NIR was identified as an inhibitor of histone acetyltransferase and it represses transcriptional activation of p53. NIR is predominantly localized in the nucleolus and known as Noc2p, which is involved in the maturation of the 60S ribosomal subunit. However, how NIR functions in the nucleolus remains undetermined. In the nucleolus, a 47S ribosomal RNA precursor (pre-rRNA) is transcribed and processed to produce 18S, 5.8S and 28S rRNAs. The 18S rRNA is incorporated into the 40S ribosomal subunit, whereas the 28S and 5.8S rRNAs are incorporated into the 60S subunit. U3 small nucleolar RNA (snoRNA) directs 18S rRNA processing and U8 snoRNA mediates processing of 28S and 5.8 S rRNAs. Functional disruption of nucleolus often causes p53 activation to inhibit cell proliferation.

Mammalian and bird ribosomes are nearly twice the mass of prokaryotic ribosomes in part because of their extraordinarily long rRNA tentacles. Human rRNA tentacles are not fully observable in current three-dimensional structures and their conformations remain to be fully resolved. In previous work we identified sequences that favor G-quadruplexes in silico and in vitro in rRNA tentacles of the human large ribosomal subunit. We demonstrated by experiment that these sequences form G-quadruplexes in vitro. Here, using a more recent motif definition, we report additional G-quadruplex sequences on surfaces of both subunits of the human ribosome. The revised sequence definition reveals expansive arrays of potential G-quadruplex sequences on LSU tentacles. In addition, we demonstrate by a variety of experimental methods that fragments of the small subunit rRNA form G-quadruplexes in vitro. Prior to this report rRNA sequences that form G-quadruplexes were confined to the large ribosomal subunit. Our combined results indicate that the surface of the assembled human ribosome contains numerous sequences capable of forming G-quadruplexes on both ribosomal subunits. The data suggest conversion between duplexes and G-quadruplexes in response to association with proteins, ions, or other RNAs. In some systems it seems likely that the integrated population of RNA G-quadruplexes may be dominated by rRNA, which is the most abundant cellular RNA.

The human WBSCR22 protein was previously shown to be up-regulated in invasive breast cancer and its ectopic expression enhances tumor cell survival in the vasculature. In the current study, we show that the WBSCR22 protein is important for cell growth. Knock-down of WBSCR22 with siRNA results in slower growth of WBSCR22-depleted cells. Treatment with siWBSCR22 causes defects in the processing of pre-rRNAs and reduces the level of free 40S ribosomal subunit, suggesting that WBSCR22 is involved in ribosome small subunit biosynthesis. The human WBSCR22 partially complements the growth of WBSCR22 yeast homologue, bud23 deletion mutant suggesting that the human WBSCR22 is a functional homologue of yeast Bud23. WBSCR22 is localized throughout the cell nucleus and is not stably associated with ribosomal subunits within the cell nucleus. We also show that the WBSCR22 protein level is decreased in lymphoblastoid cell lines derived from William-Beuren Syndrome (WBS) patients compared to healthy controls. Our data suggest that the WBSCR22 protein is a ribosome biogenesis factor involved in the biosynthesis of 40S ribosomal particles in mammalian cells.

Ribosome heterogeneity is of increasing biological significance and several examples have been described for multicellular and single cells organisms. In here we show for the first time a variation in ribose methylation within the 18S rRNA of Saccharomyces cerevisiae. Using RNA-cleaving DNAzymes, we could specifically demonstrate that a significant amount of S. cerevisiae ribosomes are not methylated at 2'-O-ribose of A100 residue in the 18S rRNA. Furthermore, using LC-UV-MS/MS of a respective 18S rRNA fragment, we could not only corroborate the partial methylation at A100, but could also quantify the methylated versus non-methylated A100 residue. Here, we exhibit that only 68% of A100 in the 18S rRNA of S.cerevisiae are methylated at 2'-O ribose sugar. Polysomes also contain a similar heterogeneity for methylated Am100, which shows that 40S ribosome subunits with and without Am100 participate in translation. Introduction of a multicopy plasmid containing the corresponding methylation guide snoRNA gene SNR51 led to an increased A100 methylation, suggesting the cellular snR51 level to limit the extent of this modification. Partial rRNA modification demonstrates a new level of ribosome heterogeneity in eukaryotic cells that might have substantial impact on regulation and fine-tuning of the translation process.

