NRF2 Promotes Tumor Maintenance by Modulating mRNA Translation in Pancreatic Cancer.

Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nfe2l2/Nrf2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.

Mitochondrial Threonyl-tRNA Synthetase TARS2 Is Required for Threonine-Sensitive mTORC1 Activation.

Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.

microRNA-induced translational control of antiviral immunity by the cap-binding protein 4EHP.

Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-ß production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-ß induction, prompting a negative feedback regulatory mechanism that represses IFN-ß expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.

The SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression.

Viruses use microRNAs (miRNAs) to impair the host antiviral response and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. miRNAs inhibit translation initiation of their target mRNAs by recruiting the GIGYF2-4EHP (or EIF4E2) translation repressor complex to the mRNA 5'-cap structure. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifnb1 mRNA that encodes the cytokine interferon ß, and thereby impairs the host antiviral response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with argonaute 2 (AGO2), the core component of the miRNA-induced silencing complex (miRISC), via GIGYF2 and enhances the translational repression mediated by natural miRNA-binding sites in the 3' untranslated region of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program by co-opting the host miRNA-mediated silencing machinery.

TPL2 kinase expression is regulated by the p38γ/p38δ-dependent association of aconitase-1 with TPL2 mRNA.

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ-/-) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 3' Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 3'UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ-/- cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology.

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-ß. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-ß production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

Mnk1/2 kinases regulate memory and autism-related behaviours via Syngap1.

MAPK interacting protein kinases 1 and 2 (Mnk1/2) regulate a plethora of functions, presumably via phosphorylation of their best characterized substrate, eukaryotic translation initiation factor 4E (eIF4E) on Ser209. Here, we show that, whereas deletion of Mnk1/2 (Mnk double knockout) impairs synaptic plasticity and memory in mice, ablation of phospho-eIF4E (Ser209) does not affect these processes, suggesting that Mnk1/2 possess additional downstream effectors in the brain. Translational profiling revealed only a small overlap between the Mnk1/2- and phospho-eIF4E(Ser209)-regulated translatome. We identified the synaptic Ras GTPase activating protein 1 (Syngap1), encoded by a syndromic autism gene, as a downstream target of Mnk1 because Syngap1 immunoprecipitated with Mnk1 and showed reduced phosphorylation (S788) in Mnk double knockout mice. Knockdown of Syngap1 reversed memory deficits in Mnk double knockout mice and pharmacological inhibition of Mnks rescued autism-related phenotypes in Syngap1+/- mice. Thus, Syngap1 is a downstream effector of Mnk1, and the Mnks-Syngap1 axis regulates memory formation and autism-related behaviours.

Mechanisms of impairment of interferon production by SARS-CoV-2.

Interferons (IFNs) are crucial components of the cellular innate immune response to viral infections. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a remarkable capacity to suppress the host IFN production to benefit viral replication and spread. Thus far, of the 28 known virus-encoded proteins, 16 have been found to impair the host's innate immune system at various levels ranging from detection and signaling to transcriptional and post-transcriptional regulation of expression of the components of the cellular antiviral response. Additionally, there is evidence that the viral genome encodes non-protein-coding microRNA-like elements that could also target IFN-stimulated genes. In this brief review, we summarise the current state of knowledge regarding the factors and mechanisms by which SARS-CoV-2 impairs the production of IFNs and thereby dampens the host's innate antiviral immune response.

Clinical, neuroradiological, and molecular characterization of mitochondrial threonyl-tRNA-synthetase (TARS2)-related disorder.

