Ribosomal Activity Research Articles

Do Streptococcus pneumoniae and respiratory Syncytial virus synergise to promote invasive disease?

Streptococcus pneumoniae (S.p.) and Respiratory Syncytial Virus (RSV) are two major pathogens commonly found to coexist in respiratory secretions in patients with acute respiratory infection. Though there is increasing evidence of a synergistic interplay between these two pathobionts, the exact mechanisms remain obscure. The aim of our study was to decipher how coinfection with RSV alter pneumococcal growth dynamics and host immune response and how this impact on the colonisation and invasive properties of S.p. Using in vivo mouse model, we made the key observation that upon coinfection with RSV, the density of pneumococcal colonisation in the nasopharynx and dissemination to the lower respiratory tract were significantly higher in mice previously colonised with S.p. These mice also presented more severe weight loss and delayed recovery compared to mono-infected animals as well as significantly heightened pro-inflammatory cytokine profiles. Measurement of in vitro transepithelial electrical resistance (TEER) showed that, upon RSV coinfection, S.p. transmigrate through the epithelial barrier without altering epithelial integrity suggesting a transcellular mechanism rather than paracellular migration. Moreover, RSV-pneumococcal coinfection of human primary nasal epithelial cell demonstrated major changes in host protein expression involved in the catalytic activity, ubiquitination, cytoskeletal organisation, and endocytosis. Simultaneously, significant upregulation observed in bacterial proteins involved in the ribosomal activity, streptococcus-induced tissue inflammation, DNA supercoiling, and bacterial viability during oxidative stress, affecting both the survival and the virulence of S.p. Our results explain the complex interactions between pneumococci, RSV and host and help towards further understanding the significance of viral-bacterial co-infection in clinical settings.

An rRNA variant to deal with stress.

Bacteria have previously been assumed to cope with environmental stress by tuning their total number of active ribosomes. Instead, a study in this issue of Nature Microbiology shows that from a heterogeneous pool of ribosomes, Vibrio vulnificus uses ribosomes with a particular ribosomal RNA variant to translate upregulated stress response mRNAs.

Artificial selection on storage protein 1 possibly contributes to increase of hatchability during silkworm domestication.

Like other domesticates, the efficient utilization of nitrogen resources is also important for the only fully domesticated insect, the silkworm. Deciphering the way in which artificial selection acts on the silkworm genome to improve the utilization of nitrogen resources and to advance human-favored domestication traits, will provide clues from a unique insect model for understanding the general rules of Darwin's evolutionary theory on domestication. Storage proteins (SPs), which belong to a hemocyanin superfamily, basically serve as a source of amino acids and nitrogen during metamorphosis and reproduction in insects. In this study, through blast searching on the silkworm genome and further screening of the artificial selection signature on silkworm SPs, we discovered a candidate domestication gene, i.e., the methionine-rich storage protein 1 (SP1), which is clearly divergent from other storage proteins and exhibits increased expression in the ova of domestic silkworms. Knockout of SP1 via the CRISPR/Cas9 technique resulted in a dramatic decrease in egg hatchability, without obvious impact on egg production, which was similar to the effect in the wild silkworm compared with the domestic type. Larval development and metamorphosis were not affected by SP1 knockout. Comprehensive ova comparative transcriptomes indicated significant higher expression of genes encoding vitellogenin, chorions, and structural components in the extracellular matrix (ECM)-interaction pathway, enzymes in folate biosynthesis, and notably hormone synthesis in the domestic silkworm, compared to both the SP1 mutant and the wild silkworm. Moreover, compared with the wild silkworms, the domestic one also showed generally up-regulated expression of genes enriched in the structural constituent of ribosome and amide, as well as peptide biosynthesis. This study exemplified a novel case in which artificial selection could act directly on nitrogen resource proteins, further affecting egg nutrients and eggshell formation possibly through a hormone signaling mediated regulatory network and the activation of ribosomes, resulting in improved biosynthesis and increased hatchability during domestication. These findings shed new light on both the understanding of artificial selection and silkworm breeding from the perspective of nitrogen and amino acid resources.

