Translation factor and RNA binding protein mRNA interactomes support broader RNA regulons for posttranscriptional control.

The regulation of translation provides a rapid and direct mechanism to modulate the cellular proteome. In eukaryotes, an established model for the recruitment of ribosomes to mRNA depends upon a set of conserved translation initiation factors. Nevertheless, how cells orchestrate and define the selection of individual mRNAs for translation, as opposed to other potential cytosolic fates, is poorly understood. We have previously found significant variation in the interaction between individual mRNAs and an array of translation initiation factors. Indeed, mRNAs can be separated into different classes based upon these interactions to provide a framework for understanding different modes of translation initiation. Here, we extend this approach to include new mRNA interaction profiles for additional proteins involved in shaping the cytoplasmic fate of mRNAs. This work defines a set of seven mRNA clusters, based on their interaction profiles with 12 factors involved in translation and/or RNA binding. The mRNA clusters share both physical and functional characteristics to provide a rationale for the interaction profiles. Moreover, a comparison with mRNA interaction profiles from a host of RNA binding proteins suggests that there are defined patterns in the interactions of functionally related mRNAs. Therefore, this work defines global cytoplasmic mRNA binding modules that likely coordinate the synthesis of functionally related proteins.

Interaction of the La-related protein Slf1 with colliding ribosomes maintains translation of oxidative-stress responsive mRNAs.

In response to oxidative stress cells reprogram gene expression to enhance levels of antioxidant enzymes and promote survival. In Saccharomyces cerevisiae the polysome-interacting La-related proteins (LARPs) Slf1 and Sro9 aid adaptation of protein synthesis during stress by undetermined means. To gain insight in their mechanisms of action in stress responses, we determined LARP mRNA binding positions in stressed and unstressed cells. Both proteins bind within coding regions of stress-regulated antioxidant enzyme and other highly translated mRNAs in both optimal and stressed conditions. LARP interaction sites are framed and enriched with ribosome footprints suggesting ribosome-LARP-mRNA complexes are identified. Although stress-induced translation of antioxidant enzyme mRNAs is attenuated in slf1Δ, these mRNAs remain on polysomes. Focusing further on Slf1, we find it binds to both monosomes and disomes following RNase treatment. slf1Δ reduces disome enrichment during stress and alters programmed ribosome frameshifting rates. We propose that Slf1 is a ribosome-associated translational modulator that stabilises stalled/collided ribosomes, prevents ribosome frameshifting and so promotes translation of a set of highly-translated mRNAs that together facilitate cell survival and adaptation to stress.

Cytosolic aspartate aminotransferase moonlights as a ribosome-binding modulator of Gcn2 activity during oxidative stress.

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors, and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of PE in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2α phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.

Quantifying the Binding of Fluorescently Labeled Guanine Nucleotides and Initiator tRNA to Eukaryotic Translation Initiation Factor 2.

The translation initiation factor eIF2 is critical for protein synthesis initiation, and its regulation is central to the integrated stress response (ISR). eIF2 is a G protein, and the activity is regulated by its GDP or GTP-binding status, such that only GTP-bound eIF2 has high affinity for initiator methionyl tRNA. In the ISR, regulatory signaling reduces the availability of eIF2-GTP and so downregulates protein synthesis initiation in cells. Fluorescence spectroscopy can be used as an analytical tool to study protein-ligand interactions in vitro. Here we describe methods to purify eIF2 and assays of its activity, employing analogs of GDP, GTP, and methionyl initiator tRNA ligands to accurately measure their binding affinities.

GTP binding to translation factor eIF2B stimulates its guanine nucleotide exchange activity.

eIF2B is the guanine nucleotide exchange factor (GEF) required for cytoplasmic protein synthesis initiation in eukaryotes and its regulation within the integrated stress response (ISR). It activates its partner factor eIF2, thereby promoting translation initiation. Here we provide evidence through biochemical and genetic approaches that eIF2B can bind directly to GTP and this can enhance its rate of GEF activity toward eIF2-GDP in vitro. GTP binds to a subcomplex of the eIF2Bγ and ε subunits. The eIF2Bγ amino-terminal domain shares structural homology with hexose sugar phosphate pyrophosphorylase enzymes that bind specific nucleotides. A K66R mutation in eIF2Bγ is especially sensitive to guanine or GTP in a range of functional assays. Taken together, our data suggest eIF2Bγ may act as a sensor of purine nucleotide availability and thus modulate eIF2B activity and protein synthesis in response to fluctuations in cellular nucleotide levels.

Overlapping regions of Caf20 mediate its interactions with the mRNA-5'cap-binding protein eIF4E and with ribosomes.