PURPOSE: Biallelic variants in TARS2, encoding the mitochondrial threonyl-tRNA-synthetase, have been reported in a small group of individuals displaying a neurodevelopmental phenotype but with limited neuroradiological data and insufficient evidence for causality of the variants. METHODS: Exome or genome sequencing was carried out in 15 families. Clinical and neuroradiological evaluation was performed for all affected individuals, including review of 10 previously reported individuals. The pathogenicity of TARS2 variants was evaluated using in vitro assays and a zebrafish model. RESULTS: We report 18 new individuals harboring biallelic TARS2 variants. Phenotypically, these individuals show developmental delay/intellectual disability, regression, cerebellar and cerebral atrophy, basal ganglia signal alterations, hypotonia, cerebellar signs, and increased blood lactate. In vitro studies showed that variants within the TARS2301-381 region had decreased binding to Rag GTPases, likely impairing mTORC1 activity. The zebrafish model recapitulated key features of the human phenotype and unraveled dysregulation of downstream targets of mTORC1 signaling. Functional testing of the variants confirmed the pathogenicity in a zebrafish model. CONCLUSION: We define the clinico-radiological spectrum of TARS2-related mitochondrial disease, unveil the likely involvement of the mTORC1 signaling pathway as a distinct molecular mechanism, and establish a TARS2 zebrafish model as an important tool to study variant pathogenicity.

microRNA-mediated translation repression through GYF-1 and IFE-4 in C. elegans development.

microRNA (miRNA)-mediated gene silencing is enacted through the recruitment of effector proteins that direct translational repression or degradation of mRNA targets, but the relative importance of their activities for animal development remains unknown. Our concerted proteomic surveys identified the uncharacterized GYF-domain encoding protein GYF-1 and its direct interaction with IFE-4, the ortholog of the mammalian translation repressor 4EHP, as key miRNA effector proteins in Caenorhabditis elegans. Recruitment of GYF-1 protein to mRNA reporters in vitro or in vivo leads to potent translation repression without affecting the poly(A) tail or impinging on mRNA stability. Loss of gyf-1 is synthetic lethal with hypomorphic alleles of embryonic miR-35-42 and larval (L4) let-7 miRNAs, which is phenocopied through engineered mutations in gyf-1 that abolish interaction with IFE-4. GYF-1/4EHP function is cascade-specific, as loss of gyf-1 had no noticeable impact on the functions of other miRNAs, including lin-4 and lsy-6. Overall, our findings reveal the first direct effector of miRNA-mediated translational repression in C. elegans and its physiological importance for the function of several, but likely not all miRNAs.

Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis.

Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.

Cap-binding protein 4EHP effects translation silencing by microRNAs.

MicroRNAs (miRNAs) play critical roles in a broad variety of biological processes by inhibiting translation initiation and by destabilizing target mRNAs. The CCR4-NOT complex effects miRNA-mediated silencing, at least in part through interactions with 4E-T (eIF4E transporter) protein, but the precise mechanism is unknown. Here we show that the cap-binding eIF4E-homologous protein 4EHP is an integral component of the miRNA-mediated silencing machinery. We demonstrate that the cap-binding activity of 4EHP contributes to the translational silencing by miRNAs through the CCR4-NOT complex. Our results show that 4EHP competes with eIF4E for binding to 4E-T, and this interaction increases the affinity of 4EHP for the cap. We propose a model wherein the 4E-T/4EHP interaction engenders a closed-loop mRNA conformation that blocks translational initiation of miRNA targets.

Loss of eIF4E Phosphorylation Engenders Depression-like Behaviors via Selective mRNA Translation.

The MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated kinase) pathway is a cardinal regulator of synaptic plasticity, learning, and memory in the hippocampus. One of major endpoints of this signaling cascade is the 5' mRNA cap binding protein eIF4E (eukaryotic Initiation Factor 4E), which is phosphorylated on Ser 209 by MNK (MAPK-interacting protein kinases) and controls mRNA translation. The precise role of phospho-eIF4E in the brain is yet to be determined. Herein, we demonstrate that ablation of eIF4E phosphorylation in male mice (4Eki mice) does not impair long-term spatial or contextual fear memory, or the late phase of LTP. Using unbiased translational profiling in mouse brain, we show that phospho-eIF4E differentially regulates the translation of a subset of mRNAs linked to inflammation, the extracellular matrix, pituitary hormones, and the serotonin pathway. Consequently, 4Eki male mice display exaggerated inflammatory responses and reduced levels of serotonin, concomitant with depression and anxiety-like behaviors. Remarkably, eIF4E phosphorylation is required for the chronic antidepressant action of the selective serotonin reuptake inhibitor fluoxetine. Finally, we propose a novel phospho-eIF4E-dependent translational control mechanism in the brain, via the GAIT complex (gamma IFN activated inhibitor of translation). In summary, our work proposes a novel translational control mechanism involved in the regulation of inflammation and depression, which could be exploited to design novel therapeutics.SIGNIFICANCE STATEMENT We demonstrate that downstream of the MAPK (mitogen-activated protein kinase) pathway, eukaryotic Initiation Factor 4E (eIF4E) Ser209 phosphorylation is not required for classical forms of hippocampal LTP and memory. We reveal a novel role for eIF4E phosphorylation in inflammatory responses and depression-like behaviors. eIF4E phosphorylation is required for the chronic action of antidepressants, such as fluoxetine in mice. These phenotypes are accompanied by selective translation of extracellular matrix, pituitary hormones, and serotonin pathway genes, in eIF4E phospho-mutant mice. We also describe a previously unidentified translational control mechanism in the brain, whereby eIF4E phosphorylation is required for inhibiting the translation of gamma IFN activated inhibitor of translation element-containing mRNAs. These findings can be used to design novel therapeutics for depression.

eIF2α phosphorylation controls thermal nociception.

A response to environmental stress is critical to alleviate cellular injury and maintain cellular homeostasis. Eukaryotic initiation factor 2 (eIF2) is a key integrator of cellular stress responses and an important regulator of mRNA translation. Diverse stress signals lead to the phosphorylation of the α subunit of eIF2 (Ser51), resulting in inhibition of global protein synthesis while promoting expression of proteins that mediate cell adaptation to stress. Here we report that eIF2α is instrumental in the control of noxious heat sensation. Mice with decreased eIF2α phosphorylation (eIF2α+/S51A) exhibit reduced responses to noxious heat. Pharmacological attenuation of eIF2α phosphorylation decreases thermal, but not mechanical, pain sensitivity, whereas increasing eIF2α phosphorylation has the opposite effect on thermal nociception. The impact of eIF2α phosphorylation (p-eIF2α) on thermal thresholds is dependent on the transient receptor potential vanilloid 1. Moreover, we show that induction of eIF2α phosphorylation in primary sensory neurons in a chronic inflammation pain model contributes to thermal hypersensitivity. Our results demonstrate that the cellular stress response pathway, mediated via p-eIF2α, represents a mechanism that could be used to alleviate pathological heat sensation.

Control of embryonic stem cell self-renewal and differentiation via coordinated alternative splicing and translation of YY2.

Translational control of gene expression plays a key role during the early phases of embryonic development. Here we describe a transcriptional regulator of mouse embryonic stem cells (mESCs), Yin-yang 2 (YY2), that is controlled by the translation inhibitors, Eukaryotic initiation factor 4E-binding proteins (4E-BPs). YY2 plays a critical role in regulating mESC functions through control of key pluripotency factors, including Octamer-binding protein 4 (Oct4) and Estrogen-related receptor-ß (Esrrb). Importantly, overexpression of YY2 directs the differentiation of mESCs into cardiovascular lineages. We show that the splicing regulator Polypyrimidine tract-binding protein 1 (PTBP1) promotes the retention of an intron in the 5'-UTR of Yy2 mRNA that confers sensitivity to 4E-BP-mediated translational suppression. Thus, we conclude that YY2 is a major regulator of mESC self-renewal and lineage commitment and document a multilayer regulatory mechanism that controls its expression.

Characterizing Cellular Responses During Oncolytic Maraba Virus Infection.

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) as being involved in the regulation of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

Pleiotropic function of SRY-related HMG box transcription factor 4 in regulation of tumorigenesis.

In addition to their critical roles in embryonic development, cell fate decision, and differentiation, members of Sox (Sry-related high-mobility group box) family of transcription factors including Sox4 have been implicated in various cancers. Multiple studies have revealed an increased expression along with specific oncogenic function of Sox4 in tumors, while others observed a reduced expression of Sox4 in different types of malignancies and suppression of tumor initiation or progression by this protein. More interestingly, the prognostic value of Sox4 is debated due to obvious differences between various reports as well as inconsistencies within specific studies. This review summarizes our current understanding of Sox4 expression pattern and its transcription-dependent, as well as transcription-independent, functions in tumor initiation or progression and its correlation with patient survival. We also discuss the existing discrepancies between different reports and their possible explanations.

