Translation Stress Response Research Articles

Lost in translation: How neurons cope with tRNA decoding.

Post-transcriptional tRNA modifications contribute to the decoding efficiency of tRNAs by supporting codon recognition and tRNA stability. Recent work shows that the molecular and cellular functions of tRNA modifications and tRNA-modifying-enzymes are linked to brain development and neurological disorders. Lack of these modifications affects codon recognition and decoding rate, promoting protein aggregation and translational stress response pathways with toxic consequences to the cell. In this review, we discuss the peculiarity of local translation in neurons, suggesting a role for fine-tuning of translation performed by tRNA modifications. We provide several examples of tRNA modifications involved in physiology and pathology of the nervous system, highlighting their effects on protein translation and discussing underlying mechanisms, like the unfolded protein response (UPR), ribosome quality control (RQC), and no-go mRNA decay (NGD), which could affect neuronal functions. We aim to deepen the understanding of the roles of tRNA modifications and the coordination of these modifications with the protein translation machinery in the nervous system.

Global translational control by the transcriptional repressor TrcR in the filamentous cyanobacterium Anabaena sp. PCC 7120

Transcriptional and translational regulations are important mechanisms for cell adaptation to environmental conditions. In addition to house-keeping tRNAs, the genome of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena) has a long tRNA operon (trn operon) consisting of 26 genes present on a megaplasmid. The trn operon is repressed under standard culture conditions, but is activated under translational stress in the presence of antibiotics targeting translation. Using the toxic amino acid analog β-N-methylamino-L-alanine (BMAA) as a tool, we isolated and characterized several BMAA-resistance mutants from Anabaena, and identified one gene of unknown function, all0854, named as trcR, encoding a transcription factor belonging to the ribbon-helix-helix (RHH) family. We provide evidence that TrcR represses the expression of the trn operon and is thus the missing link between the trn operon and translational stress response. TrcR represses the expression of several other genes involved in translational control, and is required for maintaining translational fidelity. TrcR, as well as its binding sites, are highly conserved in cyanobacteria, and its functions represent an important mechanism for the coupling of the transcriptional and translational regulations in cyanobacteria.

Inhibitors of eIF4G1–eIF1 uncover its regulatory role of ER/UPR stress-response genes independent of eIF2α-phosphorylation

During translation initiation, eIF4G1 dynamically interacts with eIF4E and eIF1. While the role of eIF4E-eIF4G1 is well established, the regulatory functions of eIF4G1-eIF1 are poorly understood. Here, we report the identification of the eIF4G1-eIF1 inhibitors i14G1-10 and i14G1-12. i14G1s directly bind eIF4G1 and inhibit translation invitro and in the cell, and their effects on translation are dependent on eIF4G1 levels. Translatome analyses revealed that i14G1s mimic eIF1 and eIF4G1 perturbations on the stringency of start codon selection and the opposing roles of eIF1-eIF4G1 in scanning-dependent and scanning-independent short 5' untranslated region (UTR) translation. Remarkably, i14G1s activate ER/unfolded protein response (UPR) stress-response genes via enhanced ribosome loading, elevated 5'UTR translation at near-cognate AUGs, and unexpected concomitant up-regulation of coding-region translation. These effects are, at least in part, independent of eIF2α-phosphorylation. Interestingly, eIF4G1-eIF1 interaction itself is negatively regulated by ER stress and mTOR inhibition. Thus, i14G1s uncover an unknown mechanism of ER/UPR translational stress response and are valuable research tools and potential drugs against diseases exhibiting dysregulated translation.

Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy

The microtubule-associated protein tau oligomerizes, but the actions of oligomeric tau (oTau) are unknown. We have used Cry2-based optogenetics to induce tau oligomers (oTau-c). Optical induction of oTau-c elicits tau phosphorylation, aggregation, and a translational stress response that includes stress granules and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau-c. The association of HNRNPA2B1 with endogenous oTau was verified in neurons, animal models, and human Alzheimer brain tissues. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine (m6A) modified RNA transcripts. Knockdown of HNRNPA2B1 prevents oTau or oTau-c from associating with m6A or from reducing protein synthesis and reduces oTau-induced neurodegeneration. Levels of m6A and the m6A-oTau-HNRNPA2B1 complex are increased up to 5-fold in the brains of Alzheimer subjects and P301S tau mice. These results reveal a complex containing oTau, HNRNPA2B1, and m6A that contributes to the integrated stress response of oTau.

