Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.     

Ribosome Assembly Factors Pwp1 and Nop12 Are Important for Folding of 5.8S rRNA during Ribosome Biogenesis in Saccharomyces cerevisiae

Previous work from our lab suggests that a group of interdependent assembly factors (A₃ factors) is necessary to create early, stable preribosomes. Many of these proteins bind at or near internal transcribed spacer 2 (ITS2), but in their absence, ITS1 is not removed from rRNA, suggesting long-range communication between these two spacers. By comparing the nonessential assembly factors Nop12 and Pwp1, we show that misfolding of rRNA is sufficient to perturb early steps of biogenesis, but it is the lack of A₃ factors that results in turnover of early preribosomes. Deletion of NOP12 significantly inhibits 27SA₃ pre-rRNA processing, even though the A₃ factors are present in preribosomes. Furthermore, pre-rRNAs are stable, indicating that the block in processing is not sufficient to trigger turnover. This is in contrast to the absence of Pwp1, in which the A₃ factors are not present and pre-rRNAs are unstable. In vivo RNA structure probing revealed that the pre-rRNA processing defects are due to misfolding of 5.8S rRNA. In the absence of Nop12 and Pwp1, rRNA helix 5 is not stably formed. Interestingly, the absence of Nop12 results in the formation of an alternative yet unproductive helix 5 when cells are grown at low temperatures.     

Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3

Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles.     

Attenuation of 40S Ribosomal Subunit Abundance Differentially Affects Host and HCV Translation and Suppresses HCV Replication

For Hepatitis C virus (HCV), initiation of translation is cap-independently mediated by its internal ribosome entry site (IRES). Unlike other IRES-containing viruses that shut off host cap-dependent translation, translation of HCV coexists with that of the host. How HCV IRES-mediated translation is regulated in the infected cells remains unclear. Here, we show that the intracellular level of 40S ribosomal subunit plays a key role in facilitating HCV translation over host translation. In a loss-of-function screen, we identified small subunit ribosomal protein 6 (RPS6) as an indispensable host factor for HCV propagation. Knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation. Such preferential suppression of HCV translation correlated well with the reduction of the abundance of 40S ribosomal subunit following knockdown of RPS6 or other RPS genes. In contrast, reduction of the amount of ribosomal proteins of the 60S subunit did not produce similar effects. Among the components of general translation machineries, only knockdowns of RPS genes caused inhibitory effects on HCV translation, pointing out the unique role of 40S subunit abundance in HCV translation. This work demonstrates an unconventional notion that the translation initiation of HCV and host possess different susceptibility toward reduction of 40S ribosomal subunit, and provides a model of selective modulation of IRES-mediated translation through manipulating the level of 40S subunit.     

Distinct Regions of Human eIF3 Are Sufficient for Binding to the HCV IRES and the 40S Ribosomal Subunit

Translation of the hepatitis C virus (HCV) genomic RNA initiates from an internal ribosome entry site (IRES) in its 5′ untranslated region and requires a minimal subset of translation initiation factors to occur, namely eukaryotic initiation factor (eIF) 2 and eIF3. Low-resolution structural information has revealed how the HCV IRES RNA binds human eIF3 and the 40S ribosomal subunit and positions the start codon for initiation. However, the exact nature of the interactions between the HCV IRES RNA and the translational machinery remains unknown. Using limited proteolysis and mass spectrometry, we show that distinct regions of human eIF3 are sufficient for binding to the HCV IRES RNA and the 40S subunit. Notably, the eIF3 subunit eIF3b is protected by HCV IRES RNA binding, yet is exposed in the complex when compared to subunits eIF3e, eIF3f, eIF3h, and eIF3l. Limited proteolysis reveals that eIF3 binding to the 40S ribosomal subunit occurs through many redundant interactions that can compensate for each other. These data suggest how the HCV IRES binds to specific regions of eIF3 to target the translational machinery to the viral genomic RNA and provide a framework for modeling the architecture of intact human eIF3.     

Nip7p Interacts with Nop8p, an Essential Nucleolar Protein Required for 60S Ribosome Biogenesis, and the Exosome Subunit Rrp43p

NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001–5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3′-to-5′ exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.     

Interaction between Ribosome Assembly Factors Krr1 and Faf1 Is Essential for Formation of Small Ribosomal Subunit in Yeast

Ribosome formation in Saccharomyces cerevisiae requires a large number of transiently associated assembly factors that coordinate processing and folding of pre-rRNA and binding of ribosomal proteins. Krr1 and Faf1 are two interacting proteins present in early 90 S precursor particles of the small ribosomal subunit. Here, we determined a co-crystal structure of the core domain of Krr1 bound to a 19-residue fragment of Faf1 at 2.8 Å resolution. The structure reveals that Krr1 consists of two packed K homology (KH) domains, KH1 and KH2, and resembles archaeal Dim2-like proteins. We show that KH1 is a divergent KH domain that lacks the RNA-binding GXXG motif and is involved in binding another assembly factor, Kri1. KH2 contains a canonical RNA-binding surface and additionally associates with an α-helix of Faf1. Specific disruption of the Krr1-Faf1 interaction impaired early 18 S rRNA processing at sites A0, A1, and A2 and caused cell lethality, but it did not prevent incorporation of the two proteins into pre-ribosomes. The Krr1-Faf1 interaction likely maintains a critical conformation of 90 S pre-ribosomes required for pre-rRNA processing. Our results illustrate the versatility of KH domains in protein interaction and provide insight into the role of Krr1-Faf1 interaction in ribosome biogenesis.     

