Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.     

Human RioK3 is a novel component of cytoplasmic pre-40S pre-ribosomal particles

Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.     

Ribosomal proteins of Tetrahymena thermophila. Correlation of one- and two-dimensional electrophoretic migration patterns and characterization of additional small and large subunit proteins

Further analysis of the protein complement of the cytoplasmic ribosome of the protozoon Tetrahymena thermophila has led to the identification and characterization of seven additional proteins, three in the small and four in the large subunit of this ribosome. Several of these proteins are poorly soluble or insoluble in the absence of high concentrations of urea and are not seen in the electrophoretic distribution patterns of ribosomal proteins in two-dimensional polyacrylamide gels unless 6 M urea is added to electrode buffers in contact with protein samples (first dimension) and first-dimension gels (second dimension). The migration patterns of the 40S and 60S subunits of the T. thermophila ribosome in one-dimensional polyacrylamide SDS gels and in two-dimensional gels prepared by means of the basic-acidic system of Kaltschmidt and Wittmann**, and the basic-SDS system of Zinker and Warner*** have been correlated.     

Complementarity of sequences in low molecular weight RNAs to regions of messenger and ribosomal RNAs.

Total low molecular weight nuclear RNAs of mouse ascites cells have been labeled in vitro and used as probes to search for complementary sequences contained in nuclear or cytoplasmic RNA. From a subset of hybridizing lmw RNAs, two major species of 58,000 and 35,000 mol. wt. have been identified as mouse 5 and 5.8S ribosomal RNA. Mouse 5 and 5.8S rRNA hybridize not only to 18 and 28S rRNA, respectively, but also to nuclear and cytoplasmic poly(A+) RNA. Northern blot analysis and oligo-dT cellulose chromatography have confirmed the intermolecular base-pairing of these two small rRNA sequences to total poly(A+) RNA as well as to purified rabbit globin mRNA. 5 and 5.8S rRNA also hybridize with positive (coding) but not negative (noncoding) strands of viral RNA. Temperature melting experiments have demonstrated that their hybrid stability with mRNA sequences is comparable to that observed for the 5S:18S and 5.8S:28S hybrids. The functional significance of 5 and 5.8S rRNA base-pairing with mRNAs and larger rRNAs is unknown, but these interactions could play important coordinating roles in ribosome structure, subunit interaction, and mRNA binding during translation.     

Active regulator of SIRT1 is required for ribosome biogenesis and function

Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD⁺-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.     

Immature small ribosomal subunits can engage in translation initiation in Saccharomyces cerevisiae

It is generally assumed that, in Saccharomyces cerevisiae, immature 40S ribosomal subunits are not competent for translation initiation. Here, we show by different approaches that, in wild‐type conditions, a portion of pre‐40S particles (pre‐SSU) associate with translating ribosomal complexes. When cytoplasmic 20S pre‐rRNA processing is impaired, as in Rio1p‐ or Nob1p‐depleted cells, a large part of pre‐SSUs is associated with translating ribosomes complexes. Loading of pre‐40S particles onto mRNAs presumably uses the canonical pathway as translation‐initiation factors interact with 20S pre‐rRNA. However, translation initiation is not required for 40S ribosomal subunit maturation. We also provide evidence suggesting that cytoplasmic 20S pre‐rRNAs that associate with translating complexes are turned over by the no go decay (NGD) pathway, a process known to degrade mRNAs on which ribosomes are stalled. We propose that the cytoplasmic fate of 20S pre‐rRNA is determined by the balance between pre‐SSU processing kinetics and sensing of ribosome‐like particles loaded onto mRNAs by the NGD machinery, which acts as an ultimate ribosome quality check point.     

Scaffolding as an organizing principle in trans-translation. The roles of small protein B and ribosomal protein S1

A eubacterial ribosome stalled on a defective mRNA can be released through a quality control mechanism referred to as trans-translation, which depends on the coordinating binding actions of transfer-messenger RNA, small protein B, and ribosome protein S1. By means of cryo-electron microscopy, we obtained a map of the complex composed of a stalled ribosome and small protein B, which appears near the decoding center. This result suggests that, when lacking a codon, the A-site on the small subunit is a target for small protein B. To investigate the role of S1 played in trans-translation, we obtained a cryo-electron microscopic map, including a stalled ribosome, transfer-messenger RNA, and small protein Bs but in the absence of S1. In this complex, several connections between the 30 S subunit and transfer-messenger RNA that appear in the +S1 complex are no longer found. We propose the unifying concept of scaffolding for the roles of small protein B and S1 in binding of transfer-messenger RNA to the ribosome during trans-translation, and we infer a pathway of sequential binding events in the initial phase of trans-translation.     

Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1

Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.     

