Ribosomal stress activates eEF2K-eEF2 pathway causing translation elongation inhibition and recruitment of Terminal Oligopyrimidine (TOP) mRNAs on polysomes

The synthesis of adequate amounts of ribosomes is an essential task for the cell. It is therefore not surprising that regulatory circuits exist to organize the synthesis of ribosomal components. It has been shown that defect in ribosome biogenesis (ribosomal stress) induces apoptosis or cell cycle arrest through activation of the tumor suppressor p53. This mechanism is thought to be implicated in the pathophysiology of a group of genetic diseases such as Diamond Blackfan Anemia which are called ribosomopathies. We have identified an additional response to ribosomal stress that includes the activation of eukaryotic translation elongation factor 2 kinase with a consequent inhibition of translation elongation. This leads to a translational reprogramming in the cell that involves the structurally defined group of messengers called terminal oligopyrimidine (TOP) mRNAs which encode ribosomal proteins and translation factors. In fact, while general protein synthesis is decreased by the impairment of elongation, TOP mRNAs are recruited on polysomes causing a relative increase in the synthesis of TOP mRNA-encoded proteins compared to other proteins. Therefore, in response to ribosomal stress, there is a change in the translation pattern of the cell which may help restore a sufficient level of ribosomes

Intralobar pulmonary sequestration in an adult female patient mimicking asthma: a case report.

Pulmonary sequestration (PS) is a rare congenital broncho-pulmonary malformation. The main feature of this disease is that partial lung tissues separate from the main lung during the embryonic period, receiving blood supply from systemic circulation arteries. Pathogenesis of PS is not clear, and categorized into congenital and acquired PS. We report a case of a 38 year old woman smoker with medical history characterized by difficult to treat asthma with frequent exacerbations and infections since childhood. CT scan showed a partial PS of left lower lobe, supplied by an abnormal artery arising from supradiaphragmatic aortic diverticulum. Surgical treatment through a lung sequestrectomy and laterobasal segment resection was performed

Depletion of ribosomal protein S19 causes a reduction of rRNA synthesis

Ribosome biogenesis plays key roles in cell growth by providing increased capacity for protein synthesis. It requires coordinated production of ribosomal proteins (RP) and ribosomal RNA (rRNA), including the processing of the latter. Here, we show that, the depletion of RPS19 causes a reduction of rRNA synthesis in cell lines of both erythroid and non-erythroid origin. A similar effect is observed upon depletion of RPS6 or RPL11. The deficiency of RPS19 does not alter the stability of rRNA, but instead leads to an inhibition of RNA Polymerase I (Pol I) activity. In fact, results of nuclear run-on assays and ChIP experiments show that association of Pol I with the rRNA gene is reduced in RPS19-depleted cells. The phosphorylation of three known regulators of Pol I, CDK2, AKT and AMPK, is altered during ribosomal stress and could be involved in the observed downregulation. Finally, RNA from patients with Diamond Blackfan Anemia (DBA), shows, on average, a lower level of 47S precursor. This indicates that inhibition of rRNA synthesis could be one of the molecular alterations at the basis of DBA.Giada Juli, Angelo Gismondi, Valentina Monteleone, Sara Caldarola, Valentina Iadevaia, Anna Aspesi, Irma Dianzani, Christopher G. Proud, Fabrizio Loren

H-Bond Self-Assembly: Folding versus Duplex Formation

Linear oligomers equipped with complementary H-bond donor (D) and acceptor (A) sites can interact via intermolecular H-bonds to form duplexes or fold via intramolecular H-bonds. These competing equilibria have been quantified using NMR titration and dilution experiments for seven systems featuring different recognition sites and backbones. For all seven architectures, duplex formation is observed for homo-sequence 2-mers (AA·DD) where there are no competing folding equilibria. The corresponding hetero-sequence AD 2-mers also form duplexes, but the observed self-association constants are strongly affected by folding equilibria in the monomeric states. When the backbone is flexible (five or more rotatable bonds separating the recognition sites), intramolecular H-bonding is favored, and the folded state is highly populated. For these systems, the stability of the AD·AD duplex is 1-2 orders of magnitude lower than that of the corresponding AA·DD duplex. However, for three architectures which have more rigid backbones (fewer than five rotatable bonds), intramolecular interactions are not observed, and folding does not compete with duplex formation. These systems are promising candidates for the development of longer, mixed-sequence synthetic information molecules that show sequence-selective duplex formation.We thank the Engineering and Physical Sciences Research Council (EP/J008044/2) and European Research Council (ERC-2012-AdG 320539-duplex) for funding

Biological Insights into the Expression of Translation Initiation Factors from Recombinant CHOK1SV Cell Lines and their Relationship to Enhanced Productivity

