Degradation of 18S and 28S ribosomal RNA in the cytoplasm of rat liver cells after inhibition of protein synthesis

Inhibition of protein synthesis (up to 95%) in starved rat liver cells after a single injection of a sublethal dose of cycloheximide (0.3 mg per 100 g of body weight) results in degradation of 18S rRNA during the first 3 hours, whereas the 28S rRNA remains unaffected. However, the increase of 28S rRNA degradation products was observed by the 6th and 12th hours. The rapid decay of 18S rRNA is due to the degradation of this RNA in 40S ribosomal subunits. In contrast to 28S rRNA the specific radioactivity of 18S rRNA is increased by the 6th hour. Presumably the synthesis and processing of 18S rRNA impaired during the 1st hour are recovered partially or completely by this time. A molecular mechanism underlying 18S rRNA degradation in 40S ribosomal subunits is proposed.     

A bifunctional archaeal protein that is a component of 30S ribosomal subunits and interacts with C/D box small RNAs

We have identified a novel archaeal protein that apparently plays two distinct roles in ribosome metabolism. It is a polypeptide of about 18 kDa (termed Rbp18) that binds free cytosolic C/D box sRNAs in vivo and in vitro and behaves as a structural ribosomal protein, specifically a component of the 30S ribosomal subunit. As Rbp18 is selectively present in Crenarcheota and highly thermophilic Euryarchaeota, we propose that it serves to protect C/D box sRNAs from degradation and perhaps to stabilize thermophilic 30S subunits.     

Relationship Between Sporulation-Specific 20S Ribonucleic Acid and Ribosomal Ribonucleic Acid Processing in Saccharomyces cerevisiae

The nature and properties of the 20S ribonucleic acid which accumulates only during the sporulation of Saccharomyces cerevisiae were examined. The 20S ribonucleic acid (RNA) has a base composition considerably different from ribosomal RNA species and is virtually unmethylated. The 20S RNA did, however, exhibit approximately 70% homology with 18S RNA by RNA-deoxyribonucleic acid filter hybridization competitions. The 20S RNA showed a hybridization saturation plateau level 30 to 40% higher than 18S, consistent with measurements of the size difference in polyacrylamide gels. Pulse-chase experiments in the presence and absence of cycloheximide indicate that the 20S RNA has a presumptive relationship to the 20S ribosomal RNA precursor normally observed only in short pulse-labeling in vegetative cells.     

Regulation of 40S ribosomal protein S6 phosphorylation in Swiss mouse 3T3 cells

Addition of serum to resting cultures of Swiss mouse 3T3 cells causes an immediate multiple phosphorylation of 40S ribosomal protein S6. After 60 min of stimulation, changing to medium containing no serum led to the net dephosphorylation of S6. During this same period, a second protein, as yet unidentified, became increasingly phosphorylated. Incubation of cells with cycloheximide prior to the addition of serum almost completely blocked the activation of protein synthesis. There was no effect on the serum-induced phosphorylation of S6. If cells were stimulated in the presence of cAMP phosphodiesterase inhibitors theophylline or SQ 20006, both S6 phosphorylation and the activation of protein synthesis were inhibited. Stimulation of cells with serum also led to an immediate drop in total intracellular cAMP levels. This was blocked by prostaglandin E1 (PGE1), which caused a 10 fold increase in total intracellular cyclic AMP. However, PGE1 had no effect on protein synthesis or S6 phosphorylation.     

An excess of the small ribosomal subunits and a higher rate of turnover of the 60 S than of the 40 S ribosomal subunits in L cells grown in suspension culture.

A considerable excess of small ribosomal subunits was observed in L cells grown in suspension culture. The ratio between the small and large ribosomal subunits in the cytoplasm was estimated to be 1.17 ± 0.05 for cells dividing every 20 to 24 hours.The 60 S ribosomal subunits were turning over much faster than the 40 S subunits. Half-lives of 155 ± 20 hours for 18 S ribosomal RNA and 82 ± 15 hours for 28 S ribosomal RNA were observed under conditions where the cell number doubled every 24 hours and the viability was 95%. By correcting for cell death the half-lives of 18 S and 28 S ribosomal RNA were estimated to be approximately 300 hours and 110 hours, respectively. During storage of isolated ribosomes the small ribosomal subunits were degraded faster than the large subunits. This shows that the degradation of 60 S subunits was not an artifact taking place during the isolation procedure.It is postulated that the small ribosomal subunits are protected by protein to a greater extent than the 60 S subunits in these rapidly growing cells in suspension culture. The protection may take place both in the nucleus during synthesis, thus avoiding degradation (“wastage”) of nascent subunit precursors, and later in the cytoplasm. A calculation has been carried out to show that the observed excess of small subunits may be accounted for on the basis of a 1:1 synthesis of the small and large ribosomal subunits in the nucleus and different degradation rates in the cytoplasm. The results do not exclude the possibility of a difference in the “wastage” of 18 S and 28 S ribosomal RNA in the nucleus in addition to the difference in the turnover rates in the cytoplasm.     

