Ribosomal proteins

Summary The number of specific binding sites for homogenous single ribosomal proteins on 16S E. coli ribosomal RNA was investigated. The capacity of each of the twenty-one 30S subunit proteins to bind to the RNA was estimated by two newly developed methods, namely immunoprecipitation and a polyacrylamide gel method. Five proteins, namely S4, S7, S8, S15 and S20 bound specifically. One, S17, bound nonspecifically. No binding of the other proteins was detected. The binding proteins bound simultaneously to the RNA, with stimulated binding of proteins S7 and S8. Evidence is provided for the similarity of the chemistry of the binding sites of the binding proteins in Escherichia coli and in Bacillus stearothermophilus ribosomes.     

Persistent Cytoplasmic Synthesis of Ribosomal Proteins during the Selective Inhibition of Ribosomal RNA Synthesis

Synthesis of ribosomal proteins takes place in the cytoplasm and is independent of nuclear synthesis of ribosomal RNA.     

Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction

Translation of the hepatitis C virus genomic RNA is mediated by an internal ribosome entry site (IRES). The 330-nt IRES RNA forms a binary complex with the small 40S ribosomal subunit as a first step in translation initiation. Here chemical probing and 4-thiouridine-mediated crosslinking are used to characterize the interaction of the HCV IRES with the HeLa 40S subunit. No IRES-18S rRNA contacts were detected, but several specific crosslinks to 40S ribosomal proteins were observed. The identity of the crosslinked proteins agrees well with available structural information and provides new insights into HCV IRES function. The protein-rich surface of the 40S subunit thus mediates the IRES-ribosome interaction.     

Ribosomal proteins in the spotlight

The assignment of specific ribosomal functions to individual ribosomal proteins is difficult due to the enormous cooperativity of the ribosome; however, important roles for distinct ribosomal proteins are becoming evident. Although rRNA has a major role in certain aspects of ribosomal function, such as decoding and peptidyl-transferase activity, ribosomal proteins are nevertheless essential for the assembly and optimal functioning of the ribosome. This is particularly true in the context of interactions at the entrance pore for mRNA, for the translation-factor binding site and at the tunnel exit, where both chaperones and complexes associated with protein transport through membranes bind.     

Ribosomal chromatin organization.

Mammalian cells contain approximately 400 copies of the ribosomal RNA genes organized as tandem, head-to-tail repeats spread among 6-8 chromosomes. Only a subset of the genes is transcribed at any given time. Experimental evidence suggests that, in a specific cell type, only a fraction of the genes exists in a conformation that can be transcribed. An increasing body of study indicates that eukaryotic ribosomal RNA genes exist in either a heterochromatic nucleosomal state or in open euchromatic states in which they can be, or are, transcribed. This review will attempt to summarize our current understanding of the structure and organization of ribosomal chromatin.     

Ribosomal history reveals origins of modern protein synthesis

The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.     

Ribosomal Proteins in the Spotlight

ABSTRACT

The assignment of specific ribosomal functions to individual ribosomal proteins is difficult due to the enormous cooperativity of the ribosome; however, important roles for distinct ribosomal proteins are becoming evident. Although rRNA has a major role in certain aspects of ribosomal function, such as decoding and peptidyl-transferase activity, ribosomal proteins are nevertheless essential for the assembly and optimal functioning of the ribosome. This is particularly true in the context of interactions at the entrance pore for mRNA, for the translation-factor binding site and at the tunnel exit, where both chaperones and complexes associated with protein transport through membranes bind.     

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.     

Ribosomal RNA pseudouridines and pseudouridine synthases

Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.     

Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.     

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.     

Elucidation of Motifs in Ribosomal Protein S9 That Mediate Its Nucleolar Localization and Binding to NPM1/Nucleophosmin

Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site.     

Ribosomal proteins: Structure,function, and evolution

The question concerning reasons for the variety of ribosomal proteins that arose for more than 40 years ago is still open. Ribosomes of modern organisms contain 50–80 individual proteins. Some are characteristic for all domains of life (universal ribosomal proteins), whereas others are specific for bacteria, archaea, or eucaryotes. Extensive information about ribosomal proteins has been obtained since that time. However, the role of the majority of ribosomal proteins in the formation and functioning of the ribosome is still not so clear. Based on recent data of experiments and bioinformatics, this review presents a comprehensive evaluation of structural conservatism of ribosomal proteins from evolutionarily distant organisms. Considering the current knowledge about features of the structural organization of the universal proteins and their intermolecular contacts, a possible role of individual proteins and their structural elements in the formation and functioning of ribosomes is discussed. The structural and functional conservatism of the majority of proteins of this group suggests that they should be present in the ribosome already in the early stages of its evolution.     

Ribosomal protein interactions in yeast. Protein L15 forms a complex with the acidic proteins

Protein L15 from Saccharomyces cerevisiae ribosomes has been shown to interact in solution with acidic ribosomal proteins L44, L44' and L45 by different methods. Thus, the presence of the acidic proteins changes the elution characteristics of protein L15 from CM-cellulose and DEAE-cellulose columns and from reverse-phase HPLC columns. Moreover, immunoprecipitation using anti-L15 specific monoclonal antibodies coprecipitates the acidic proteins, too. Conversely, antibodies raised against the acidic proteins immunoprecipitate protein L15. This coprecipitation seems to be specific since it does not involve other ribosomal proteins present in the sample. Similarly, plastic-adsorbed antibodies specific for one of the components in the L15--acidic-protein complex are able to retain the other component of the complex but cannot bind unrelated proteins. Moreover, protein L15 can be chemically cross-linked to the acidic proteins in solution. These results indicate that protein L15 might be equivalent to bacterial ribosomal protein L10 in forming a complex with the acidic proteins. Since, on the other hand, protein L15 has been shown to be immunologically related to bacterial protein L11 [Juan Vidales et al. (1983) Eur. J. Biochem. 136, 276-281] and to interact with the same region of the large ribosomal RNA as does protein L11 [El-Baradi et al. (1987) J. Mol. Biol. 195, 909-917], these results suggest strongly that protein L15 plays the same role in the yeast ribosome as proteins L10 and L11 do in the bacterial particles.     

Mutant Ribosomal Protein with Defective RNA Binding Site

THE 30S ribosomal subunits of Escherichia coli contain twenty-one different proteins^1–4, which together with 16S RNA can reassemble in vitro to form functional 30S particles⁵. Five proteins can individually bind to specific sites on the 16S RNA^6–8 and these are S4, S7, S8, S15 and S20 (in the nomenclature recently adopted by several laboratories to report results with the E. coli system⁹). We report here the first identification of a mutation that affects a ribosomal protein-nucleic acid interaction.