Depressed translational activity in the androgen sensitive levator ani muscle of the rat

The androgen-dependent levator ani (LA) muscle of the rat provides a suitable model to explore the molecular mechanism of steroid hormone action in target tissues. The objective of the present series of experiments was to study the effect of gonadectomy (GDX) and androgen replacement therapy on the in vitro protein synthetic capacity of the LA muscle. The incorporation of labeled methionine into the contractile protein fraction of the LA muscle maintained in organ culture decreases in a time-dependent manner following GDX. Translation of total polyadenylated mRNA in the rabbit reticulocyte translation system revealed that the decrease in protein synthetic capacity was not associated with differences in the template activity of the mRNA derived from GDX tissue. However, when polyribosomes were used to direct the same in vitro synthesis system, a significant time-dependent loss of translational activity was observed following GDX. The polyribosomes of the LA muscle of control and GDX rats were shown to contain equivalent amount of rRNA and mRNA of comparable translation efficiency. Collectively the results of these experiments indicate that the decrease in protein synthetic capacity of the LA muscle in androgen deficient rats is due, in part, to a repression of the translation process associated to the functional integrity of polyribosomes.     

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

Metabolism of mRNA in regenerating mouse liver: acceleration of the processing and decay of mRNA]

The kinetics of accumulation of poly(A+)mRNA in polyribosomes and the ratio: poly(A+)mRNA/(poly A-)mRNA were studied in regenerating mouse liver. It has been found, that the ratio: (poly A+)mRNA/(poly A-)mRNA was associated with the function of the cells: (poly A+)mRNA fraction has been decreased to 7% at 7 hours after partial hepatectomy and then reached the original value (25%) at 30-40 hours. The kinetics of accumulation of (poly A+)mRNA in polyribosomes during the transition from resting to growing state has revealed that both the lifetime and the presumable time of processing of the mRNAs of free and membranebound polyribosomes were decreased as compared to resting liver cells.     

Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland.

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.     

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.     

Effect of post-ischaemic recovery on albumin synthesis and relative amount of translatable albumin messenger RNA in rat liver.

In liver cells recovering from reversible ischaemia, total protein synthesis by postmitochondrial supernatant and membrane-bound and free polyribosomes is not different from that in sham-operated controls. However, the relative proportion of specific proteins is changed, since the incorporation of [3H]leucine in vivo into liver albumin, relative to incorporation into total protein, as determined by precipitation of labelled albumin with the specific antibody, decreases by 40-50% in post-ischaemic livers. Cell-free synthesis by membrane-bound polyribosomes and poly(A)-enriched RNA isolated from unfractionated liver homogenate shows that the decrease in albumin synthesis in liver of rats recovering from ischaemia is due to the relative decrease in translatable albumin mRNA.     

The metabolism of a poly(A) minus mRNA fraction in HeLa cells

About 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. Our method of RNA fractionation precludes contamination of the poly(A)^? mRNA with large amounts of poly(A)⁺ sequences. The poly(A)^? species is associated with polyribosomes, has an average sedimentation value equal to or greater than poly(A)⁺ mRNA, and behaves like the poly(A)⁺ mRNA in its sensitivity to EDTA and puromycin release from polyribosomes. There is very little, if any, hybridization at Rot values characteristic of abundant RNA sequences between the poly(A)^? RNA fractions from total cytoplasm or from polyribosomes and ³H-cDNA made to poly(A)⁺ RNA. This indicates that poly(A)^? mRNA does not arise from poly(A)⁺ mRNA by nonadenylation, deadenylation, or degradation of random abundant mRNA sequences. The rate of accumulation of poly(A)^? mRNA larger than 9S in the cytoplasm parallels the accumulation of poly(A)^? mRNA. The poly(A)^? mRNA is maintained as approximately 30% of the total labeled mRNA in a short (90 min) and in a long (20 hr) time period. These data indicate that poly(A)^? mRNA is not short-lived nuclear or cytoplasmic heterogeneous RNA contamination, and that the half-life of the poly(A)^? mRNA may parallel that of the poly(A)⁺ mRNA. Cordycepin appears to almost completely (95%) inhibit poly(A)⁺ mRNA while only partially (60%) inhibiting the poly(A)^? mRNA. The origin of the cordycepin-insensitive mRNA has not been ascertained.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

Changes in Polysomes and RNA in Developing Seeds of Chickpea (Cicer arietinum L.)

In the present investigation changes in polyribosomes and RNAs in the developing seeds of chickpea (Cicer arietinum L.) have been studied. The total polysome yield was higher in the early stages of development and declined at the later stages. The maximum level of polyribosomes was obtained at 18 days after flowering and a drastic decrease was noticed at maturity. The total RNA yield correlated with the polysomal yield. Northern hybridization with a heterologous probe (pea legumin cDNA) gave distinct hybridization with the mRNA coding for legumin proteins at different stages of seed development. Hybridization showed a direct relation between mRNA levels and seed weight accumulation.     