Quantitative proteomics reveals systematic dysregulations of liver protein metabolism in sucralose-treated mice

Sucralose, one kind of “sugar-free” artificial sweeteners, is widely used as food and drinks additives. It is generally considered that sucralose is safe because majority of ingested sucralose is not metabolized and absorbed by human body. However, increasing evidence shows the negative effects of artificial sweeteners in perturbations of gut microbiota which plays an important role in a variety of processes related to host health such as immune system development. Specifically, sucralose uptake can alter the homeostasis of mouse gut microbiota, resulting in the significant changes of gut bacterial genera diversity, metabolic patterns, and fecal metabolite profiles as well as inducing host liver inflammation. Therefore, there is a need to study liver proteome changes which may be potentially affected by sucralose-induced dysbiosis. In this study, isobaric labeling-based quantitative proteomics was performed to reveal the liver functional proteome changes in male C57BL/6J mice with sucralose administration in drinking water for six-month period. The labeled tryptic peptides were off-line fractionated before LC-MS/MS analysis to improve proteome coverage detected. SignificanceWe demonstrated the first quantitative proteomics for mice liver proteome to evaluate the effect of sucralose consumption. In total, >5700 protein groups were identified from 18 mouse liver tissues (9 from control group; 9 from sucralose-treated group), and 4327 protein groups were quantified in all samples without any missing values. Among them, 113 protein groups were identified with statistical significance (q value <0.05) as differentially expressed proteins. Bioinformatics analysis revealed the systematic dysregulations of protein metabolism after sucralose treatment. Importantly, our findings proposed that enhanced inflammation may be triggered by ribosomal inactivation in sucralose treated mice liver.

Structural basis for neutralization of cytotoxic abrin by monoclonal antibody D6F10.

Abrin, an extremely cytotoxic Type II ribosome-inactivating protein (RIP), is a potential bio-warfare agent. Abrin A-chain (ABA) depurinates an adenosine of sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, thereby arresting protein synthesis and leading to cell death. Monoclonal antibody (mAb) D6F10 is the only known antibody that neutralizes ABA's activity in cell-free systems as well as abrin's toxicity invitro and invivo. However, how binding of mAb D6F10 to abrin interferes with abrin's catalytic activity at ribosome is still poorly understood. To provide structural basis for mAb D6F10-mediated rescue of ribosome inactivation by abrin, we determined crystal structures of ABA with and without substrate analogs. The structures of ABA-substrate analogs and ribosome were used in an experiment-guided computational protocol, to construct the ABA-Ribosome complex. A homology model of the variable region (Fv ) of mAb D6F10 was generated and docked with the apo-ABA structure to construct the ABA-D6F10 Fv complex. Structural superposition of ABA common to ABA-D6F10 Fv and ABA-Ribosome complexes reveals steric hindrance as the primary mechanism by which mAb D6F10 neutralizes abrin. In contrast to ABA alone, ABA bound to mAb D6F10 is unable to access the SRL on the ribosome owing to steric clashes of mAb D6F10 with the ribosome. Crystal structures of ABA also reveal a catalytic water molecule implicated in hydrolyzing N-glycosidic bond of the susceptible adenosine by RIPs. Furthermore, our strategy provides structural details of steric hindrance important for neutralization of ricin, another RIP, by mAb 6C2 and hence is of wide applicability. ENZYME: EC3.2.2.22. DATABASE: Structural data have been deposited in the Protein Data Bank (PDB) under the accession numbers 5Z37, 5Z3I, and 5Z3J.

