Has1p, a member of the DEAD-box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae

The Has1 protein, a member of the DEAD-box family of ATP-dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre-60S ribosomal complexes. Here, we show that Has1p is an essential trans-acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature-sensitive has1-1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre-rRNA processing by pulse-chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre-rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre-rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high-molecular-weight complex sedimenting at positions equivalent to 60S and pre-60S ribosomal particles. A mutation in the ATP-binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.     

In vivo and in vitro phosphorylation of ribosomal proteins by protein kinases from Saccharomyces cerevisiae.

From the high salt wash of the ribosomes of the yeast Saccharomyces cerevisiae, three protein kinases have been isolated and separated by DEAE-cellulose chromatography. The three kinases differ in their abilities to phosphorylate substrates such as histones (calf thymus), casein, and S. cerevisiae ribosomes; two of the kinases showed increased activity in the presence of cyclic adenosine 3',5'-monophosphate when histones and 40S ribosomal subunits were used as substrates. The protein kinases catalyzed phosphorylation of certain proteins of the 40S and 60S ribosomal subunits, and 80S ribosomes in vitro. Nine proteins of the 80S ribosome, seven proteins of the 40S subunit, and eleven of the 60S subunit were phosphorylated; different proteins were modified to various extents when different kinases were used. We have identified several proteins of 40S and 60S ribosomal subunits which are not available to the kinases in the 80S particles. Ribosomes isolated from S. cerevisiae cells growing in logarithmic phase of growth were found to contain a number of phosphorylated proteins. Studies by two-dimensional polyacrylamide gel electrophoresis indicated that the ribosomal proteins phosphorylated in vivo correspond with those phosphorylated in vitro. The relationship of in vivo phsophorylation of ribosomes to the growth and physiology of S. cerevisiae is not known.     

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.     

A kinetic light-scattering study of the binding of wheat germ protein synthesis initiation factor 3 to 40S ribosomal subunits and 80S ribosomes

The rate constants for eucaryotic initiation factor 3 (eIF3) association and dissociation with 40S ribosomal subunits and 80S monosomes have been determined. These rate constants were determined by laser light scattering with unmodified eIF3. The affinity of eIF3 for 40S subunits is about 30-fold greater than for 80S ribosomes. This difference in affinity resides mainly in the association rate constants. Rate constants of 8.8 X 10(7) and 7.3 X 10(6) M-1 s-1 were obtained for eIF3 binding to 40S subunits and 80S ribosomes, respectively. From thermodynamic cycles, the affinity of eIF3-40S subunits for 60S subunits is about 30-fold lower than free 40S subunits for 60S subunits. A calculation shows that under these conditions and assuming simple equilibria, approximately 12% of ribosomal subunits would associate via a reaction of 40S-eIF3 with 60S subunits as opposed to a path where eIF3 dissociates from the 40S subunits prior to association with 60S subunits.     

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.     

Binding of the IRES of hepatitis C virus RNA to the 40S ribosomal subunit: Role of p40

Ribosomal protein p40 is a structural component of the eukaryotic 40S ribosomal subunit, is partly homologous to prokaryotic ribosomal protein S2, and has a long eukaryote-specific C-terminal region. The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA was tested for the binding to 40S ribosomal subunits deficient in p40, saturated with recombinant p40, or pretreated with monoclonal antibody (MAB) 4F6 against p40. The apparent association constant of HCV IRES binding to 40S subunits was shown to directly depend on the p40 content in the subunits. MAB 4F6 prevented HCV IRES binding to 40S subunits and blocked translation of IRES-containing RNA in a cell-free translation system. The results implicate p40 in the binding of the HCV IRES to the ribosome and, therefore, in translation initiation on HCV RNA.     

Immunochemical analysis of the structure of eukaryotic ribosomes

Summary Antibodies were prepared in rabbits and sheep to rat liver ribosomes, ribosomal subunits, and to mixtures of proteins from the particles. The antisera were characterized by quantitative immunoprecipitation, by passive hemagglutination, by immunodiffusion on Ouchterlony plates, and by immunoelectrophoresis. While all the antisera contained antibodies specific for ribosomal proteins, none had precipitating antibodies against ribosomal RNA. Rat liver ribosomal proteins were more immunogenic in sheep than rabbits, and the large ribosomal subunit and its proteins were more immunogenic than those of the 40S subparticle. Antisera specific for one or the other ribosomal subunit could be prepared; thus it is unlikely that there are antigenic determinants common to the proteins of the two subunits. When ribosomes, ribosomal subunits, or mixtures of proteins were used as antigens the sera contained antibodies directed against a large number of the ribosomal proteins.Abbreviations TP total proteins—used to designate mixtures of proteins from ribosomal particles, hence TP80 is a mixtures of all the proteins from 80S ribosomes - TP60 the proteins from 60S subunits - TP40 the proteins from 40S particles     

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.     

Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.     

A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.     

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.     

A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits

Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.     

Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

Ribosome biogenesis requires >100 nonribosomal proteins, which are associated with different preribosomal particles. The substrates, the interacting partners, and the timing of action of most of these proteins are largely unknown. To elucidate the functional environment of the putative ATP-dependent RNA helicase Dbp6p from Saccharomyces cerevisiae, which is required for 60S ribosomal subunit assembly, we have previously performed a synthetic lethal screen and thereby revealed a genetic interaction network between Dbp6p, Rpl3p, Nop8p, and the novel Rsa3p. In this report, we extended the characterization of this functional network by performing a synthetic lethal screen with the rsa3 null allele. This screen identified the so far uncharacterized Npa1p (YKL014C). Polysome profile analysis indicates that there is a deficit of 60S ribosomal subunits and an accumulation of halfmer polysomes in the slowly growing npa1-1 mutant. Northern blotting and primer extension analysis shows that the npa1-1 mutation negatively affects processing of all 27S pre-rRNAs and the normal accumulation of both mature 25S and 5.8S rRNAs. In addition, 27SA(2) pre-rRNA is prematurely cleaved at site C(2). Moreover, GFP-tagged Npa1p localizes predominantly to the nucleolus and sediments with large complexes in sucrose gradients, which most likely correspond to pre-60S ribosomal particles. We conclude that Npa1p is required for ribosome biogenesis and operates in the same functional environment of Rsa3p and Dbp6p during early maturation of 60S ribosomal subunits.     

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.     

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.     

Ebp2p, the yeast homolog of Epstein-Barr virus nuclear antigen 1-binding protein 2, interacts with factors of both the 60 S and the 40 s ribosomal subunit assembly

Ebp2p, the yeast homolog of human Epstein-Barr virus nuclear antigen 1-binding protein 2, is essential for biogenesis of the 60 S ribosomal subunit. Two-hybrid screening exhibited that, in addition to factors necessary for assembly of the 60 S subunit, Ebp2p interacts with Rps16p, ribosomal protein S16, and the 40 S ribosomal subunit assembly factor, Utp11p, as well as Yil019w, the function of which was previously uncharacterized. Depletion of Yil019w resulted in reduction in levels of both of 18 S rRNA and 40 S ribosomal subunit without affecting levels of 25 S rRNA and 60 S ribosomal subunits. 35 S pre-rRNA and aberrant 23 S RNA accumulated, indicating that pre-rRNA processing at sites A(0)-A(2) is inhibited when Yil019w is depleted. Each combination from Yil019w, Utp11p, and Rps16p showed two-hybrid interaction.     

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2.GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNA(f) remains bound. The released eIF-2.GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNA(f).eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2.GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2.GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNA(f)l.eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2.eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2.GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2.GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2.GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2.GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2.eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2.GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.