The loss of the large ribosomal protein L9 causes a reduction in translation fidelity by an unknown mechanism. To identify pathways affected by L9, we identified mutants of E. coli that require L9 for fitness. In a prior study, we characterized L9-dependent mutations in the essential GTPase Der (EngA). Here, we describe a second class of L9-dependent mutations that either compromise or inactivate elongation factor P (EF-P, eIF5A in eukaryotes). Without L9, Δefp cells are practically inviable. Cell fractionation studies revealed that, in both the Der and EF-P mutant cases, L9's activity reduces immature 16S rRNA in 30S particles and partially restores the abundance of monosomes. Inspired by these findings, we discovered that L9 also enhances 16S maturation in wild-type cells. Surprisingly, although the amount of immature 16S in 30S particles was found to be elevated in ΔrplI cells, the amount in polysomes was low and inversely correlated with the immature 16S abundance. These findings provide an explanation for the observed fitness increases afforded by L9 in these mutants and reveal particular physiological conditions in which L9 becomes critical. Additionally, L9 may affect the partitioning of small subunits containing immature 16S rRNA.

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.

Ribosome biogenesis is required for protein synthesis and cell proliferation. Ribosome subunits are assembled in the nucleolus following transcription of a 47S ribosome RNA precursor by RNA polymerase I and rRNA processing to produce mature 18S, 28S and 5.8S rRNAs. The 18S rRNA is incorporated into the ribosomal small subunit, whereas the 28S and 5.8S rRNAs are incorporated into the ribosomal large subunit. Pol I transcription and rRNA processing are coordinated processes and this coordination has been demonstrated to be mediated by a subset of U3 proteins known as t-UTPs. Up to date, five t-UTPs have been identified in humans but the mechanism(s) that function in the t-UTP(s) activation of Pol I remain unknown. In this study we have identified 1A6/DRIM, which was identified as UTP20 in our previous study, as a t-UTP. In the present study, we investigated the function and mechanism of 1A6/DRIM in Pol I transcription.

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex.

The control of ribosomal RNA biogenesis is essential for the regulation of protein synthesis in eukaryotic cells. Here, we report the characterization of NOF1 that encodes a putative nucleolar protein involved in the control of rRNA expression in Arabidopsis. The gene has been isolated by T-DNA tagging and its function verified by the characterization of a second allele and genetic complementation of the mutants. The nof1 mutants are affected in female gametogenesis and embryo development. This result is consistent with the detection of NOF1 mRNA in all tissues throughout plant life's cycle, and preferentially in differentiating cells. Interestingly, the closely related proteins from zebra fish and yeast are also necessary for cell division and differentiation. We showed that the nof1-1 mutant displays higher rRNA expression and hypomethylation of rRNA promoter. Taken together, the results presented here demonstrated that NOF1 is an Arabidopsis gene involved in the control of rRNA expression, and suggested that it encodes a putative nucleolar protein, the function of which may be conserved in eukaryotes.

