A growth-dependent excess of 40 S ribosomal subunits in plasmacytoma cells grown in suspension culture

When the plasmacytoma cell line MPC-11 was grown at high cell density in suspension culture (limited feeding) an approximately stoichiometric ratio between ribosomal 60S and 40S subunits was found in the cytoplasm. When grown in suspension culture at low cell density (well-fed cells) the ratio of 40–60S ribosomal subunits was higher than stoichiometric. A transfer from high cell density (poor growth conditions) to low cell density (good growth conditions) by addition of fresh medium strongly favoured accumulation of native 40S subunits in the cytoplasm compared with the accumulation of native 60S subunits. Both absorbance measurements and rRNA labelling experiments suggested the presence of an excess of 40S subunits under these conditions. The ratio between the ribosomal subunits in the cytoplasm of this transformed cell line, therefore, is strongly influenced by the growth conditions.     

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.     

The metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were 3H-labeled in vitro and injected back into X. laevis oocytes, most 3H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, 3H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of 3H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide, a cysteine protease inhibitor, resulting in the accumulation of 3H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.     

Disproportionate accumulation of 18S and 28S ribosomal RNA in cultured normal rat kidney cells treated with picolinic acid or 5-methylnicotinamide

Normal rat kidney cells treated with the pyridine derivative picolinic acid, or 5-methylnicotinamide, an inhibitor of ADP-ribosylation, are unable to process 28S rRNA and accumulate 60S ribosomal subunits in the cytoplasm. Synthesis of polyA(+) RNA, rRNA precursors, and the processing of 18S rRNA into 40S ribosomal subunits are almost unaffected. Serum starvation and treatment of cells with histidinol, cycloleucine, nicotinic acid, or 1,10-phenanthroline do not elicit this alteration in rRNA metabolism. Ribosomal subunits synthesized before picolinic acid addition have different stabilities after picolinic acid treatment. The 40S subunits are degraded while the 60S subunits are more stable, demonstrating that a compensatory mechanism exists to maintain preferentially existing subunits when they are no longer being synthesized. The results suggest that ADP-ribosylation is necessary for proper processing of 28S rRNA and therefore for formation of mature 60S ribosomal subunits.     

Comparison of native and KCl treated 40S ribosomal subunits from the silkmoth Antherea pernyi and mammals

Native 40S ribosomal subunits and 18S ribosomal RNA from ovarian follicles of the silkmoth A. pernyi showed a lower sedimentation coefficient in comparison to ascites cells, in contrast to the KCl treated 40S ribosomal subunits where no difference was observed in both tissues. Moreover the silkmoth native 40S ribosomal subunits--in contrast to the KCl treated ones--could not reassociate with radioactive ascites cell 60S ribosomal subunits. These results, combined with the great similarities in the two dimensional electrophoretic patterns of 40S ribosomal proteins from silkmoth follicles and other mammalian cells lead to the possibility of the existence of a specific RNase associated with the 40S ribosomal subunit.     

Interaction of the intermediate filament protein vimentin with ribosomal subunits and ribosomal RNA in vitro

If in a low ionic strength extract of Triton X-100-resistant residual cell structures derived from Ehrlich ascites tumour (EAT) cells Mg²⁺ was chelated by EDTA, vimentin became associated with unfolded ribosomal subunits. The first molecular characterization of this association has shown that (1) vimentin binds to the RNA moiety of the ribosomes, (2) vimentin has a higher affinity for unfolded small ribosomal subunits or 18S rRNA than for unfolded large ribosomal subunits or 28S rRNA, (3) the limited degradation of vimentin by the vimentin-specific, Ca²⁺-activated proteinase, with the formation of a 48 Kd breakdown product, abolishes its affinity for rRNA, (4) the association products are rather sensitive to moderate concentrations of KCl and Mg²⁺, and (5) reductive alkylation of vimentin with pyridoxal-5-phosphate and NaBH₄ has no effect on the affinity of vimentin for rRNA. Actin and tubulin do not interact with EAT cell rRNA under the above ionic conditions.     

Effect of ribonuclease T1 on ribosomal subunits of rat liver.

The accessibility of 28S RNA within the ribosomal subunits to ribonuclease T1 was studied, in comparing results obtained after enzyme treatment of compact, K+ deficient 60S subunits and of EDTA-treated 60S subunits. RNA, extracted from the subunits, using a mixture of sodium dodecyl sulfate and phenol was analyzed on sucrose gradients. The RNA from active subunits was only degraded in high enzyme concentrations. In the K+ deficient subunits, RNA is more accessible since it breaks down into 6 well-defined fragments, sedimenting between 4S and 18.5S. Within the EDTA-subunits, there is no more protection of the RNA. In fact, it is degraded by weak enzyme concentrations, as is the free 28S RNA, giving heterogeneous fragments. Comparison of the melting curves of subunits and free 28S RNA showed that it is only in EDTA subunits that proteins do not stabilize the secondary structure of RNA. In the case of 40S subunits, the action of ribonuclease T1 combines with the action of the endogenous nuclease which makes the degradation process more difficult to analyze.     