Hibernation factors directly block ribonucleases from entering the ribosome in response to starvation.

Ribosome hibernation is a universal translation stress response found in bacteria as well as plant plastids. The term was coined almost two decades ago and despite recent insights including detailed cryo-EM structures, the physiological role and underlying molecular mechanism of ribosome hibernation has remained unclear. Here, we demonstrate that Escherichia coli hibernation factors RMF, HPF and RaiA (HFs) concurrently confer ribosome hibernation. In response to carbon starvation and resulting growth arrest, we observe that HFs protect ribosomes at the initial stage of starvation. Consistently, a deletion mutant lacking all three factors (ΔHF) is severely inhibited in regrowth from starvation. ΔHF cells increasingly accumulate 70S ribosomes harbouring fragmented rRNA, while rRNA in wild-type 100S dimers is intact. RNA fragmentation is observed to specifically occur at HF-associated sites in 16S rRNA of assembled 70S ribosomes. Surprisingly, degradation of the 16S rRNA 3′-end is decreased in cells lacking conserved endoribonuclease YbeY and exoribonuclease RNase R suggesting that HFs directly block these ribonucleases from accessing target sites in the ribosome.

HNRNPA2B1 Mediates the Association of Oligomeric Tau with N <sup>6</sup>-Methyladenosine and Neurodegeneration

We combine optogenetics with proteomics for time-resolved identification of tau oligomer (oTau) protein interactomes. Optically induced tau oligomerization is elicits a translational stress response exhibiting increased cytoplasmic localization of N6-methyladenosine (m6A) modified transcripts that partially co-localize with oTau, stress granules composed of HNRNPA2B1 and TIA1 but not G3BP1. Appearance of the cytoplasmic m6A complex occurs with a concomitant reduction in protein synthesis. Persistent tau oligomerization elicits neurodegeneration. We show that cytoplasmic translocation of m6A labeled transcripts is a prominent neuropathological feature of tauopathy in P301S tau mice and Alzheimer’s disease brain. Characterization of the oTau proteome identifies the RNA binding protein, HNRNPA2B1 as a principle target of oTau. Knockdown of HNRNPA2B1 abrogates the association of oTau with m6A-labeled transcripts, preventing the oTau-induced reduction in protein synthesis and reducing neurodegeneration. These results suggest that oTau elicits stress and toxicity through binding to HNRNPA2B1, which functions as a reader of m6A-labeled transcripts.

Tau Oligomers and Fibrils Exhibit Differential Patterns of Seeding and Association With RNA Binding Proteins.

Tau aggregates are pleiotropic and exhibit differences in conformation, structure, and size. These aggregates develop endogenously but are also propagated among neurons in disease. We explored the actions of two distinct types of tau aggregates, tau oligomers (oTau) and tau fibrils (fTau), using a seeding assay in primary neuron cultures expressing human 4R0N tau. We find that oTau and fTau elicit distinct patterns of tau inclusions in the neurons and distinct molecular interactions. The exogenously applied oTau and fTau both clear rapidly from the neurons, but both also seed intracellular inclusions composed of endogenously produced tau. The two types of seeds elicit differential dose–response relationships for seed uptake and the number of resulting intracellular inclusions. Immunocytochemical studies show that co-localization with RNA binding proteins associated with stress granules is much greater for seeds composed of oTau than fTau. Conversely, co-localization with p62/SQSTM1 and thioflavine S is much greater for fTau than oTau. These results suggest that oTau seeds inclusions that modulate the translational stress response and are physiologically active, whereas fTau seeds inclusions that are fibrillar and shunted to the autolysosomal cascade.

A Complex Containing HNRNPA2B1 and N <sup>6</sup>-Methyladenosine Modified Transcripts Mediates Actions of Toxic Tau Oligomers

The microtubule associated protein tau oligomerizes in response to stress and disease, but the function of oligomeric tau (oTau) and the ultimate mechanisms of toxicity are unknown. To gain insights, we have now used Cry2-based optogenetics to induce tau oligomers (oTau-c) in neuronal cultures. oTau-c can seed tau aggregation and biochemical fractionatesin a manner similar to oTau. Optical induction of oTau elicits a translational stress response that includes cytoplasmic translocation of the TIA1, abundant stress granules(SGs) and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau. Imaging and immunoprecipitation verify the HNRNPA2B1 association with endogenous oTau in neurons, animal models and human Alzheimer brain tissue. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine modified transcripts (m6A). Knockdown of HNRNPA2B1 prevents oTau from associating with m6A, prevents oTau-induced reductions in protein synthesis and reduces oTau-induced toxicity. Finally, we show striking increases in m6A-oTau and -HNRNPA2B1 complexes in brains of Alzheimer subjects and P301S tau mice. These results reveal a novel complex containing oTau, HNRNPA2B1 and m6A that contributes to the integrated stress response of oTau.