4 - A ZEBRAFISH MODEL OF CLOZAPINE EXPOSURE: DRUG-INDUCED TRANSCRIPTOMIC CHANGES IN THE BRAIN

Background Schizophrenia (SZ) is a severe neuropsychiatric disorder characterized by episodic psychosis and altered cognitive function. About 20% of SZ patients are resistant to the commonly-prescribed antipsychotic medications used to treat disease symptoms. Clozapine is an atypical antipsychotic drug often prescribed to treatment-resistant SZ patients, although the functional pathways mediating its action are not well understood. Importantly, 40–70% of patients treated with clozapine show an inadequate response and there are sever side-effects. Understanding the molecular pathways involved in antipsychotic response will help in the development of new improved therapeutics that act on pathogenicity rather than just treating the acute manifestations of SZ. Methods We exposed wild type zebrafish to high and low doses of the antipsychotic drug clozapine over 72 hours. Relevant controls were included at all stages of the experiment. We recorded the animals and analysed changes in behavior associated with clozapine exposure. We then performed RNA-sequencing in the brain and identified changes in gene expression in response to clozapine exposure. Network analysis was performed to identify modules of co-expressed genes sensitive to clozapine exposure and pathway analysis was employed to identify overrepresentation of GO terms in each of the clozapine exposure-associated modules Results The fish showed a dose-sensitive shift in behaviour in response to clozapine. We identified five genes showing significant gene expression changes in response to clozapine, including ODC1, a gene previously implicated in SZ which shows changes in expression during brain development. Using network analysis, we identified fifteen modules of co-expressed genes which present a striking change in module connectivity in response to the drug. These modules show overrepresentation of key gene ontology terms, implicating processes such as calcium ion transport, cell-cell signalling, G-protein coupled receptor activity, ribosomal activity, and the regulation of transcription. Discussion Our study highlights the utility of zebrafish as a model for assessing the molecular consequences of antipsychotic medications. Our data show marked behavioural effects induced by clozapine and significant transcriptomic alterations in important functional pathways in the brain.

Detection and expression analysis of tet(B) in Streptococcus oralis

ABSTRACTTetracycline resistance can be achieved through tet genes, which code for efflux pumps, ribosomal protection proteins and inactivation enzymes. Some of these genes have only been described in either Gram-positive or Gram-negative bacteria. This is the case of tet(B), which codes for an efflux pump and, so far, had only been found in Gram-negative bacteria. In this study, tet(B) was detected in two clinical Streptococcus oralis strains isolated from the gingival sulci of two subjects. In both cases, the gene was completely sequenced, yielding 100% shared identity and coverage with other previously published sequences of tet(B). Moreover, we studied the expression of tet(B) using RT-qPCR in the isolates grown with and without tetracycline, detecting constitutive expression in only one of the isolates, with no signs of expression in the other one.This is the first time that the presence and expression of the tet(B) gene has been confirmed in Gram-positive bacteria, which highlights the potential of the genus Streptococcus to become a reservoir and a disseminator of antibiotic resistance genes in an environment so prone to horizontal gene transfer as is the oral biofilm.

Activity of nucleolar organizers in endometrial glandular and surface epithelial cells in patients with external genital endometriosis

Hypothesis/aims of study. Endometriosis is a disease characterized by proliferation of tissue that is similar in morphology and function to the endometrium, outside the uterine cavity. Thanks to the theory of J. Sampson (1927), which explains endometrioid heterotopia formation by bits of endometrial tissue migrating through the fallopian tubes during menstruation and their subsequent implantation in the pelvis, research has begun on the biological properties of eutopic endometrial cells in women with endometriosis, which is still relevant today. The present study was aimed to assess the activity of nucleolus organizers in the endometrium in women with external genital endometriosis and without it at different stages of the menstrual cycle.&#x0D; Study design, materials, and methods. Paraffin blocks of endometrial biopsy specimens were taken from 68 women with and without external genital endometriosis. The activity of nucleolar organizers was evaluated by the method of J. Crocker (1983).&#x0D; Results. The data obtained showed a significant increase in ribosomal activity in the cells of the endometrial glandular and surface epithelium in women with external genital endometriosis at the stages of late proliferation, as well as early and middle secretions. There was also a significant decrease in the activity of the nucleolar organizers of endometrial epithelial cells at the stage of late secretion in women with external genital endometriosis.&#x0D; Conclusion. The obtained differences in regulation of ribosomal synthesis in the cells of the integument and glandular epithelium of the endometrium in women with external genital endometriosis, in our opinion, can provide an increase in the total amount of epitheliocytes of the endometrium, increase their adhesive and invasive abilities, and lengthen the life cycle of cells. This contributes to the formation of endometrioid heterotopies and the development of external genital endometriosis. Changes in the activity of nucleolar organizers in the endometrium in women with external genital endometriosis may be of clinical interest for the diagnosis of this pathology.