Metabolism of poly(A+)mRNA during ontogenesis: changes in half-life and processing time in rat brain cortical cells

The values of half-life (t 1/2) and processing (to) time of poly(A+) mRNA were calculated from the kinetics of label accumulation by free and membrane-bound polyribosomes of brain cortical cells of 1-, 7-, 30-, 180-, 360-, 540-, and 720-day-old rats and of 17- and 21-day-old embryos. The membrane-bound polyribosomes contain only one class of poly(A+) mRNA, whereas the free ones--predominantly two classes. The most pronounced changes in poly(A+) mRNA metabolism are observed in the case of free polyribosomes. It was found that poly(A+) mRNA of both polyribosomal classes is generally stabilized during late embryogenesis and neonatal period, after which the values of t 1/2 are decreased, showing a drastic fall upon ageing. The changes in to are inversely related to those of t 1/2. Free polyribosomes, beginning with the 30th postnatal day, reveal a short-living rapidly turned over poly(A+) mRNA, whose parameters remain practically unchanged throughout the ontogenesis period under study.     

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.     

Modification of the half-life of poly (A+)mRNA in liver and brain cells of the rat during embryogenesis and postnatal development

The poly(A+)/poly(A-)mRNA ratio and the half-life time of poly(A+)mRNA for mRNA metabolism in the liver and brain of rat in the course of ontogensis, late embryogenesis, postnatal development and upon ageing were determined. It was shown that in the course of ontogenesis both the ratio of poly(A+)/poly(A-)mRNA of free and membrane-bound polyribosomes and the half-life time of poly(A+)mRNA determined from the degradation kinetics in the presence of actinomycin D are changed. A possible role of poly(A) sequences in the regulation of mRNA life-time is discussed.     

Membrane-bound polyribosomes in HeLa cells: association of polyadenylic acid with membranes

Polyadenylic acid of membrane-bound polyribosomes is shown to be associated with rapidly sedimenting membrane structures. Most of this poly(A) remains attached to membranes after extensive degradation of polyribosomal messenger RNA with pancreatic ribonuclease. Previously, it was shown that exposure to EDTA removes up to 40% of the membrane-associated mRNA. In our experiments, 57% of the membrane-associated poly(A) still sediments with membrane structures after treatment with pancreatic ribonuclease followed by the addition of EDTA. This indicates that the association of about 60% of the membrane-bound poly(A) is EDTA-resistant, while the remainder is labile after removal of magnesium ions. Reconstruction experiments suggest that poly(A) from detergent-treated, membrane-derived polyribosomes is not trapped by other membrane structures. The poly(A)-containing RNA fragment that remains associated with the membranes after pancreatic ribonuclease treatment is shown to be a single peak at about 7 S, or about the size of cellular poly(A). Thus, the attachment site is almost pure poly(A). The poly(A)-containing, membrane-bound mRNA appears to be of a larger average size than total cellular poly(A)-containing RNA, as judged by its greater sedimentation value.     

Inhibition of host protein synthesis by vaccinia virus: fate of cell mRNA and synthesis of small poly (A)-rich polyribonucleotides in the presence of actinomycin D.

Purified vaccinia virus rapidly inhibited HeLa cell protein synthesis in the presence of actinomycin D. Under these conditions host polyribosomes were extensively degraded but the mRNA was stable as indicated by a greater than 90% recovery of prelabeled polyadenylylated RNA. Although actinomycin D prevented the synthesis of host mRNA and poly(A) in uninfected cells, incorporation of adenosine into poly(A) was inhibited by less than 50% in infected cells. Further analysis indicated that there was little or no normal size viral mRNA but that a unique class of small poly(A)-rich RNA was made in the presence of actinomycin D. From measurements of the RNase resistance and base composition of the RNA, approximately 40% of the nucleotide sequence was estimated to be poly(A). The poly(A)-rich RNA was found associated with small polyribosomes and monoribosomes that were inactive in protein synthesis. It was suggested that the poly(A) segment of the RNA is formed by the poly(A) polymerase previously found in vaccinia virus cores and that the inactive RNA, by competing with host mRNA, may contribute to the virus-mediated inhibition of host protein synthesis observed in the presence of actinomycin D.     

Association of the polyadenylate segment of messenger RNA with other polynucleotide sequences in mouse sarcoma 180 polyribosomes.

Limited digestion of polysomal RNA with pancreatic ribonuclease releases a structure consisting of poly(A) associated with other polyribonucleotide sequences. This complex can be purified by oligo(dT)-cellulose chromatography. Heating for formamide treatment causes the dissociation of fragments free of poly(A) from the poly(A)-containing components. The two types of fragments tend to reassociate under annealing conditions, and this association is prevented by poly(U). Control experiments indicate that this structure is not an artifact generated during the manipulations. The same structure can be obtained by limited RNase digestion of polyribosomes, followed by deproteinization. The results suggest that the mRNA in polyribosomes may have a defined configuration caused by the interaction of the poly(A) sequence with another segment of the RNA.     

Effect of the androgenic status on the phenotype of the plantaris muscle of the rat

1. The early demonstration of specific androgen binding sites in skeletal muscle suggested that androgen might be involved in the homeostasis of the tissue in adult mammals. 2. Following five weeks of androgen withdrawal, a noticeable increase in the plantaris (PLT) myosin heavy chain cleavage occurs. No other alteration of the contractile proteins pattern was detected. 3. No effect on the template activity of major mRNAs associated with the polysomal fraction was observed in GDX or GDX + TP group compared to control rats. 4. As opposed to the PLT muscle, the template activity of polyribosomes isolated from the androgen sensitive levator ani muscle displays a severe alteration following GDX. 5. Force-frequency analysis of PLT indicated however that the MHC modification was of no functional significance.