Protein synthesis of the sponge Geodia cydonium: characterization of the system

The ribosomal population of the sponge Geodia cydonium has been examined. The monosomes have a sedimentation constant of 80 S, the sizes of the subunits are approximately 60 S and 45 S respectively. The polyribosomes contain up to 40 ribosomal units. Cell free protein synthesizing systems (cell homogenate as well as reconstituted system) have been prepared and characterized with respect to Mg²⁺, KCI and ATP concentrations, temperature, pH and time course of the reaction. In the cell-free system and in the cellular system the protein biosynthesis is inhibited by chloramphenicol. It is not affected by cycloheximide.     

Individual ribosomal protein pool size and turnover rate in Escherichia coli

The pool size of free individual ribosomal proteins present in the cell sap of Escherichia coli has been determined by pulse-labelling a culture before a chase with cold marker.Ribosomes plus ribosomal precursor particles were prepared together and the proteins from this fraction purified. The specific radioactivity of each 30 S and 50 S protein was measured at the time of pulse and at the various times of chase: unequal labelling was already observed at the time of pulse; the kinetics of chase of most 30 S proteins reached a plateau very rapidly; the kinetics of 50 S proteins were more variable. Precise calculation of individual pool size was carried out using the mathematical model described in the Appendix. Almost all ribosomal 30 S proteins have a pool size close to zero. Only four 30 S proteins (S10, S16, S17 and S18) have a sizeable pool (2 to 6% of the corresponding ribosomal protein). Most 50 S proteins have a small pool size (1 to 2%). The free ribosomal proteins of the pool are transferred to mature ribosomes; the half-life of these proteins in the pool has been calculated (0 to 1·4 min). Finally, as judged from the kinetic data, no degradation of ribosome-bound protein was apparent. The significance of the results is discussed with respect to the function of ribosome and the process of ribosome biogenesis.     

Release of ribosome-bound 5S rRNA upon cleavage of the phosphodiester bond between nucleotides A54 and A55 in 5S rRNA

Reticulocyte lysates contain ribosome-bound and free populations of 5S RNA. The free population is sensitive to nuclease cleavage in the internal loop B, at the phosphodiester bond connecting nucleotides A54 and A55. Similar cleavage sites were detected in 5S rRNA in 60S subunits and 80S ribosomes. However, 5S rRNA in reticulocyte polysomes is insensitive to cleavage unless ribosomes are salt-washed. This suggests that a translational factor protects the backbone surrounding A54 from cleavage in polysomes. Upon nuclease treatment of mouse 60S subunits or reticulocyte lysates a small population of ribosomes released its 5S rRNA together with ribosomal protein L5. Furthermore, rRNA sequences from 5.8S, 28S and 18S rRNA were released. In 18S rRNA the sequences mainly originate from the 630 loop and stem (helix 18) in the 5' domain, whereas in 28S rRNA a majority of fragments is derived from helices 47 and 81 in domains III and V, respectively. We speculate that this type of rRNA-fragmentation may mimic a ribosome degradation pathway.     

Methionine-Dependent Synthesis of Ribosomal Ribonucleic Acid During Sporulation and Vegetative Growth of Saccharomyces cerevisiae