By interacting with the mRNA 5' cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells. Caf20 is a member of the family of proteins found across eukaryotes termed 4E-BPs, which compete with eIF4G for interaction with eIF4E. Caf20 independently interacts with ribosomes. Thus, Caf20 modulates the mRNA selection process via poorly understood mechanisms. Here we performed unbiased mutagenesis across Caf20 to characterise which regions of Caf20 are important for interaction with eIF4E and with ribosomes. Caf20 binding to eIF4E is entirely dependent on a canonical motif shared with other 4E-BPs. However, binding to ribosomes is weakened by mutations throughout the protein, suggesting an extended binding interface that partially overlaps with the eIF4E-interaction region. By using chemical crosslinking, we identify a potential ribosome interaction region on the ribosome surface that spans both small and large subunits and is close to a known interaction site of eIF3. The function of ribosome binding by Caf20 remains unclear.

Impaired eIF5A function causes a Mendelian disorder that is partially rescued in model systems by spermidine.

The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.

The structural basis of translational control by eIF2 phosphorylation.

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.

Translational regulation in response to stress in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae must dynamically alter the composition of its proteome in order to respond to diverse stresses. The reprogramming of gene expression during stress typically involves initial global repression of protein synthesis, accompanied by the activation of stress-responsive mRNAs through both translational and transcriptional responses. The ability of specific mRNAs to counter the global translational repression is therefore crucial to the overall response to stress. Here we summarize the major repressive mechanisms and discuss mechanisms of translational activation in response to different stresses in S. cerevisiae. Taken together, a wide range of studies indicate that multiple elements act in concert to bring about appropriate translational responses. These include regulatory elements within mRNAs, altered mRNA interactions with RNA-binding proteins and the specialization of ribosomes that each contribute towards regulating protein expression to suit the changing environmental conditions.

Regulation of translation initiation factor eIF2B at the hub of the integrated stress response.

Phosphorylation of the translation initiation factor eIF2 is one of the most widely used and well-studied mechanisms cells use to respond to diverse cellular stresses. Known as the integrated stress response (ISR), the control pathway uses modulation of protein synthesis to reprogram gene expression and restore homeostasis. Here the current knowledge of the molecular mechanisms of eIF2 activation and its control by phosphorylation at a single-conserved phosphorylation site, serine 51 are discussed with a major focus on the regulatory roles of eIF2B and eIF5 where a current molecular view of ISR control of eIF2B activity is presented. How genetic disorders affect eIF2 or eIF2B is discussed, as are syndromes where excess signaling through the ISR is a component. Finally, studies into the action of recently identified compounds that modulate the ISR in experimental systems are discussed; these suggest that eIF2B is a potential therapeutic target for a wide range of conditions. This article is categorized under: Translation > Translation Regulation.

Archetypal transcriptional blocks underpin yeast gene regulation in response to changes in growth conditions.

The transcriptional responses of yeast cells to diverse stresses typically include gene activation and repression. Specific stress defense, citric acid cycle and oxidative phosphorylation genes are activated, whereas protein synthesis genes are coordinately repressed. This view was achieved from comparative transcriptomic experiments delineating sets of genes whose expression greatly changed with specific stresses. Less attention has been paid to the biological significance of 1) consistent, albeit modest, changes in RNA levels across multiple conditions, and 2) the global gene expression correlations observed when comparing numerous genome-wide studies. To address this, we performed a meta-analysis of 1379 microarray-based experiments in yeast, and identified 1388 blocks of RNAs whose expression changes correlate across multiple and diverse conditions. Many of these blocks represent sets of functionally-related RNAs that act in a coordinated fashion under normal and stress conditions, and map to global cell defense and growth responses. Subsequently, we used the blocks to analyze novel RNA-seq experiments, demonstrating their utility and confirming the conclusions drawn from the meta-analysis. Our results provide a new framework for understanding the biological significance of changes in gene expression: 'archetypal' transcriptional blocks that are regulated in a concerted fashion in response to external stimuli.

Protein Synthesis Initiation in Eukaryotic Cells.

This review summarizes our current understanding of the major pathway for the initiation phase of protein synthesis in eukaryotic cells, with a focus on recent advances. We describe the major scanning or messenger RNA (mRNA) m7G cap-dependent mechanism, which is a highly coordinated and stepwise regulated process that requires the combined action of at least 12 distinct translation factors with initiator transfer RNA (tRNA), ribosomes, and mRNAs. We limit our review to studies involving either mammalian or budding yeast cells and factors, as these represent the two best-studied experimental systems, and only include a reference to other organisms where particular insight has been gained. We close with a brief description of what we feel are some of the major unknowns in eukaryotic initiation.

Dynamic changes in eIF4F-mRNA interactions revealed by global analyses of environmental stress responses.