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

Although most eggs are intensely predated, the aerial egg clutches from the aquatic snail Pomacea canaliculata have only one reported predator due to unparalleled biochemical defenses. These include two storage-proteins: ovorubin that provides a conspicuous (presumably warning) coloration and has antinutritive and antidigestive properties, and PcPV2 a neurotoxin with lethal effect on rodents. We sequenced PcPV2 and studied whether it was able to withstand the gastrointestinal environment and reach circulation of a potential predator. Capacity to resist digestion was assayed using small-angle X-ray scattering (SAXS), fluorescence spectroscopy and simulated gastrointestinal proteolysis. PcPV2 oligomer is antinutritive, withstanding proteinase digestion and displaying structural stability between pH 4.0-10.0. cDNA sequencing and protein domain search showed that its two subunits share homology with membrane attack complex/perforin (MACPF)-like toxins and tachylectin-like lectins, a previously unknown structure that resembles plant Type-2 ribosome-inactivating proteins and bacterial botulinum toxins. The protomer has therefore a novel AB toxin combination of a MACPF-like chain linked by disulfide bonds to a lectin-like chain, indicating a delivery system for the former. This was further supported by observing PcPV2 binding to glycocalix of enterocytes in vivo and in culture, and by its hemaggutinating, but not hemolytic activity, which suggested an interaction with surface oligosaccharides. PcPV2 is able to get into predator's body as evidenced in rats and mice by the presence of circulating antibodies in response to sublethal oral doses. To our knowledge, a lectin-pore-forming toxin has not been reported before, providing the first evidence of a neurotoxic lectin in animals, and a novel function for ancient and widely distributed proteins. The acquisition of this unique neurotoxic/antinutritive/storage protein may confer the eggs a survival advantage, opening new perspectives in the study of the evolution of animal defensive strategies.

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.

The structural constituents of the large eukaryotic ribosomal subunit are 3 ribosomal RNAs, namely the 25S, 5.8S and 5S rRNA and about 46 ribosomal proteins (r-proteins). They assemble and mature in a highly dynamic process that involves more than 150 proteins and 70 small RNAs. Ribosome biogenesis starts in the nucleolus, continues in the nucleoplasm and is completed after nucleo-cytoplasmic translocation of the subunits in the cytoplasm. In this work we created 26 yeast strains, each of which conditionally expresses one of the large ribosomal subunit (LSU) proteins. In vivo depletion of the analysed LSU r-proteins was lethal and led to destabilisation and degradation of the LSU and/or its precursors. Detailed steady state and metabolic pulse labelling analyses of rRNA precursors in these mutant strains showed that LSU r-proteins can be grouped according to their requirement for efficient progression of different steps of large ribosomal subunit maturation. Comparative analyses of the observed phenotypes and the nature of r-protein-rRNA interactions as predicted by current atomic LSU structure models led us to discuss working hypotheses on i) how individual r-proteins control the productive processing of the major 5' end of 5.8S rRNA precursors by exonucleases Rat1p and Xrn1p, and ii) the nature of structural characteristics of nascent LSUs that are required for cytoplasmic accumulation of nascent subunits but are nonessential for most of the nuclear LSU pre-rRNA processing events.

Pno1 is a protein that plays a role in proteasome and ribosome neogenesis in yeast. So far, its functions in mammalian cells have not been investigated. To understand its function in mammals, we performed in situ hybridization analysis of Pno1 expression in different development stages and generated Pno1 gene knockout (KO) and transgenic (Tg) mice lineages. The results showed early lethality of homozygous Pno1 KO lineage caused, as demonstrated in parallel by ex vivo experiments, by arrest of embryo development before compaction stage. Though, heterozygous (HET) mice with 50% of normal Pno1 mRNA concentration were fertile and showed no obvious anomalies. The lymphoid organs of HET mice were normal in size, weight and cellularity, with normal T and B cell subpopulations. TCR-triggered activation and proliferation of HET T cells were normal. Proteasome activities in HET organs were uncompromised. Tg mice with actin promoter-driven Pno1 expression were also fertile, with no apparent anomalies, although they expressed 2-5-fold higher Pno1 mRNA levels. The lymphoid organs of Tg mice were of normal size, weight and cellularity with normal T and B cell sub-populations. TCR-triggered activation and proliferation of Tg T cells were normal. Tg organs and tissues presented normal proteasome activity as did their wild type counterparts. Tagged Pno1 over-expression in L cells and density gradient fractionation established that Pno1 existed in large complexes with sedimentation rates between 20S and 26S, bigger than mature 26S proteasomes. Pno1 in fractions did not coincide with 40S or 60S ribosome subunits. Our study indicates that Pno1 is essential for cellular functions, but only a small percentage of its normal level is sufficient, and excessive amounts are neither harmful nor useful. The nature of the large complexes it associates with remains to be identified, but it is certain that they are not mature proteasomes or ribosomes.