细胞周期决定核糖体蛋白S6在真核细胞核仁中的聚积

核糖体蛋白S6（rpS6）是核糖体40S小亚基的核心组成蛋白之一。研究表明,rpS6可以通过核定位信号进入细胞核中,在核仁中参与核糖体的组装。在该研究中发现,rpS6在高等真核细胞核仁中的聚积与细胞周期有关,rpS6在S期中晚期开始在核仁中聚积,G2期含量达到最高,M期核仁分解时消失。推测,rpS6在核仁中的这种分布特性可能与核糖体的合成随细胞周期变化有关。     

Ski6p Is a Homolog of RNA-Processing Enzymes That Affects Translation of Non-Poly(A) mRNAs and 60S Ribosomal Subunit Biogenesis

We mapped and cloned SKI6 of Saccharomyces cerevisiae, a gene that represses the copy number of the L-A double-stranded RNA virus, and found that it encodes an essential 246-residue protein with homology to a tRNA-processing enzyme, RNase PH. The ski6-2 mutant expressed electroporated non-poly(A) luciferase mRNAs 8- to 10-fold better than did the isogenic wild type. No effect of ski6-2 on expression of uncapped or normal mRNAs was found. Kinetics of luciferase synthesis and direct measurement of radiolabeled electroporated mRNA indicate that the primary effect of Ski6p was on efficiency of translation rather than on mRNA stability. Both ski6 and ski2 mutants show hypersensitivity to hygromycin, suggesting functional alteration of the translation apparatus. The ski6-2 mutant has normal amounts of 40S and 60S ribosomal subunits but accumulates a 38S particle containing 5′-truncated 25S rRNA but no 5.8S rRNA, apparently an incomplete or degraded 60S subunit. This suggests an abnormality in 60S subunit assembly. The ski6-2 mutation suppresses the poor expression of the poly(A)⁻ viral mRNA in a strain deficient in the 60S ribosomal protein L4. Thus, a ski6 mutation bypasses the requirement of the poly(A) tail for translation, allowing better translation of non-poly(A) mRNA, including the L-A virus mRNA which lacks poly(A). We speculate that the derepressed translation of non-poly(A) mRNAs is due to abnormal (but full-size) 60S subunits.     

Perturbation of 60 S Ribosomal Biogenesis Results in Ribosomal Protein L5- and L11-dependent p53 Activation

Ribosomal proteins play an important role in p53 activation in response to nucleolar stress. Multiple ribosomal proteins, including L5, L11, L23, and S7, have been shown to bind to and inhibit MDM2, leading to p53 activation. However, it is not clear whether ribosomal protein regulation of MDM2 is specific to some, but not all ribosomal proteins. Here we show that L29 and L30, two ribosomal proteins from the 60 S ribosomal subunit, do not bind to MDM2 and do not inhibit MDM2-mediated p53 suppression, indicating that the ribosomal protein regulation of the MDM2-p53 feedback loop is specific. Interestingly, direct perturbation of the 60 S ribosomal biogenesis by knocking down either L29 or L30 drastically induced the level and activity of p53, leading to p53-depedent cell cycle arrest. This p53 activation was drastically inhibited by knockdown of L11 or L5. Consistently, knockdown of L29 or L30 enhanced the interaction of MDM2 with L11 and L5 and markedly inhibited MDM2-mediated p53 ubiquitination, suggesting that direct perturbation of 60 S ribosomal biogenesis activates p53 via L11- and L5-mediated MDM2 suppression. Mechanistically, knockdown of L30 or L29 significantly increased the NEDDylation and nuclear retention of L11. Knocking down endogenous NEDD8 suppressed p53 activation induced by knockdown of L30. These results demonstrate that NEDDylation of L11 plays a critical role in mediating p53 activation in response to perturbation of ribosomal biogenesis.     

Azithromycin and Clarithromycin Inhibition of 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells

The ID₅₀ values for azithromycin and clarithromycin inhibition of translation and of 50S assembly in Staphylococcus aureus cells have been measured. For clarithromycin, 50% inhibition of growth occurred at 0.075 μg/ml, and the effects on translation and 50S formation were equivalent at 0.15 μg/ml. The inhibition of these processes by azithromycin was less effective, with an ID₅₀ of 2.5 μg/ml for growth and 5 μg/ml for inhibition of translation and 50S formation. The additive effects of each of these drugs on translation and 50S formation account quantitatively for their observed influence on cellular growth rates. In macrolide-treated cells, there was also a direct relationship between the loss of ribosomal RNA from the 50S subunit and its accumulation as oligoribonucleotides. These results are compared with the previously described effects of erythromycin on these same processes. Received: 30 June 1997 / Accepted: 12 August 1997     