The Pathophysiology of Tau and Stress Granules in Disease.

This chapter discusses the relationship between tau, RNA binding proteins and stress granules, which exhibit an intimate bidirectional relationship affecting the functions of both tau and the translational stress response. We describe how tau becomes hyperphosphorylated and oligomerized as part of an endogenous mechanism to promote the translational stress response through interaction with RNA binding proteins. Prior studies demonstrate that dysfunction of RNA binding proteins biology is sufficient to cause neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia. Emerging evidence indicates that tau-mediated neurodegeneration also occurs through a mechanism that is mediated by RNA binding proteins and the translational stress response. Discovery of the role of RNA metabolism in tauopathy opens a wide variety of novel therapeutic approaches. Multiple studies have already shown that approaches reducing the levels of selected RNA binding proteins or inhibiting the translational stress response can intervene in the pathophysiology of motoneuron diseases. Emerging studies show that reducing the levels of selected RNA binding proteins or inhibiting the translational stress response also reduces neurodegeneration in models of tauopathy and Aβ mediated degeneration. The combined impact of these studies indicate that RNA binding proteins and RNA metabolism represent a valuable new frontier for the investigation and treatment tauopathies.

PI3K\u03b4 activates E2F1 synthesis in response to mRNA translation stress

The c-myc oncogene stimulates ribosomal biogenesis and protein synthesis to promote cellular growth. However, the pathway by which cells sense and restore dysfunctional mRNA translation and how this is linked to cell proliferation and growth is not known. We here show that mRNA translation stress in cis triggered by the gly-ala repeat sequence of Epstein–Barr virus (EBV)-encoded EBNA1, results in PI3Kδ-dependent induction of E2F1 mRNA translation with the consequent activation of c-Myc and cell proliferation. Treatment with a specific PI3Kδ inhibitor Idelalisib (CAL-101) suppresses E2F1 and c-Myc levels and causes cell death in EBNA1-induced B cell lymphomas. Suppression of PI3Kδ prevents E2F1 activation also in non-EBV-infected cells. These data illustrate an mRNA translation stress–response pathway for E2F1 activation that is exploited by EBV to promote cell growth and proliferation, offering new strategies to treat EBV-carrying cancers.

Reducing the RNA binding protein TIA1 protects against tau-mediated neurodegeneration in vivo.

SUMMARYEmerging studies suggest a role for tau in regulating the biology of RNA binding proteins (RBPs). We now show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo protects against neurodegeneration and prolongs survival in transgenic P301S tau mice. Biochemical fractionation shows co-enrichment and co-localization of tau oligomers and RBPs in transgenic P301S tau mice. Reducing TIA1 decreases the number and size of granules co-localizing with stress granule markers. Decreasing TIA1 also inhibits the accumulation of tau oligomers at the expense of increasing neurofibrillary tangles (NFTs). Despite the increase in NFTs, TIA1 reduction increases neuronal survival and rescues behavioral deficits and lifespan. These data provide in vivo evidence that TIA1 plays a key role in mediating toxicity, and further suggest that RBPs direct the pathway of tau aggregation and the resulting neurodegeneration. We propose a paradigm in which dysfunction of the translational stress response leads to tau-mediated pathology.

PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer.

Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR.