Methionine limitation during growth and sporulation of a methionine-requiring diploid of Saccharomyces cerevisiae causes two significant changes in the normal synthesis of ribonucleic acid (RNA). First, whereas 18S ribosomal RNA is produced, there is no significant accumulation of either 26S ribosomal RNA or 5.8S RNA. The effect of methionine on the accumulation of these RNA species occurs after the formation of a common 35S precursor molecule which is still observed in the absence of methionine. During sporulation, diploid strains of S. cerevisiae produce a stable, virtually unmethylated 20S RNA which has previously been shown to be largely homologous to methylated 18S ribosomal RNA. The appearance of this species is not affected by the presence or absence of methionine from sporulation medium. However, when exponentially growing vegetative cells are starved for methionine, unmethylated 20S RNA is found. The 20S RNA, which had previously been observed only in cells undergoing sporulation, accumulates at the same time as a methylated 18S RNA. These effects on ribosomal RNA synthesis are specific for methionine limitation, and are not observed if protein synthesis is inhibited by cycloheximide or if cells are starved for a carbon source or for another amino acid. The phenomena are not marker specific as analogous results have been obtained for both a methionine-requiring diploid homozygous for met13 and a diploid homozygous for met2. The results demonstrate that methylation of ribosomal RNA or other methionine-dependent events plays a critical role in the recognition and processing of ribosomal precursor RNA to the final mature species.     

The effect of ethionine on ribonucleic acid synthesis in rat liver.

1. By 1h after administration of ethionine to the female rat the appearance of newly synthesized 18SrRNA in the cytoplasm is completely inhibited. This is not caused by inhibition of RNA synthesis, for the synthesis of the large ribosomal precursor RNA (45S) and of tRNA continues. Cleavage of 45S RNA to 32S RNA also occurs, but there was no evidence for the accumulation of mature or immature rRNA in the nucleus. 2. The effect of ethionine on the maturation of rRNA was not mimicked by an inhibitor of protein synthesis (cycloheximide) or an inhibitor of polyamine synthesis [methylglyoxal bis(guanylhydrazone)]. 3. Unlike the ethionine-induced inhibition of protein synthesis, this effect was not prevented by concurrent administration of inosine. A similar effect could be induced in HeLa cells by incubation for 1h in a medium lacking methionine. The ATP concentration in these cells was normal. From these two observations it was concluded that the effect of etionine on rRNA maturation is not caused by an ethionine-induced lack of ATP. It is suggested that ethionine, by lowering the hepatic concentration of S-adenosylmethionine, prevents methylation of the ribosomal precursor. The methylation is essential for the correct maturation of the molecule; without methylation complete degradation occurs.     

Phenol Extraction Op 28 S Ribosomal RNA Prom Rat Liver Cytoplasm

A simple and reproducible phenol method for the isolation of 28 S ribosomal RNA from rat liver cytoplasm, free from poly(A)-RNA is described. The procedure is based on the observation that at lower pH of the homogenate (pH 5.5) 28 S ribosomal RNA is extracted, while 18 S ribosomal RNA remains in the interphase layer.

Isolation of pure 28 S or 18 S ribosomal RNA in preparative amounts requires density gradient cen-trifugation or preparative gel electrophoresis. In this communication a rapid and reproducible method for the isolation of 28 S ribosomal RNA is proposed.     

Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes

Summary Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the durg were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis.Hybrid 80S ribosomes from subunits of mutant and the ocrresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.Abbreviations CHM Cycloheximide - CHO Chinese hamster ovarien - FBS foetal bovine serum - Eagle MEM Eagle minimal essential medium - EMS Ethyl-metansulfonate - NMU N-nitrosomethylurea     

Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae

RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with 125I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.     

Secondary structure of the 5'-terminal region of the 18S ribosomal RNA5.3S fragment from the rat liver

Two small RNA fragments, 5,3S and 4,7S, were observed in gel electrophoretic analysis of RNA of the 40S ribosomal subunit of rat liver. 5,3S RNA (134-136 nucleotides long) proved to be 5'-terminal fragment of 18S ribosomal RNA, whereas 4,7 RNA is the degradation product of 5,3S RNA with 27-28 5'-terminal nucleotides lost. The secondary structure of 5,3S RNA was probed with two structure-specific nucleases, S1 nuclease and the double-strand specific cobra venom endoribonuclease. The nuclease digestion data agree well with the computer generated secondary structure model for 5,3S RNA. This model predicts that the 5'-terminal part of rat liver ribosomal 18S RNA forms an independent structural domain. The affinity chromatography experiments with the immobilized 5,3S fragment show that 5,3S RNA does not bind rat liver ribosomal proteins.     

Translation and M1 double-stranded RNA propagation: MAK18 = RPL41B and cycloheximide curing.