BACKGROUND: Translation factors eIF4E and eIF4G form eIF4F, which interacts with the messenger RNA (mRNA) 5' cap to promote ribosome recruitment and translation initiation. Variations in the association of eIF4F with individual mRNAs likely contribute to differences in translation initiation frequencies between mRNAs. As translation initiation is globally reprogrammed by environmental stresses, we were interested in determining whether eIF4F interactions with individual mRNAs are reprogrammed and how this may contribute to global environmental stress responses. RESULTS: Using a tagged-factor protein capture and RNA-sequencing (RNA-seq) approach, we have assessed how mRNA associations with eIF4E, eIF4G1 and eIF4G2 change globally in response to three defined stresses that each cause a rapid attenuation of protein synthesis: oxidative stress induced by hydrogen peroxide and nutrient stresses caused by amino acid or glucose withdrawal. We find that acute stress leads to dynamic and unexpected changes in eIF4F-mRNA interactions that are shared among each factor and across the stresses imposed. eIF4F-mRNA interactions stabilised by stress are predominantly associated with translational repression, while more actively initiating mRNAs become relatively depleted for eIF4F. Simultaneously, other mRNAs are insulated from these stress-induced changes in eIF4F association. CONCLUSION: Dynamic eIF4F-mRNA interaction changes are part of a coordinated early translational control response shared across environmental stresses. Our data are compatible with a model where multiple mRNA closed-loop complexes form with differing stability. Hence, unexpectedly, in the absence of other stabilising factors, rapid translation initiation on mRNAs correlates with less stable eIF4F interactions.

Fail-safe control of translation initiation by dissociation of eIF2α phosphorylated ternary complexes.

Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and contributes to the cellular integrated stress response. Key to controlling the activity of eIF2 are translation factors eIF2B and eIF5, thought to primarily function with eIF2-GDP and TC respectively. Using a steady-state kinetics approach with purified proteins we demonstrate that eIF2B binds to eIF2 with equal affinity irrespective of the presence or absence of competing guanine nucleotides. We show that eIF2B can compete with Met-tRNAi for eIF2-GTP and can destabilize TC. When TC is formed with unphosphorylated eIF2, eIF5 can out-compete eIF2B to stabilize TC/eIF5 complexes. However when TC/eIF5 is formed with phosphorylated eIF2, eIF2B outcompetes eIF5 and destabilizes TC. These data uncover competition between eIF2B and eIF5 for TC and identify that phosphorylated eIF2-GTP translation initiation intermediate complexes can be inhibited by eIF2B.

Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma.

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

eIF2ß is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control.

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2ß that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2ß mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2ß mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2ß acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation.

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae.

In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs.

Enhanced translation initiation factor 4G levels correlate with production levels of monoclonal antibodies in recombinant CHO cell lines.

Using cells to manufacture protein-based therapeutics or biopharmaceuticals is a rapidly expanding industrial activity. Chinese hamster ovary (CHO) cells are the most frequently used mammalian host-expression system for the manufacture of biopharmaceuticals. Over the past ∼30 years academic and industrial researchers have studied cell expression characteristics with aims to improve product yield, quality, scalability and reproducibility. Although many steps in the gene expression and secretion pathways have been optimized, little attention has been paid to optimizing protein synthesis factors and regulators during this process. A new study in Biochemical Journal by Mead et al., provides a first systematic study of several protein synthesis factors and finds that the expression level of eIF4G1 correlates with the level of recombinant protein expressed in cultures. Optimizing levels and activities of protein synthesis factors may help to enhance recombinant protein expression of biopharmaceuticals.

Integrated multi-omics analyses reveal the pleiotropic nature of the control of gene expression by Puf3p.

The PUF family of RNA-binding proteins regulate gene expression post-transcriptionally. Saccharomyces cerevisiae Puf3p is characterised as binding nuclear-encoded mRNAs specifying mitochondrial proteins. Extensive studies of its regulation of COX17 demonstrate its role in mRNA decay. Using integrated genome-wide approaches we define an expanded set of Puf3p target mRNAs and quantitatively assessed the global impact of loss of PUF3 on gene expression using mRNA and polysome profiling and quantitative proteomics. In agreement with prior studies, our sequencing of affinity-purified Puf3-TAP associated mRNAs (RIP-seq) identified mRNAs encoding mitochondrially-targeted proteins. Additionally, we also found 720 new mRNA targets that predominantly encode proteins that enter the nucleus. Comparing transcript levels in wild-type and puf3∆ cells revealed that only a small fraction of mRNA levels alter, suggesting Puf3p determines mRNA stability for only a limited subset of its target mRNAs. Finally, proteomic and translatomic studies suggest that loss of Puf3p has widespread, but modest, impact on mRNA translation. Taken together our integrated multi-omics data point to multiple classes of Puf3p targets, which display coherent post-transcriptional regulatory properties and suggest Puf3p plays a broad, but nuanced, role in the fine-tuning of gene expression.

The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation.

Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5'cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3' UTR motif. Caf20p binds all tested motif-containing 3' UTRs. Caf20p and the 3'UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3'UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E.