Inferring the Ancient History of the Translation Machinery and Genetic Code via Recapitulation of Ribosomal Subunit Assembly Orders

Universally conserved positions in ribosomal proteins have significant biases in amino acid usage, likely indicating the expansion of the genetic code at the time leading up to the most recent common ancestor(s) (MRCA). Here, we apply this principle to the evolutionary history of the ribosome before the MRCA. It has been proposed that the experimentally determined order of assembly for ribosomal subunits recapitulates their evolutionary chronology. Given this model, we produce a probabilistic evolutionary ordering of the universally conserved small subunit (SSU) and large subunit (LSU) ribosomal proteins. Optimizing the relative ordering of SSU and LSU evolutionary chronologies with respect to minimizing differences in amino acid usage bias, we find strong compositional evidence for a more ancient origin for early LSU proteins. Furthermore, we find that this ordering produces several trends in specific amino acid usages compatible with models of genetic code evolution.     

Yeast Ribosomal Protein L40 Assembles Late into Precursor 60 S Ribosomes and Is Required for Their Cytoplasmic Maturation

Most ribosomal proteins play important roles in ribosome biogenesis and function. Here, we have examined the contribution of the essential ribosomal protein L40 in these processes in the yeast Saccharomyces cerevisiae. Deletion of either the RPL40A or RPL40B gene and in vivo depletion of L40 impair 60 S ribosomal subunit biogenesis. Polysome profile analyses reveal the accumulation of half-mers and a moderate reduction in free 60 S ribosomal subunits. Pulse-chase, Northern blotting, and primer extension analyses in the L40-depleted strain clearly indicate that L40 is not strictly required for the precursor rRNA (pre-rRNA) processing reactions but contributes to optimal 27 SB pre-rRNA maturation. Moreover, depletion of L40 hinders the nucleo-cytoplasmic export of pre-60 S ribosomal particles. Importantly, all these defects most likely appear as the direct consequence of impaired Nmd3 and Rlp24 release from cytoplasmic pre-60 S ribosomal subunits and their inefficient recycling back into the nucle(ol)us. In agreement, we show that hemagglutinin epitope-tagged L40A assembles in the cytoplasm into almost mature pre-60 S ribosomal particles. Finally, we have identified that the hemagglutinin epitope-tagged L40A confers resistance to sordarin, a translation inhibitor that impairs the function of eukaryotic elongation factor 2, whereas the rpl40a and rpl40b null mutants are hypersensitive to this antibiotic. We conclude that L40 is assembled at a very late stage into pre-60 S ribosomal subunits and that its incorporation into 60 S ribosomal subunits is a prerequisite for subunit joining and may ensure proper functioning of the translocation process.     

Assembly of the Yeast Exoribonuclease Rrp6 with Its Associated Cofactor Rrp47 Occurs in the Nucleus and Is Critical for the Controlled Expression of Rrp47

Rrp6 is a key catalytic subunit of the nuclear RNA exosome that plays a pivotal role in the processing, degradation, and quality control of a wide range of cellular RNAs. Here we report our findings on the assembly of the complex involving Rrp6 and its associated protein Rrp47, which is required for many Rrp6-mediated RNA processes. Recombinant Rrp47 is expressed as a non-globular homodimer. Analysis of the purified recombinant Rrp6·Rrp47 complex revealed a heterodimer, suggesting that Rrp47 undergoes a structural reconfiguration upon interaction with Rrp6. Studies using GFP fusion proteins show that Rrp6 and Rrp47 are localized to the yeast cell nucleus independently of one another. Consistent with this data, Rrp6, but not Rrp47, is found associated with the nuclear import adaptor protein Srp1. We show that the interaction with Rrp6 is critical for Rrp47 stability in vivo; in the absence of Rrp6, newly synthesized Rrp47 is rapidly degraded in a proteasome-dependent manner. These data resolve independent nuclear import routes for Rrp6 and Rrp47, reveal a structural reorganization of Rrp47 upon its interaction with Rrp6, and demonstrate a proteasome-dependent mechanism that efficiently suppresses the expression of Rrp47 in the absence of Rrp6.     

Analysis of the Ribosome Large Subunit Assembly and 23 S rRNA Stabilityin Vivo

The ability of mutant 23 S ribosomal RNA to form particles with proteins of the large ribosomal subunitin vivowas studied. A series of overlapping deletions covering the entire 23 S rRNA, were constructed in the plasmid copy of anE. coli23 S rRNA gene. The mutant genes were expressedin vivousing an inducibletacpromoter. Mutant species of 23 S rRNA, containing deletions between positions 40 and 2773, were incorporated into stable ribonucleoprotein particles. In contrast, if one end of the 23 S rRNA was deleted, the mutant rRNA was unstable and did not form ribosomal particles. Protein composition of the mutant particles was specific; the presence of the primary rRNA-binding proteins corresponded to their known binding sites. Furthermore, several previously unknown ribosomal protein binding sites in 23 S rRNA were identified. Implications of the results on ribosome assembly are discussed.