Ribosomal protein eL42 contributes to the catalytic activity of the yeast ribosome at the elongation step of translation

The GGQ minidomain of the ribosomal protein eL42 was previously shown to contact the CCA-arm of P-site bound tRNA in human ribosome, indicating a possible involvement of the protein in the catalytic activity. Here, using Schizosaccharomyces pombe (S. pombe) cells, we demonstrate that the GGQ minidomain and neighboring region of eL42 is critical for the ribosomal function. Mutant eL42 proteins containing amino acid substitutions within or adjacent to the GGQ minidomain failed to complement the function of wild-type eL42, and expression of the mutant eL42 proteins led to severe growth defects. These results suggest that the mutations in eL42 interfere with the ribosomal function in vivo. Furthermore, we show that some of the mutations associated with the conserved GGQ region lead to reduced activities in the poly(Phe) synthesis and/or in the peptidyl transferase reaction with respect to puromycin, as compared with those of the wild-type ribosomes. A pK value of 6.95 was measured for the side chain of Lys-55/Arg-55, which is considerably less than that of a Lys or Arg residue. Altogether, our findings suggest that eL42 contributes to the 80S ribosome's peptidyl transferase activity by promoting the course of the elongation cycle.

Deletion 6q Drives T-cell Leukemia Progression by Ribosome Modulation.

Deletion of chromosome 6q is a well-recognized abnormality found in poor-prognosis T-cell acute lymphoblastic leukemia (T-ALL). Using integrated genomic approaches, we identified two candidate haploinsufficient genes contiguous at 6q14, SYNCRIP (encoding hnRNP-Q) and SNHG5 (that hosts snoRNAs), both involved in regulating RNA maturation and translation. Combined silencing of both genes, but not of either gene alone, accelerated leukemogeneis in a Tal1/Lmo1/Notch1-driven mouse model, demonstrating the tumor-suppressive nature of the two-gene region. Proteomic and translational profiling of cells in which we engineered a short 6q deletion by CRISPR/Cas9 genome editing indicated decreased ribosome and mitochondrial activities, suggesting that the resulting metabolic changes may regulate tumor progression. Indeed, xenograft experiments showed an increased leukemia-initiating cell activity of primary human leukemic cells upon coextinction of SYNCRIP and SNHG5. Our findings not only elucidate the nature of 6q deletion but also highlight the role of ribosomes and mitochondria in T-ALL tumor progression. SIGNIFICANCE: The oncogenic role of 6q deletion in T-ALL has remained elusive since this chromosomal abnormality was first identified more than 40 years ago. We combined genomic analysis and functional models to show that the codeletion of two contiguous genes at 6q14 enhances malignancy through deregulation of a ribosome-mitochondria axis, suggesting the potential for therapeutic intervention.This article is highlighted in the In This Issue feature, p. 1494.