Removal of a ribosome small subunit-dependent GTPase confers salt resistance on Escherichia coli cells

RsgA is a unique GTP hydrolytic protein in which GTPase activity is significantly enhanced by the small ribosomal subunit. Deletion of RsgA causes slow cell growth as well as defects in subunit assembly of the ribosome and 16S rRNA processing, suggesting its involvement in maturation of the small subunit. In this study, we found that removal of RsgA or inactivation of its ribosome small subunit-dependent GTPase activity provides Escherichia coli cells with resistance to high salt stress. Salt stress suppressed the defects in subunit assembly of the ribosome and processing of 16S rRNA as well as truncation of the 3′ end of 16S rRNA in RsgA-deletion cells. In contrast, salt stress transiently impaired subunit assembly of the ribosome and processing of 16S rRNA and induced 3′ truncation of 16S rRNA in wild-type cells. These results suggest that the action of RsgA on the ribosome, which usually facilitates maturation of the small subunit, disturbs it under a salt stress condition. Consistently, there was a drastic but transient decrease in the intracellular amount of RsgA after salt shock. Salt shock would make the pathway of maturation of the ribosome small subunit RsgA independent.     

Nucleophosmin serves as a rate-limiting nuclear export chaperone for the Mammalian ribosome

Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.     

A novel GTPase activated by the small subunit of ribosome

The GTPase activity of Escherichia coli YjeQ, here named RsgA (ribosome small subunit-dependent GTPase A), has been shown to be significantly enhanced by ribosome or its small subunit. The enhancement of GTPase activity was inhibited by several aminoglycosides bound at the A site of the small subunit, but not by a P site-specific antibiotic. RsgA stably bound the small subunit in the presence of GDPNP, but not in the presence of GTP or GDP, to dissociate ribosome into subunits. Disruption of the gene for RsgA from the genome affected the growth of the cells, which predominantly contained the dissociated subunits having only a weak activation activity of RsgA. We also found that 17S RNA, a putative precursor of 16S rRNA, was contained in the small subunit of the ribosome from the RsgA-deletion strain. RsgA is a novel GTPase that might provide a new insight into the function of ribosome.     

Thiostrepton, an Inhibitor of 50S Ribosome Subunit Function

Thiostrepton inhibits (14)C-leucine incorporation by intact cells of Bacillus megaterium as well as (14)C-phenylalanine incorporation by a poly U-directed extract of Escherichia coli. Extracts of E. coli which are pretreated by incubation with thiostrepton cannot be reactivated by dialysis to more than 5% of their former activity. The 50S ribosome subunit appears to be the site of thiostrepton action, since protein-synthesizing activity can be restored to dialyzed pretreated extracts by supplementation with 50S ribosome subunits but not with 30S ribosome subunits. This technique also provides a simple sensitive method for detection of the biological activity of very small amounts of 50S ribosome subunits.     

Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus subtilis

The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ～55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.     

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.     

The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity

The acidic proteins, A-proteins, from the large ribosomal subunit of Saccharomyces cerevisiae grown under different conditions have been quantitatively estimated by ELISA tests using rabbit sera specific for these polypeptides. It has been found that the amount of A-protein present in the ribosome is not constant and depends on the metabolic state of the cell. Ribosomes from exponentially growing cultures have about 40% more of these proteins than those from stationary phase. Similarly, the particles forming part of the polysomes are enriched in A-proteins as compared with the free 80 S ribosomes. The cytoplasmic pool of A-protein is considerably high, containing as a whole as much protein as the total ribosome population. These results are compatible with an exchanging process of the acidic proteins during protein synthesis that can regulate the activity of the ribosome. On the other hand, cells inhibited with different metabolic inhibitors produce a very low yield of ribosomes that contain, however, a surprisingly high amount of acidic proteins while the cytoplasmic pool is considerably reduced, suggesting that under stress conditions the ribosome and the A-protein may aggregate, forming complex structures that are not recovered by the standard preparation methods.     

Bevorzugter Einbau markierten Uridins in die Vorläufer von Chloroplasten-Ribosomen in Algenzellen

Summary After short time pulses with 5-[3H]uridine have been given to Chlorella cells, most of the radioactivity of the ribosome fractions is neither in the polysomes nor in the cytoplasmic ribosomes. Peaks with sedimentation of about 50 S and 30 S are found which are comparable in sedimentation to ribosomal subunits of Escherichia coli. During chase treatment with the one-hundred-fold amount of unlabelled uridine, the radioactivity shifts into the 70 S region. The RNA of the rapidly labelled 50 S and 30 S particles is shown to have 23 S, 14 S and 5 S, respectively.In contrast to this, radioactive inorganic phosphate and amino acids are mainly incorporated into the cytoplasmic ribosomes with 80 S and into, their polysomes.The chloroplast-damaged mutant of Chlorella, Nr.125 of Schwarze, shows no uridine incorporation into particles of 50 S and of 30 S, but some very weak labelling of the 80 S cytoplasmic monosomes.Nitrogen deficient Chlorella cells also incorporate uridine mainly into the 50 S and 30 S particles. When chase treatment with unlabelled uridine is performed under recovering conditions, the label shifts into the 70 S particles as well as into the 80 S cytoplasmic ribosomes.The results indicate that in Chlorella, uridine is incorporated into chloroplast ribosome precursors rather than into particles of nuclear origin.     

Different mechanisms for pseudouridine formation in yeast 5S and 5.8S rRNAs

The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.