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAb) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop; eukaryotic initiation factors (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A) binding protein (PABP) 1 and PABP interacting protein 1 (PAIP1) across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilise knowledge of the amounts of these factors to build predictive models for, and use cluster analysis to identify, high-producing cell lines. This study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity

Generation of ribosome imprinted polymers for sensitive detection of translational responses

Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of “smart materials”, namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity. Furthermore, ribosome imprinted polymers enabled the sensitive measurement of an mRNA translational regulatory event, requiring 1,000-fold less cells than current methodologies. These results provide first evidence for the suitability of MIPs to selectively recover ribonucleoprotein complexes such as ribosomes, founding a novel means for sensitive detection of gene regulation

Molecular networks of human muscle adaptation to exercise and age

Physical activity and molecular ageing presumably interact to precipitate musculoskeletal decline in humans with age. Herein, we have delineated molecular networks for these two major components of sarcopenic risk using multiple independent clinical cohorts. We generated genome-wide transcript profiles from individuals (n = 44) who then undertook 20 weeks of supervised resistance-exercise training (RET). Expectedly, our subjects exhibited a marked range of hypertrophic responses (3% to +28%), and when applying Ingenuity Pathway Analysis (IPA) up-stream analysis to ~580 genes that co-varied with gain in lean mass, we identified rapamycin (mTOR) signaling associating with growth (P = 1.4×10−30). Paradoxically, those displaying most hypertrophy exhibited an inhibited mTOR activation signature, including the striking down-regulation of 70 rRNAs. Differential analysis found networks mimicking developmental processes (activated all-trans-retinoic acid (ATRA, Z-score = 4.5; P = 6×10−13) and inhibited aryl-hydrocarbon receptor signaling (AhR, Z-score = −2.3; P = 3×10−7)) with RET. Intriguingly, as ATRA and AhR gene-sets were also a feature of endurance exercise training (EET), they appear to represent “generic” physical activity responsive gene-networks. For age, we found that differential gene-expression methods do not produce consistent molecular differences between young versus old individuals. Instead, utilizing two independent cohorts (n = 45 and n = 52), with a continuum of subject ages (18–78 y), the first reproducible set of age-related transcripts in human muscle was identified. This analysis identified ~500 genes highly enriched in post-transcriptional processes (P = 1×10−6) and with negligible links to the aforementioned generic exercise regulated gene-sets and some overlap with ribosomal genes. The RNA signatures from multiple compounds all targeting serotonin, DNA topoisomerase antagonism, and RXR activation were significantly related to the muscle age-related genes. Finally, a number of specific chromosomal loci, including 1q12 and 13q21, contributed by more than chance to the age-related gene list (P = 0.01–0.005), implying possible epigenetic events. We conclude that human muscle age-related molecular processes appear distinct from the processes regulated by those of physical activity

Disease-Associated Mutations That Alter the RNA Structural Ensemble

Genome-wide association studies (GWAS) often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs) from the Human Gene Mutation Database (HGMD) that map to the untranslated regions (UTRs) of a gene. Rather than using minimum free energy approaches (e.g. mFold), we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, β-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD), and Hypertension), we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5′ UTRs of FTL and RB1) SNP–induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a “RiboSNitch,” that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble

Computational Modeling and Analysis of Insulin Induced Eukaryotic Translation Initiation

Insulin, the primary hormone regulating the level of glucose in the bloodstream, modulates a variety of cellular and enzymatic processes in normal and diseased cells. Insulin signals are processed by a complex network of biochemical interactions which ultimately induce gene expression programs or other processes such as translation initiation. Surprisingly, despite the wealth of literature on insulin signaling, the relative importance of the components linking insulin with translation initiation remains unclear. We addressed this question by developing and interrogating a family of mathematical models of insulin induced translation initiation. The insulin network was modeled using mass-action kinetics within an ordinary differential equation (ODE) framework. A family of model parameters was estimated, starting from an initial best fit parameter set, using 24 experimental data sets taken from literature. The residual between model simulations and each of the experimental constraints were simultaneously minimized using multiobjective optimization. Interrogation of the model population, using sensitivity and robustness analysis, identified an insulin-dependent switch that controlled translation initiation. Our analysis suggested that without insulin, a balance between the pro-initiation activity of the GTP-binding protein Rheb and anti-initiation activity of PTEN controlled basal initiation. On the other hand, in the presence of insulin a combination of PI3K and Rheb activity controlled inducible initiation, where PI3K was only critical in the presence of insulin. Other well known regulatory mechanisms governing insulin action, for example IRS-1 negative feedback, modulated the relative importance of PI3K and Rheb but did not fundamentally change the signal flow