CD4+ T Cell Fate in Multiple Myeloma

The average incidence of multiple myeloma (MM) is in the 7th decade that coincides with the development of immunosenescence and thymic atrophy, meaning that lymphocyte recovery after lymphopenia-inducing therapies (most notably autologous stem cell transplant, ASCT) is largely reliant on homeostatic proliferation of peripheral T cells rather than replenishing the T cell pool with new thymic emigrants. We have previously shown that there is a significant reduction in circulating naïve T cells with a reciprocal expansion of antigen-experienced cells from newly diagnosed MM (NDMM) to relapsed/refractory disease (RRMM). This results in a reduced TCR repertoire and the accumulation of senescence-associated secretory phenotype cytotoxic T cells, which maintain the ability to produce IFNγ but lose proliferative potential. A reduction in CD4:8 ratio is also a characteristic finding in MM with disease progression, which can be explained by high IL-15 levels in lymphopenic states that preferentially drive expansion of CD8+ memory T cells.We wanted to further evaluate what changes were occurring in the CD4+ T cell population with disease progression in MM. We analyzed paired peripheral blood (PB) samples from patients with NDMM and RRMM, and compared with age-matched normal donors (ND). In the NDMM cohort, we examined T cells from PB samples at baseline, after 4 cycles of lenalidomide and dexamethasone (len/dex), and after ASCT; and in the RRMM cohort samples from baseline and after 6 cycles of len/dex.We firstly confirmed in flow cytometric analysis of T cells at serial intervals in NDMM patients that the reduction in circulating naïve T cells and in CD4:8 ratio occurs post ASCT and does not recover by time of last follow-up.We next utilised RNA-seq to analyse differences in CD4+ T cells from NDMM, RRMM and ND. CD4+ T cells from RRMM showed downregulation of cytosolic ribosomal activity but maintenance of mitochondrial ribosomal activity and significant upregulation of pathways involved with calcium signalling. To this end, we evaluated mitochondrial biogenesis and metabolic pathways involved with mitochondrial respiration. Flow cytometric analysis of mitochondrial mass showed a marked increase in RRMM compared with ND, in keeping with a shift towards memory phenotype. Key rate-limiting enzymes in fatty acid β-oxidation (CPT1-A, ACAA2 and ACADVL) were all significantly increased in RRMM compared with ND.To analyse whether these cells were metabolically active, we also measured mitochondrial membrane potential and reactive oxygen species (ROS), gating on cells with high mitochondrial mass. Mitochondrial membrane potential was significantly increased in RRMM compared with ND, although ROS was reduced. The significance of this is not clear, as ROS are not only implicated in cell senescence and activation-induced cell death, but are also positively involved in tyrosine kinase and PI3K-signalling pathways.PD-1 has been shown to play a role in transitioning activated CD4+ T cells from glycolysis to FAO metabolism, and elevating ROS in activated CD8+ T cells. We analysed PD-1 expression on T cells in RRMM and at treatment intervals in NDMM (as described earlier). The proportion of CD4+ and CD8+ T cells expressing PD-1 was increased 4-6 months post-ASCT and remained elevated in CD4+ T cells 9-12 months post-ASCT, but normalised to baseline levels in CD8+ T cells. Increased PD-1 expressing CD4+ T cells was also evident in RRMM patient samples. This may suggest that in the lymphopenic state, PD-1 expression enhances longevity in a subset of CD4+ T cells by promoting reliance on mitochondrial respiration; however, their ability to undergo homeostatic proliferation is impaired. In CD8+ T cells, high PD-1 expression may lead to cell death via ROS accumulation, and these cells do not persist.ASCT remains a backbone of myeloma treatment in medically fit patients. However, this leads to significant permanent defects in the T cell repertoire, which may have unintended adverse outcomes. Additionally, T cells post-ASCT may not be metabolically adequate for the production of CAR-T cells, nor respond to checkpoint blockade therapies. DisclosuresQuach:Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sanofi Genzyme: Research Funding; Janssen Cilag: Consultancy. Harrison:Janssen-Cilag: Other: Scientific advisory board. Prince:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Identification of Tipifarnib Sensitivity Biomarkers in T-Cell Tumor Cell Lines