RNA Polymerase I (Pol I) has recently been recognized as a cancer therapeutic target. The activity of this enzyme is essential for ribosome biogenesis and is universally activated in cancers. The enzymatic activity of this multi-subunit complex resides in its catalytic core composed of RPA194, RPA135, and RPA12, a subunit with functions in RNA cleavage, transcription initiation and elongation. Here we explore whether RPA12 influences the regulation of RPA194 in human cancer cells. We use a specific small-molecule Pol I inhibitor BMH-21 that inhibits transcription initiation, elongation and ultimately activates the degradation of Pol I catalytic subunit RPA194. We show that silencing RPA12 causes alterations in the expression and localization of Pol I subunits RPA194 and RPA135. Furthermore, we find that despite these alterations not only does the Pol I core complex between RPA194 and RPA135 remain intact upon RPA12 knockdown, but the transcription of Pol I and its engagement with chromatin remain unaffected. The BMH-21-mediated degradation of RPA194 was independent of RPA12 suggesting that RPA12 affects the basal expression, but not the drug-inducible turnover of RPA194. These studies add to knowledge defining regulatory factors for the expression of this Pol I catalytic subunit.

Shewanella algae, which produces tetrodotoxin and exists in various seafoods, can cause human diseases, such as spondylodiscitis and bloody diarrhea. In the present study, we focused on the temporal, dynamic process in salt-stressed S. algae by monitoring the gene transcript levels at different time points after high salt exposure. Transcript changes in amino acid metabolism, carbohydrate metabolism, energy metabolism, membrane transport, regulatory functions, and cellular signaling were found to be important for the high salt response in S. algae. The most common strategies used by bacteria to survive and grow in high salt environments, such as Na+ efflux, K+ uptake, glutamate transport and biosynthesis, and the accumulation of compatible solutes, were also observed in S. algae. In particular, genes involved in peptidoglycan biosynthesis and DNA repair were highly and steadily up-regulated, accompanied by rapid and instantaneous enhancement of the transcription of large- and small-ribosome subunits, which suggested that the structural changes in the cell wall and some stressful responses occurred in S. algae. Furthermore, the transcription of genes involved in the tricarboxylic acid (TCA) cycle and the glycolytic pathway was decreased, whereas the transcription of genes involved in anaerobic respiration was increased. These results, demonstrating the multi-pathway reactions of S. algae in response to salt stress, increase our understanding of the microbial stress response mechanisms.

Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of different mycobacterial species indicates the presence of multiple P-loop GTPases that exhibit highly conserved motifs. However, an exact function of most of these GTPases in mycobacteria remains elusive. In the present study we characterized the function of a P-loop GTPase in mycobacteria by employing an EngA homologue from Mycobacterium smegmatis, encoded by an open reading frame, designated as MSMEG_3738. Amino acid sequence alignment and phylogenetic analysis suggest that MSMEG_3738 (termed as EngA(MS)) is highly conserved in mycobacteria. Homology modeling of EngA(MS) reveals a cloverleaf structure comprising of α/β fold typical to EngA family of GTPases. Recombinant EngA(MS) purified from E. coli exhibits a GTP hydrolysis activity which is inhibited by the presence of GDP. Interestingly, the EngA(MS) protein is co-eluted with 16S and 23S ribosomal RNA during purification and exhibits association with 30S, 50S and 70S ribosomal subunits. Further studies demonstrate that GTP is essential for interaction of EngA(MS) with 50S subunit of ribosome and specifically C-terminal domains of EngA(MS) are required to facilitate this interaction. Moreover, EngA(MS) devoid of N-terminal region interacts well with 50S even in the absence of GTP, indicating a regulatory role of the N-terminal domain in EngA(MS)-50S interaction.