MAK18 is one of nearly 30 chromosomal genes of Saccharomyces cerevisiae necessary for propagation of the killer toxin-encoding M1 double-stranded RNA satellite of the L-A double-stranded RNA virus. We have cloned and sequenced MAK18 and find that it is identical to RPL41B, one of the two genes encoding large ribosomal subunit protein L41. The mak18-1 mutant is deficient in 60S subunits, which we suggest results in a preferential decrease in translation of viral poly(A)-deficient mRNA. We have reexamined the curing of M1 by low concentrations of cycloheximide (G. R. Fink and C. A. Styles, Proc. Natl. Acad. Sci. USA 69:2846-2849, 1972), which is known to act on ribosomal large subunit protein L29. We find that when M1 is supported by L-A proteins made from the poly(A)+ mRNA of a cDNA clone of L-A, cycloheximide does not decrease the M1 copy number, consistent with our hypothesis.     

Identification of proteins crosslinked to RNA in 40S ribosomal subunits of Saccharomyces cerevisiae

RNA-protein crosslinks were introduced into the 40S ribosomal subunits from Saccharomyces cerevisiae by mild UV treatment. Proteins crosslinked to the 18S rRNA molecule were separated from free proteins by repeated extraction of the treated subunits and centrifugation in glycerol gradients. After digestion with RNase to remove the RNA molecules, proteins were radio-labeled with ¹²⁵I and identified by electrophoresis on two-dimensional polyacrylamide gels with carrier total 40S ribosomal proteins and autoradiography. Proteins S2, S7, S13, S14, S17/22/27, and S18 were linked to the 18S rRNA. A shorter period of irradiation resulted in crosslinking of S2 and S17/22/27 only. Several of these proteins were previously demonstrated to be present in ribosomal core particles or early assembled proteins.     

Phosphopeptide patterns of the ribosomal protein S6 following stimulation of guinea pig parotid glands by secretagogues involving either cAMP or calcium as second messenger

Stimulation of secretion in exocrine cells is associated with the incorporation of up to 3 to 4 phosphates into the ribosomal protein S6. This occurs with secretagogues involving either cAMP or free calcium as second messenger. An analysis of the phosphorylation pattern of S6 from stimulated guinea pig parotid glands reveals 3 phosphopeptides (termed A,B,C). The phosphopeptide pattern was identical for cAMP- or calcium-mediated stimulation, whereas phosphorylation of the S6 protein in vitro with catalytic subunit of cAMP-dependent protein kinase resulted only in the formation of phosphopeptides A and C. Therefore, secretagogue-mediated phosphorylation is not or not exclusively catalyzed by cAMP-dependent protein kinase even when cAMP is the second messenger.     

Synthesis of ribosomal proteins in developing Dictyostelium discoideum cells is controlled by the methylation of proteins S24 and S31.

Ribosomal protein mRNAs left over from growth are selectively excluded from polyribosomes in the first half of Dictyostelium discoideum development. This is due to the fact that they are sequestered by a class of free 40S ribosomal subunits, characterized by possessing a methylated S24 protein. At the time of formation of tight cell aggregates, the methylated S24 is substituted by an unmethylated S24, while protein S31 of the same or other 40S subunits becomes methylated. This leads to a rapid degradation of the ribosomal protein mRNAs.     

ABERRANT INTRANUCLEOLAR MATURATION OF RIBOSOMAL PRECURSORS IN THE ABSENCE OF PROTEIN SYNTHESIS

To help elucidate the role of protein in the maturation of ribosomal RNA in cultured L cells, we have studied the effects of cycloheximide upon the maturation process and upon the intranucleolar ribonucleoprotein particles containing the "preribosomal RNA's." Five parameters of these particles were analyzed: (a) extractability, (b) sedimentation characteristics in sucrose gradients, (c) RNA composition, (d) buoyant density in CsCl gradients, and (e) effects of increased ionic strength on the buoyant density. When protein synthesis is inhibited, the rate of conversion of the precursor 45S ribosomal RNA is rapidly diminished, falling to less than 30% of the control rate within 1 hr. Nevertheless, in terms of the first three parameters there is no difference between control and cycloheximide nucleolar particles. However, the cycloheximide particles have a lower and more heterogeneous buoyant density and a more variable response to increased ionic strength. The results imply that the protein composition of the cycloheximide particles is different from that of particles from control cells, and that the entire protein complement is not necessary for the first cleavages in the maturation process, although it is necessary for the normal rate of processing and for the eventual appearance of both 18S and 28S rRNA in mature ribosomes.