Membrane-bound ribosomes of myeloma cells. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane- bound ribosomal particles

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.     

Stabilization of 30 S ribosomal subunits of Bacillus subtilis W168 by spermidine and magnesium ions

An explanation for the fragility of 30 S ribosomal subunits of Bacillus subtilis has been studied. Degradation of 16 S ribosomal RNA, rather than degradation of ribosomal proteins, was found to cause the inactivation of 30 S subunits. Although RNAases were bound specifically to 30 S ribosomal subunits, the RNAases were able to function. Spermidine was found to contribute to the stabilization of 30 S ribosomal subunits by inhibiting the degradation of 16 S ribosomal RNA. A high concentration of Mg²⁺ also stabilized the 30 S ribosomal subunits of Bacillus subtilis. The polypeptide synthetic activity of 30 S ribosomal subunits prepared in the presence of spermidine was at least 4-times greater than that of 30 S ribosomal subunits prepared in the absence of spermidine; this activity was maintained without any loss for 3 months at ?70°C.     

A nuclear AAA-type ATPase (Rix7p) is required for biogenesis and nuclear export of 60S ribosomal subunits.

Ribosomal precursor particles are assembled in the nucleolus before export into the cytoplasm. Using a visual assay for nuclear accumulation of 60S subunits, we have isolated several conditional-lethal strains with defects in ribosomal export (rix mutants). Here we report the characterization of a mutation in an essential gene, RIX7, which encodes a novel member of the AAA ATPase superfamily. The rix7-1 temperature-sensitive allele carries a point mutation that causes defects in pre-rRNA processing, biogenesis of 60S ribosomal subunits, and their subsequent export into the cytoplasm. Rix7p, which associates with 60S ribosomal precursor particles, localizes throughout the nucleus in exponentially growing cells, but concentrates in the nucleolus in stationary phase cells. When cells resume growth upon shift to fresh medium, Rix7p-green fluorescent protein exhibits a transient perinuclear location. We propose that a nuclear AAA ATPase is required for restructuring nucleoplasmic 60S pre-ribosomal particles to make them competent for nuclear export.     

Nuclear retention of 18S ribosomal RNA by human myeloma cells

Summary Normal quiescent lymphocytes regulate their ribosome content by selectively degrading newly synthesized 18S ribosomal RNA. Unlike actively dividing HeLa cells, lymphocytes retain 18 S ribosomal RNA in the nucleus after synthesis instead of immediately transporting it to the cytoplasm. Subcellular fractionation of the highly differentiated human neoplastic lymphocyte RPMI-8226 reveals that this cell line also retains 18 S ribosomal RNA in the nucleus, a trait not displayed by the less differentiated human lymphoblastoid cell line RPMI-4265. These observations suggest that neoplastic cells can be phenotypically characterized by their ribosomal RNA processing patterns.Operated by Union Carbide Corporation with the Department of Energy     

Rapid peptide bond formation on isolated 50S ribosomal subunits

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.     

Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran.GTP. Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation. The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1-Ran.GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.     

Carbodiimide-induced protein--RNA crosslinking in mammalian ribosomal subunits

RNA--protein interactions in the 60 S subunits of rat liver ribosomes were studied using 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC) under conditions which neither changed the sedimentation coefficient of subunits nor the intactness of their rRNA. EDC induced RNA--protein and protein--protein crosslinkings. Proteins crosslinked to 28 S RNA were identified by two-dimensional gel electrophoreses as L17, L19, L23a and L37a, shown to react with 28 S RNA when using a low dose of UV radiation. Attempts have also been made to use EDC for the studies of RNA--protein interactions in 40 S ribosomal subunits.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Electrophoretic characterization of ribosomal subunits and proteins in apoptosis: specific downregulation of S11 in staurosporine-treated human breast carcinoma cells.

Stimulation of death receptors (Fas on human T-cell leukemia Jurkat cells and tumor necrosis factor receptor-1 on human monoblastic leukemia U937 cells) triggers the specific degradation of 28S ribosomal RNA, and this process may contribute to cell death through the inhibition of protein synthesis. We have developed an analytical method using a polyacrylamide-agarose composite gel to evaluate ribosomal subunits in apoptotic cells (human breast carcinoma MCF-7 cells treated with staurosporine and human 293T cells irradiated with ultraviolet light were used in addition to the two apoptosis systems described above). No alterations were detected by this method, suggesting that apoptosis, including the process of ribosomal RNA degradation, does not cause fragmentation or extensive conformational changes in the ribosome. We also examined the status of 21 different ribosomal proteins in apoptotic cells by immunoblotting with polyclonal antibodies. S11 was specifically downregulated in apoptotic MCF-7 cells and in other apoptotic breast carcinoma cells. Previous studies have shown that S11 is heterogeneously expressed in cancer cells. Taken together, it appears that particular intracellular environments regulate the expression of S11 protein. However, the mechanism by which this process is modulated is as yet unknown. Furthermore, we have demonstrated that our composite gel electrophoresis system can efficiently detect ubiquitination of ribosomal subunits.     

A functional network involved in the recycling of nucleocytoplasmic pre-60S factors

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation.