Paralogous translation factors target distinct mRNAs to differentially regulate tolerance to oxidative stress in yeast.

Translation initiation factor 4G (eIF4G) is an integral component of the eIF4F complex which is key to translation initiation for most eukaryotic mRNAs. Many eIF4G isoforms have been described in diverse eukaryotic organisms but we currently have a poor understanding of their functional roles and whether they regulate translation in an mRNA specific manner. The yeast Saccharomyces cerevisiae expresses two eIF4G isoforms, eIF4G1 and eIF4G2, that have previously been considered as functionally redundant with any phenotypic differences arising due to alteration in eIF4G expression levels. Using homogenic strains that express eIF4G1 or eIF4G2 as the sole eIF4G isoforms at comparable expression levels to total eIF4G, we show that eIF4G1 is specifically required to mediate the translational response to oxidative stress. eIF4G1 binds the mRNA cap and remains associated with actively translating ribosomes during oxidative stress conditions and we use quantitative proteomics to show that eIF4G1 promotes oxidative stress-specific proteome changes. eIF4G1, but not eIF4G2, binds the Slf1 LARP protein which appears to mediate the eIF4G1-dependent translational response to oxidative stress. We show similar isoform specific roles for eIF4G in human cells suggesting convergent evolution of multiple eIF4G isoforms offers significant advantages especially where translation must continue under stress conditions.

The pleiotropic roles of eIF5A in cellular life and its therapeutic potential in cancer.

Protein synthesis is dysregulated in the majority of cancers and this process therefore provides a good therapeutic target. Many novel anti-cancer agents are directed to target the initiation stage of translation, however, translation elongation also holds great potential as a therapeutic target. The elongation factor eIF5A that assists the formation of peptidyl bonds during the elongation process is of considerable interest in this regard. Overexpression of eIF5A has been linked with the development of a variety of cancers and inhibitors of the molecule have been proposed for anti-cancer clinical applications. eIF5A is the only protein in the cell that contains the post-translational modification hypusine. Hypusination is a two-step enzymatic process catalysed by the Deoxyhypusine Synthase (DHPS) and Deoxyhypusine Hydroxylase (DOHH). In addition, eIF5A can be acetylated by p300/CBP-associated factor (PCAF) which leads to translocation of the protein to the nucleus and its deactivation. In addition to the nucleus, eIF5A has been found in the mitochondria and the endoplasmic reticulum (ER) with eIF5A localisation related to function from regulation of mitochondrial activity and apoptosis to maintenance of ER integrity and control of the unfolded protein response (UPR). Given the pleiotropic functions of eIF5A and by extension the hypusination enzymes, this system is being considered as a target for a range of cancers including multiple myeloma, B-Cell lymphoma, and neuroblastoma. In this review, we explore the role of eIF5A and discuss the therapeutic strategies that are currently developing both in the pre- and the clinical stage.

Aberrant protein synthesis and cancer development: The role of canonical eukaryotic initiation, elongation and termination factors in tumorigenesis.

In tumourigenesis, oncogenes or dysregulated tumour suppressor genes alter the canonical translation machinery leading to a reprogramming of the translatome that, in turn, promotes the translation of selected mRNAs encoding proteins involved in proliferation and metastasis. It is therefore unsurprising that abnormal expression levels and activities of eukaryotic initiation factors (eIFs), elongation factors (eEFs) or termination factors (eRFs) are associated with poor outcome for patients with a wide range of cancers. In this review we discuss how RNA binding proteins (RBPs) within the canonical translation factor machinery are dysregulated in cancers and how targeting such proteins is leading to new therapeutic avenues.

The pathogenesis of mesothelioma is driven by a dysregulated translatome.

Malignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no 'druggable' driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease.

Control of translation elongation in health and disease.

Regulation of protein synthesis makes a major contribution to post-transcriptional control pathways. During disease, or under stress, cells initiate processes to reprogramme protein synthesis and thus orchestrate the appropriate cellular response. Recent data show that the elongation stage of protein synthesis is a key regulatory node for translational control in health and disease. There is a complex set of factors that individually affect the overall rate of elongation and, for the most part, these influence either transfer RNA (tRNA)- and eukaryotic elongation factor 1A (eEF1A)-dependent codon decoding, and/or elongation factor 2 (eEF2)-dependent ribosome translocation along the mRNA. Decoding speeds depend on the relative abundance of each tRNA, the cognate:near-cognate tRNA ratios and the degree of tRNA modification, whereas eEF2-dependent ribosome translocation is negatively regulated by phosphorylation on threonine-56 by eEF2 kinase. Additional factors that contribute to the control of the elongation rate include epigenetic modification of the mRNA, coding sequence variation and the expression of eIF5A, which stimulates peptide bond formation between proline residues. Importantly, dysregulation of elongation control is central to disease mechanisms in both tumorigenesis and neurodegeneration, making the individual key steps in this process attractive therapeutic targets. Here, we discuss the relative contribution of individual components of the translational apparatus (e.g. tRNAs, elongation factors and their modifiers) to the overall control of translation elongation and how their dysregulation contributes towards disease processes.

The cell stress response: extreme times call for post-transcriptional measures.

Following cell stress, a wide range of molecular pathways are initiated to orchestrate the stress response and enable adaptation to an environmental or intracellular perturbation. The post-transcriptional regulation strategies adopted during the stress response result in a substantial reorganization of gene expression, designed to prepare the cell for either acclimatization or programmed death, depending on the nature and intensity of the stress. Fundamental to the stress response is a rapid repression of global protein synthesis, commonly mediated by phosphorylation of translation initiation factor eIF2α. Recent structural and biochemical information have added unprecedented detail to our understanding of the molecular mechanisms underlying this regulation. During protein synthesis inhibition, the translation of stress-specific mRNAs is nonetheless enhanced, often through the interaction between RNA-binding proteins and specific RNA regulatory elements. Recent studies investigating the unfolded protein response (UPR) provide some important insights into how posttranscriptional events are spatially and temporally fine-tuned in order to elicit the most appropriate response and to coordinate the transition from an early, acute stage into the chronic state of adaptation. Importantly, cancer cells are known to hi-jack adaptive stress response pathways, particularly the UPR, to survive and proliferate in the unfavorable tumor environment. In this review, we consider the implications of recent research into stress-dependent post-transcriptional regulation and make the case for the exploration of the stress response as a strategy to identify novel targets in the development of cancer therapies. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution Translation > Translation Mechanisms > Translation Regulation.

Correction to: The mTOR-S6 kinase pathway promotes stress granule assembly.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The mTOR-S6 kinase pathway promotes stress granule assembly.

Stress granules are cytoplasmic mRNA-protein complexes that form upon the inhibition of translation initiation and promote cell survival in response to environmental insults. However, they are often associated with pathologies, including neurodegeneration and cancer, and changes in their dynamics are implicated in ageing. Here we show that the mTOR effector kinases S6 kinase 1 (S6K1) and S6 kinase 2 (S6K2) localise to stress granules in human cells and are required for their assembly and maintenance after mild oxidative stress. The roles of S6K1 and S6K2 are distinct, with S6K1 having a more significant role in the formation of stress granules via the regulation of eIF2α phosphorylation, while S6K2 is important for their persistence. In C. elegans, the S6 kinase orthologue RSKS-1 promotes the assembly of stress granules and its loss of function sensitises the nematodes to stress-induced death. This study identifies S6 kinases as regulators of stress granule dynamics and provides a novel link between mTOR signalling, translation inhibition and survival.

Ribonucleoprotein bodies are phased in.

Intracellular compartments are necessary for the regulation of many biochemical processes that ensure cell survival, growth and proliferation. Compartmentalisation is commonly achieved in organelles with defined lipid membranes, such as mitochondria, endoplasmic reticulum or the Golgi apparatus. While these organelles are responsible for many localised biochemical processes, recent evidence points to another class of compartments that lack membrane boundaries. The structure and content of these bodies depend on their function and subcellular localisation, but they mainly incorporate proteins and RNA. Examples of these ribonucleoprotein bodies (RNPBs) include eukaryotic mRNA processing bodies (P-bodies) and stress granules (SGs). While most of these structures have been widely studied for their capacity to bind, store and process mRNAs under different conditions, their biological functions and physical properties are poorly understood. Recent intriguing data suggest that liquid-liquid phase separation (LLPS) represents an important mechanism seeding the formation and defining the function of RNPBs. In this review, we discuss how LLPS is transforming our ideas about the biological functions of SGs and P-bodies and their link to diseases.

Development of a Novel Experimental In Vitro Model of Isothiocyanate-induced Apoptosis in Human Malignant Melanoma Cells.

BACKGROUND: Isothiocyanates are constituents of cruciferous vegetables which have been associated with reduced cancer risk partially through their ability to induce apoptosis in malignant cells including melanoma. MATERIALS AND METHODS: We have utilized human malignant melanoma (A375), epidermoid carcinoma (A431) and immortalized keratinocyte (HaCaT) cells exposed to various isothiocyanates, under different experimental conditions. RESULTS: An experimental in vitro model utilizing low isothiocyanate concentrations (0.1-5 µM for 48 h with all treatments being refreshed after 24h) was shown to be (i) most efficient in exerting an anti-cancer effect when compared to higher concentrations (5-100 µM for 24 or 48 h added as a single bolus) and (ii) specific to A375 cells while A431 and HaCaT cells remained unaffected. Such effect involved the activation of several caspases including (iii) initiator caspases 8, 9, 4 (indicating the involvement of intrinsic, extrinsic and endoplasmic reticulum-based pathways) and (iv) effector caspases 3, 7 and 6. CONCLUSION: Utilization of low isothiocyanate concentrations (under the conditions described herein) exerts an anti-cancer effect specific to human malignant melanoma cells thus providing a therapeutic basis for their utilization in management of the disease.

A Novel Role of Silibinin as a Putative Epigenetic Modulator in Human Prostate Carcinoma.

Silibinin, extracted from milk thistle (Silybum marianum L.), has exhibited considerable preclinical activity against prostate carcinoma. Its antitumor and chemopreventive activities have been associated with diverse effects on cell cycle, apoptosis, and receptor-dependent mitogenic signaling pathways. Here we hypothesized that silibinin's pleiotropic effects may reflect its interference with epigenetic mechanisms in human prostate cancer cells. More specifically, we have demonstrated that silibinin reduces gene expression levels of the Polycomb Repressive Complex 2 (PRC2) members Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste Homolog 12 (SUZ12), and Embryonic Ectoderm Development (EED) in DU145 and PC3 human prostate cancer cells, as evidenced by Real Time Polymerase Chain Reaction (RT-PCR). Furthermore immunoblot and immunofluorescence analysis revealed that silibinin-mediated reduction of EZH2 levels was accompanied by an increase in trimethylation of histone H3 on lysine (Κ)-27 residue (H3K27me3) levels and that such response was, in part, dependent on decreased expression levels of phosphorylated Akt (ser473) (pAkt) and phosphorylated EZH2 (ser21) (pEZH2). Additionally silibinin exerted other epigenetic effects involving an increase in total DNA methyltransferase (DNMT) activity while it decreased histone deacetylases 1-2 (HDACs1-2) expression levels. We conclude that silibinin induces epigenetic alterations in human prostate cancer cells, suggesting that subsequent disruptions of central processes in chromatin conformation may account for some of its diverse anticancer effects.