TAU-INDUCED NEURODEGENERATION IS MEDIATED BY RNA BINDING PROTEINS

The classic model of protein aggregation posits that aggregation of tau occurs as a random event. However, this model over-looks emerging the biology of RNA binding proteins (RBPs) and tau in producing RNA granules, which control RNA metabolism. RBPs readily aggregate through a tightly regulated mechanism involving liquid-liquid phase separation. Mutations in RBPs linked to neurological diseases increase the propensity of RBPs to aggregate, which can become irreversible with time. Recent cell culture studies show that the association of tau with RBPs promotes tau aggregation, while reducing T cell intracellular antigen 1 (TIA1), a core nucleating RBP, protects against tau-mediated toxicity. PS19 P301S tau mice were crossed with TIA1-/- to generate P301S tau x TIA1+/- and P301S tau x TIA1 -/- mice. Reducing TIA1 levels in vivo strongly protects against neurodegeneration and prolongs survival. PS19 x TIA1+/- mice exhibit a 26% increase in lifespan. The protection is associated with rescue of deficits in RNA metabolism, including a striking decrease in the number of cytoplasmic stress granules, a corresponding increase in nuclear TIA1, and rescue of dysfunctional RNA splicing. There is a correspondingly increased binding of tau to microtubules and microtubule stabilization. The mice show strongly improved neuronal/brain function, including protection against synaptic and neuronal loss, and improved spatial working memory. Remarkably, this protection occurs despite increased neurofibrillary tangles. Biochemical studies show that PS19 x TIA1+/- mice exhibit a striking reduction of soluble tau oligomers with a corresponding increase in levels of insoluble tau fibrils. These results point to dysfunction of RBPs and RNA metabolism as important factors contributing to tauopathy. We propose a new paradigm in which tau pathology is (in part) driven by the role of tau in the translational stress response, which is mediated by the interaction with RBPs and RNA granules. Excess aggregation of RBPs produces pathological stress granules, which promote tau aggregation and cause RNA metabolism dysfunction. Tau / RBPs interactions appear to enhance the accumulation of tau in an oligomeric state that is more toxic than fibrillar tau. These results suggest that RBPs could provide compelling targets for therapeutic development.

Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

Highly Predictive Reprogramming of tRNA Modifications Is Linked to Selective Expression of Codon-Biased Genes.

Cells respond to stress by controlling gene expression at several levels, with little known about the role of translation. Here, we demonstrate a coordinated translational stress response system involving stress-specific reprogramming of tRNA wobble modifications that leads to selective translation of codon-biased mRNAs representing different classes of critical response proteins. In budding yeast exposed to four oxidants and five alkylating agents, tRNA modification patterns accurately distinguished among chemically similar stressors, with 14 modified ribonucleosides forming the basis for a data-driven model that predicts toxicant chemistry with >80% sensitivity and specificity. tRNA modification subpatterns also distinguish SN1 from SN2 alkylating agents, with SN2-induced increases in m3C in tRNA mechanistically linked to selective translation of threonine-rich membrane proteins from genes enriched with ACC and ACT degenerate codons for threonine. These results establish tRNA modifications as predictive biomarkers of exposure and illustrate a novel regulatory mechanism for translational control of cell stress response.

The bacterial translation stress response.

Throughout their life, bacteria need to sense and respond to environmental stress. Thus, such stress responses can require dramatic cellular reprogramming, both at the transcriptional as well as the translational level. This review focuses on the protein factors that interact with the bacterial translational apparatus to respond to and cope with different types of environmental stress. For example, the stringent factor RelA interacts with the ribosome to generate ppGpp under nutrient deprivation, whereas a variety of factors have been identified that bind to the ribosome under unfavorable growth conditions to shut-down (RelE, pY, RMF, HPF and EttA) or re-program (MazF, EF4 and BipA) translation. Additional factors have been identified that rescue ribosomes stalled due to stress-induced mRNA truncation (tmRNA, ArfA, ArfB), translation of unfavorable protein sequences (EF-P), heat shock-induced subunit dissociation (Hsp15), or antibiotic inhibition (TetM, FusB). Understanding the mechanism of how the bacterial cell responds to stress will not only provide fundamental insight into translation regulation, but will also be an important step to identifying new targets for the development of novel antimicrobial agents.

The VCP/p97 system at a glance: connecting cellular function to disease pathogenesis.

The ATPase valosin-containing protein (VCP)/p97 has emerged as a central and important element of the ubiquitin system. Together with a network of cofactors, it regulates an ever-expanding range of processes that stretch into almost every aspect of cellular physiology. Its main role in proteostasis and key functions in signaling pathways are of relevance to degenerative diseases and genomic stability. In this Cell Science at a Glance and the accompanying poster, we give a brief overview of this complex system. In addition, we discuss the pathogenic basis for VCP/p97-associated diseases and then highlight in more detail new exciting links to the translational stress response and RNA biology that further underscore the significance of the VCP/p97 system.