BackgroundT-cell leukemia and lymphoma (TCL) is a very aggressive and heterogeneous group of hematological malignancies with poor prognosis and inadequate response to current therapies. The standard first-line treatments have resulted in unsatisfactory patient outcomes and unfortunately, targeted treatments are still at a preliminary phase. The RAS/MAPK pathway is crucial for TCR signaling of T-cells and it is deregulated in TCL. We aimed to determine the therapeutic value of farnesyl transferase inhibitor (FTIs) tipifarnib in TCL cell lines. Effects on cell viability, apoptosis, cell cycle and gene expression were evaluated. Tipifarnib blocks the localization of some farnesylated proteins, including some RAS family members, to cellular membranes, thereby inhibiting their activation. Tipifarnib is a potent and specific FTI with prominent anti-proliferative effects.MethodsWe tested tipifarnib in 18 T-cell leukemia and 4 T-cell lymphoma cell lines for in vitro sensitivity and for biomarker discovery, both genomic and immunohistochemical. We selected cell lines with available genomic data from COSMIC, CCLE or generated by our group. The MAPK, NFAT, NFKB and JAK/STAT pathways were tested by immunohistochemistry analysis under basal conditions. The range of drug concentrations to perform IC50 analysis was established between 0-10,000 nM (ten points), using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) following manufacturer´s instructions at 0h, 48h and 96h. All experiments were done in sextuplet and all numerical data were expressed as the average of the values ± the standard error of the mean. IC50 analyses were performed with GraphPad Prism v5. Clinically-relevant drug sensitivity was defined as IC50 <100nM at 96h (defined as sensitive). RNAseq was performed in 4 sensitive cell lines with tipifarnib and DMSO (vehicle) in three biological replicates at 72h. Differential gene expression (DE) analysis was carried out under these two conditions. Pathway analysis was done using Gene Set Enrichment Analysis (GSEA). Induction of apoptosis and cell viability were tested by flow cytometry in sensitive cell lines.Results59.1% (n=13) of cell lines were sensitive to tipifarnib at concentrations which are readily achievable in the clinic. RAS, RAS-guanine nucleotide exchange factors (GEFs) and RAS-GTPase activating proteins (GAPs) genes were mutated in 45.5%, 50% and 27.3%, of cell lines respectively, but their mutational status was not associated with drug sensitivity. We found TP53 and DNMT3 mutated in 80% and 33.3% in sensitive cell lines respectively, and in 63.6% and 0% in resistant cell lines. The mutational state of NOTCH1 was associated with tipifarnib sensitivity; 66.7% in sensitive cell lines versus 9.1% in resistant cell lines (p=0.005). ERK activation (MAPK pathway) was significantly associated with drug sensitivity (p=0.046). Conversely, RelB (NFκB pathway) was associated with drug resistance (p=0.007). GSEA analysis showed that tipifarnib downregulated cell cycle, protein localization to membranes, metabolism, and ribosome and mitochondrial activity. The effect of tipifarnib on cell cycle progression and apoptosis was validated by flow cytometry assays. Tipifarnib decreased cellular viability, increased apoptosis and blocked cell cycle progression in G1 phase. GSEA of molecular pathways regulated by tipifarnib identified downregulation of MYC targets and mTOR and MAPK pathways. MYC is a target of NOTCH1, mutated in sensitive cases and associated with tipifarnib sensitivity.ConclusionsIn vitro response to tipifarnib in T-cell lymphoma and leukemia cell lines can be predicted using several biomarkers. The mutational NOTCH1 status, together with p-ERK (MAPK activation) were associated with increased sensitivity while RelB expression was associated with resistance. DisclosuresGualberto:Kura Oncology: Employment, Equity Ownership. Piris:Takeda: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Kura: Honoraria.

Cellular metabolism constrains innate immune responses in early human ontogeny

Pathogen immune responses are profoundly attenuated in fetuses and premature infants, yet the mechanisms underlying this developmental immaturity remain unclear. Here we show transcriptomic, metabolic and polysome profiling and find that monocytes isolated from infants born early in gestation display perturbations in PPAR-γ-regulated metabolic pathways, limited glycolytic capacity and reduced ribosomal activity. These metabolic changes are linked to a lack of translation of most cytokines and of MALT1 signalosome genes essential to respond to the neonatal pathogen Candida. In contrast, they have little impact on house-keeping phagocytosis functions. Transcriptome analyses further indicate a role for mTOR and its putative negative regulator DNA Damage Inducible Transcript 4-Like in regulating these metabolic constraints. Our results provide a molecular basis for the broad susceptibility to multiple pathogens in these infants, and suggest that the fetal immune system is metabolically programmed to avoid energetically costly, dispensable and potentially harmful immune responses during ontogeny.

Translating the Game: Ribosomes as Active Players.

Ribosomes have been long considered as executors of the translational program. The fact that ribosomes can control the translation of specific mRNAs or entire cellular programs is often neglected. Ribosomopathies, inherited diseases with mutations in ribosomal factors, show tissue specific defects and cancer predisposition. Studies of ribosomopathies have paved the way to the concept that ribosomes may control translation of specific mRNAs. Studies in Drosophila and mice support the existence of heterogeneous ribosomes that differentially translate mRNAs to coordinate cellular programs. Recent studies have now shown that ribosomal activity is not only a critical regulator of growth but also of metabolism. For instance, glycolysis and mitochondrial function have been found to be affected by ribosomal availability. Also, ATP levels drop in models of ribosomopathies. We discuss findings highlighting the relevance of ribosome heterogeneity in physiological and pathological conditions, as well as the possibility that in rate-limiting situations, ribosomes may favor some translational programs. We discuss the effects of ribosome heterogeneity on cellular metabolism, tumorigenesis and aging. We speculate a scenario in which ribosomes are not only executors of a metabolic program but act as modulators.