Dysregulated ribosome quality control in human diseases.

Precise regulation of mRNA translation is of fundamental importance for maintaining homeostasis. Conversely, dysregulated general or transcript-specific translation, as well as abnormal translation events, have been linked to a multitude of diseases. However, driven by the misconception that the transient nature of mRNAs renders their abnormalities inconsequential, the importance of mechanisms that monitor the quality and fidelity of the translation process has been largely overlooked. In recent years, there has been a dramatic shift in this paradigm, evidenced by several seminal discoveries on the role of a key mechanism in monitoring the quality of mRNA translation - namely, Ribosome Quality Control (RQC) - in the maintenance of homeostasis and the prevention of diseases. Here, we will review recent advances in the field and emphasize the biological significance of the RQC mechanism, particularly its implications in human diseases.

JWA inhibits melanoma angiogenesis by suppressing ILK signaling and is an independent prognostic biomarker for melanoma.

Melanoma is the deadliest cutaneous malignancy because of its high incidence of metastasis. Melanoma growth and metastasis relies on sustained angiogenesis; therefore, inhibiting angiogenesis is a promising approach to treat metastatic melanoma. JWA is a novel microtubule-associated protein and our previous work revealed that JWA inhibited melanoma cell invasion and metastasis. However, the role of JWA in melanoma angiogenesis and the prognostic value are still unknown. Here, we report that JWA in melanoma cells significantly inhibited the tube formation of endothelial cells. In addition, JWA regulated integrin-linked kinase (ILK) through integrin αVß3 and such regulation was achieved through the transcription factor Sp1. Notably, both in vitro and in vivo angiogenesis assays revealed that JWA dramatically suppressed melanoma angiogenesis by inhibiting ILK signaling. Furthermore, we examined the expression of JWA protein in a large set of melanocytic lesions (n = 505) at different stages by tissue microarray and found an inverse correlation between JWA expression and melanoma progression (P = 5 × 10(-6)). Importantly, reduced JWA expression was correlated with a poorer overall, and disease-specific 5 year survival of patients (P = 0.001 and 0.007, respectively). Multivariate Cox regression analyses indicated that JWA was an independent prognostic marker for melanoma patients. Moreover, we found a significant negative correlation between JWA and ILK in melanoma biopsies, and their concomitant expression was closely correlated with melanoma patient survival (P = 0.004), further indicating the regulation of ILK expression by JWA is critical in melanoma. Taken together, our data highlight the function of JWA in melanoma angiogenesis and reveal the clinical prognostic value of JWA.

Regulation of p53 by ING family members in suppression of tumor initiation and progression.

The INhibitor of Growth (ING) family is an evolutionarily conserved set of proteins, implicated in suppression of initiation and progression of cancers in various tissues. They promote cell cycle arrest, cellular senescence and apoptosis, participate in stress responses, regulate DNA replication and DNA damage responses, and inhibit cancer cell migration, invasion, and angiogenesis of the tumors. At the molecular level, ING proteins are believed to participate in chromatin remodeling and transcriptional regulation of their target genes. However, the best known function of ING proteins is their cooperation with p53 tumor suppressor protein in tumor suppression. All major isoforms of ING family members can promote the transactivition of p53 and the majority of them are shown to directly interact with p53. In addition, ING proteins are thought to interact with and modulate the function of auxiliary members of p53 pathway, such as MDM2, ARF , p300, and p21, indicating their widespread involvement in the regulation and function of this prominent tumor suppressor pathway. It seems that p53 pathway is the main mechanism by which ING proteins exert their functions. Nevertheless, regulation of other pathways which are not relevant to p53, yet important for tumorigenesis such as TGF-ß and NF-κB, by ING proteins is also observed. This review summarizes the current understanding of the mutual interactions and cooperation between different members of ING family with p53 pathway and implications of this cooperation in the suppression of cancer initiation and progression.