hatching, when the rate of cell increase is maximal, the proportion of poly(A)-containing RNA in polyribosomes is low. After hatching a new class of large polyribosomes appears and the amount of poly(A)-containing polyribosomal RNA increases. Cordycepin, an inhibitor of RNA adenylylation, prevents the appearance of the large polyribosomes after hatching as well as the increase in poly(A)-containing polyribosomal RNA.     

The control of the form and function of the ribosomes in androgen-dependent tissues by testosterone

1. The ribosome content of the rat ventral prostate gland is controlled by the concentrations of circulating androgens and the polyribosomal complement of the total population of ribosomes is acutely dependent on androgenic stimulation. After the administration of testosterone to castrated rats in vivo, there is a pronounced increase in the amounts of heavy (150-240S) polyribosomes. 2. These results are consistent with a pronounced increase in the mRNA and rRNA content of the prostate gland after the administration of testosterone in vivo. 3. From studies conducted both in vitro, the heavy prostate polyribosomes formed after androgenic stimulation are particularly active in protein synthesis. 4. The androgen-stimulated increase in the formation of prostate polyribosomes has a mandatory requirement for sustained RNA and protein synthesis. 5. Since the androgen-mediated increase in prostate polyribosomes may also be suppressed by the concomitant administration of certain anti-androgenic steroids in vivo, the response in polyribosome formation is probably initiated by the binding of a metabolite of testosterone, 5alpha-dihydrotestosterone, in the prostate gland. 6. The relevance of these findings to the pronounced increase in protein synthesis in androgen-dependent tissues after hormonal stimulation is discussed.     

Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study

The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3′-UTR derived from non-coding plasmid sequence, also led to the formation of circular polyribosomes in similar proportion (40%). Moreover, the polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5′- and 3′-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field.     

Characterization of Short Time Labeled Adenosine Monophosphate-rich Ribonucleic Acids of Soybean

The total population of newly synthesized ³²P-AMP-rich RNA has been separated into two major types based on repeated fractionation on methylated albumin-kieselguhr columns. The purified D-RNA which elutes, under our experimental conditions, primarily in the salt gradient has a GMP/AMP ratio of about 0.8 and an AMP + UMP content of about 56 mole per cent. The purified TB-RNA which preferentially remains bound to the column in the salt gradient has a GMP/AMP ratio of about 0.4 to 0.45 and an AMP + UMP content of about 65 mole per cent. In addition to being distinguished by their fractionation on the methylated albumin-kieselguhr column and base composition analysis, purified D-RNA and TB-RNA have different size distributions on sucrose gradient and acrylamide gel fractionation, are differentially associated with polyribosomes and have different stabilities in the tissue.     

Determination of the time of polypeptide chain synthesis in animal experiments]

A method is worked out for the estimation of the time of polypeptide chain synthesis in animals in vivo. The time of the synthesis of "middle" polypeptide was found to be 1.45 min. in mouse liver cells under normal conditions, while in the presence of translation inhibitors, cycloheximide (20 mg/kg) and aurintricarboxilic acid (1 g/mg) the time of the synthesis increased in 2.7 and 2.5 times respectively. The time of polypeptide synthesis linearly increased with the increase of aurintricarboxilic acid dose.     

The kinetics of the incorporation of newly synthesized ribonucleic acid and protein into the ribosomes of the uterus of the oestrogen-stimulated immature rat.

The kinetics of the synthesis of the components of polyribosomes was investigated in the uterus of the immature rat responding to the administration of oestradiol-17 beta. The hormone brings about a rapid stimulation of the association of newly synthesized mRNA with uterine ribosomes, which is maximal 2-4 h after oestradiol administration and causes the aggregation of pre-existing ribosomes into polyribosomes. Despite the striking stimulation of rRNA synthesis 2-4 h after hormone treatment [Knowler & Smellie (1971) Biochem. J. 125, 605-614], the accumulation of new rRNA into ribosomes does not reach a peak until 12 h after administration. At this time, the incorporation of new ribosomal protein is also maximal. A second peak of incorporation of newly synthesized mRNA into polyribosomes follows the peak of ribosome synthesis and coincides with the oestrogen-activated synthesis of DNA.     

Distribution of mRNAS of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of alpha-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5-6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2-3-fold). There were no alpha-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Kinetics of synthesis of cytoplasmic messenger-like RNA not associated with ribosomes in HeLa cells

The turn-over of cytoplasmic messenger-like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin-D-chase experiments, cytoplasmic messenger-like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients. The half-life of free m1RNA was found to be of 1--2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short-lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non-polyadenylated fractions showed that this short-lived minor component of half-life less than 1 h is non-polyadenylated.     

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17β and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes

1. The effects of graded doses of oestradiol-17beta and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [(14)C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10mug of oestradiol-17beta administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250mug of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10mug of oestradiol-17beta restored to the control value the inhibitory effect of a dose of either 50 or 125mug of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60-100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125mug of actinomycin D, administered to animals along with 10mug of oestradiol-17beta for 6-36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750mug of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17beta in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.     

TRANSPORT OF VIRAL RNA IN KB CELLS INFECTED WITH ADENOVIRUS TYPE 2

Messenger RNA transport was studied in KB cells infected with the nuclear DNA virus adenovirus type 2. Addition of 0.04 µg/ml of actinomycin completes the inhibition of ribosome synthesis normally observed late after infection and apparently does not alter the pattern of viral RNA synthesis: Hybridization-inhibition experiments indicate that similar viral RNA sequences are transcribed in cells treated or untreated with actinomycin. The polysomal RNA synthesized during a 2 hr labeling period in the presence of actinomycin is at least 60% viral specific. Viral messenger RNA transport can occur in the absence of ribosome synthesis. When uridine-³H is added to a late-infected culture pretreated with actinomycin, viral RNA appears in the cytoplasm at 10 min, but the polysomes do not receive viral RNA-³H until 30 min have elapsed. Only 25% of the cytoplasmic viral RNA is in polyribosomes even when infected cells have been labeled for 150 min. The nonpolysomal viral RNA in cytoplasmic extracts sediments as a broad distribution from 10S to 80S and does not include a peak cosedimenting with 45S ribosome subunits. The newly formed messenger RNA that is ribosome associated is not equally distributed among the ribosomes; by comparison to polyribosomes, 74S ribosomes are deficient at least fivefold in receipt of new messenger RNA molecules.