Active presynaptic ribosomes in the mammalian brain, and altered transmitter release after protein synthesis inhibition.

Presynaptic neuronal activity requires the localization of thousands of proteins that are typically synthesized in the soma and transported to nerve terminals. Local translation for some dendritic proteins occurs, but local translation in mammalian presynaptic nerve terminals is difficult to demonstrate. Here, we show an essential ribosomal component, 5.8S rRNA, at a glutamatergic nerve terminal in the mammalian brain. We also show active translation in nerve terminals, in situ, in brain slices demonstrating ongoing presynaptic protein synthesis in the mammalian brain. Shortly after inhibiting translation, the presynaptic terminal exhibits increased spontaneous release, an increased paired pulse ratio, an increased vesicle replenishment rate during stimulation trains, and a reduced initial probability of release. The rise and decay rates of postsynaptic responses were not affected. We conclude that ongoing protein synthesis can limit excessive vesicle release which reduces the vesicle replenishment rate, thus conserving the energy required for maintaining synaptic transmission.

Structures of translationally inactive mammalian ribosomes

The cellular levels and activities of ribosomes directly regulate gene expression during numerous physiological processes. The mechanisms that globally repress translation are incompletely understood. Here, we use electron cryomicroscopy to analyze inactive ribosomes isolated from mammalian reticulocytes, the penultimate stage of red blood cell differentiation. We identify two types of ribosomes that are translationally repressed by protein interactions. The first comprises ribosomes sequestered with elongation factor 2 (eEF2) by SERPINE mRNA binding protein 1 (SERBP1) occupying the ribosomal mRNA entrance channel. The second type are translationally repressed by a novel ribosome-binding protein, interferon-related developmental regulator 2 (IFRD2), which spans the P and E sites and inserts a C-terminal helix into the mRNA exit channel to preclude translation. IFRD2 binds ribosomes with a tRNA occupying a noncanonical binding site, the 'Z site', on the ribosome. These structures provide functional insights into how ribosomal interactions may suppress translation to regulate gene expression.

The polyproline-motif of S6K2: eIF5A translational dependence and importance for protein-protein interactions.

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2∆Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

Active Ribosome Profiling with RiboLace

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both invitro and invivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

Detection and quantification of ricin-mediated 28S ribosomal depurination by digital droplet PCR

Ricin acts to damage cells by producing a site of depurination in 28S ribosomal RNA. This depurination results in ribosome inactivation which inhibits protein synthesis and ultimately leads to cell death. We have developed a multiplexed digital droplet polymerase chain reaction assay that enables the objective measurement of toxin activity through quantitation of depurinated 28S rRNA molecules. This assay demonstrates the first use of digital PCR technology to measure ribotoxin-mediated damage. Depurination events were detected in ricin-treated lung cell cultures as early as 1 h, and within 9 h of exposure the maximum ribosomal damage of 70% was reached and was sustained for at least 24 h post-exposure.

RNA-seq-based monitoring of gene expression changes of viable but non-culturable state of Vibrio cholerae induced by cold seawater.

Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the acute diarrheal disease cholera. Entry into a viable but non-culturable (VBNC) state is a survival strategy by which V. cholerae withstands natural stresses and is important for the transition between the aquatic and host environments during the V. cholerae life cycle. In this study, the formation of VBNC V. cholerae induced by cold seawater exposure was investigated using RNA sequencing (RNA-seq). The analysis revealed that the expression of 1420 genes was changed on VBNC state formation. In the VBNC cells, genes related to biofilm formation, chitin utilization and stress responses were upregulated, whereas those related to cell division, morphology and ribosomal activity were mainly downregulated. The concurrent acquisition of a carbon source and the arrest of cell division in cells with low metabolic activity help bacteria increase their resistance to unfavourable environments. Moreover, two transcriptional regulators, SlmA and MetJ, were found to play roles in both VBNC formation and intestinal colonization, suggesting that some genes may function in both processes. This acquired knowledge will improve our understanding of the molecular mechanisms of stress tolerance and may help control future cholera infections and outbreaks.