Publications by authors named "Nahum Sonenberg"

360 Publications

Membrane-dependent relief of translation elongation arrest on pseudouridine- and N1-methyl-pseudouridine-modified mRNAs.

Nucleic Acids Res 2021 Dec 22. Epub 2021 Dec 22.

Department of Biochemistry, McGill University, Montréal, Québec H3A 1A3, Canada.

Expression of therapeutically important proteins has benefited dramatically from the advent of chemically modified mRNAs that feature decreased lability and immunogenicity. This had a momentous effect on the rapid development of COVID-19 mRNA vaccines. Incorporation of the naturally occurring pseudouridine (Ψ) or N1-methyl-pseudouridine (N1mΨ) into in vitro transcribed mRNAs prevents the activation of unwanted immune responses by blocking eIF2α phosphorylation, which inhibits translation. Here, we report that Ψs in luciferase (Luc) mRNA exacerbate translation pausing in nuclease-untreated rabbit reticulocyte lysate (uRRL) and promote the formation of high-order-ribosome structures. The major deceleration of elongation occurs at the Ψ-rich nucleotides 1294-1326 of Ψ-Luc mRNA and results in premature termination of translation. The impairment of translation is mainly due to the shortage of membranous components. Supplementing uRRL with canine microsomal membranes (CMMs) relaxes the impediments to ribosome movement, resolves collided ribosomes, and greatly enhances full-size luciferase production. CMMs also strongly stimulated an extremely inefficient translation of N1mΨ-Luc mRNA in uRRL. Evidence is presented that translational pausing can promote membrane recruitment of polysomes with nascent polypeptides that lack a signal sequence. Our results highlight an underappreciated role of membrane binding to polysomes in the prevention of ribosome collision and premature release of nascent polypeptides.
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http://dx.doi.org/10.1093/nar/gkab1241DOI Listing
December 2021

High-risk human papillomavirus-18 uses an mRNA sequence to synthesize oncoprotein E6 in tumors.

Proc Natl Acad Sci U S A 2021 10;118(41)

Unit of Biomedical Research on Cancer, Laboratory of Translation and Cancer, National Institute of Cancer (Instituto Nacional de Cancerología), Mexico City 14080, Mexico;

Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.
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http://dx.doi.org/10.1073/pnas.2108359118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8522272PMC
October 2021

Assessing eukaryotic initiation factor 4F subunit essentiality by CRISPR-induced gene ablation in the mouse.

Cell Mol Life Sci 2021 Oct 24;78(19-20):6709-6719. Epub 2021 Sep 24.

Department of Biochemistry, McGill University, Montreal, QC, H3G 1Y6, Canada.

Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphoma-prone Eμ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e/Eμ-Myc or Eif4a1/Eμ-Myc mice show increased sensitivity to the chemotherapeutic agent doxorubicin, in vivo. Our study reveals that eIF4A2 and eIF4G3 play non-essential roles in gene expression regulation during embryogenesis; whereas reductions in eIF4E or eIF4A1 levels are protective against tumor development in a murine Myc-driven lymphoma setting.
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http://dx.doi.org/10.1007/s00018-021-03940-5DOI Listing
October 2021

Lesch-Nyhan disease causes impaired energy metabolism and reduced developmental potential in midbrain dopaminergic cells.

Stem Cell Reports 2021 07 1;16(7):1749-1762. Epub 2021 Jul 1.

Psychiatric Genetics Group, McGill University, Montreal, QC, Canada; Department of Psychiatry, McGill University and Douglas Hospital Research Institute, 6875 LaSalle Boulevard, Frank Common Building, Room 2101.2, Montreal, QC H4H 1R3, Canada. Electronic address:

Mutations in HPRT1, a gene encoding a rate-limiting enzyme for purine salvage, cause Lesch-Nyhan disease which is characterized by self-injury and motor impairments. We leveraged stem cell and genetic engineering technologies to model the disease in isogenic and patient-derived forebrain and midbrain cell types. Dopaminergic progenitor cells deficient in HPRT showed decreased intensity of all developmental cell-fate markers measured. Metabolic analyses revealed significant loss of all purine derivatives, except hypoxanthine, and impaired glycolysis and oxidative phosphorylation. real-time glucose tracing demonstrated increased shunting to the pentose phosphate pathway for de novo purine synthesis at the expense of ATP production. Purine depletion in dopaminergic progenitor cells resulted in loss of RHEB, impairing mTORC1 activation. These data demonstrate dopaminergic-specific effects of purine salvage deficiency and unexpectedly reveal that dopaminergic progenitor cells are programmed to a high-energy state prior to higher energy demands of terminally differentiated cells.
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http://dx.doi.org/10.1016/j.stemcr.2021.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282463PMC
July 2021

mRNA translation is a therapeutic vulnerability necessary for bladder epithelial transformation.

JCI Insight 2021 06 8;6(11). Epub 2021 Jun 8.

Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Using genetically engineered mouse models, this work demonstrates that protein synthesis is essential for efficient urothelial cancer formation and growth but dispensable for bladder homeostasis. Through a candidate gene analysis for translation regulators implicated in this dependency, we discovered that phosphorylation of the translation initiation factor eIF4E at serine 209 is increased in both murine and human bladder cancer, and this phosphorylation corresponds with an increase in de novo protein synthesis. Employing an eIF4E serine 209 to alanine knock-in mutant mouse model, we show that this single posttranslational modification is critical for bladder cancer initiation and progression, despite having no impact on normal bladder tissue maintenance. Using murine and human models of advanced bladder cancer, we demonstrate that only tumors with high levels of eIF4E phosphorylation are therapeutically vulnerable to eFT508, the first clinical-grade inhibitor of MNK1 and MNK2, the upstream kinases of eIF4E. Our results show that phospho-eIF4E plays an important role in bladder cancer pathogenesis, and targeting its upstream kinases could be an effective therapeutic option for bladder cancer patients with high levels of eIF4E phosphorylation.
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http://dx.doi.org/10.1172/jci.insight.144920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262354PMC
June 2021

BAD regulates mammary gland morphogenesis by 4E-BP1-mediated control of localized translation in mouse and human models.

Nat Commun 2021 05 19;12(1):2939. Epub 2021 May 19.

Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.

Elucidation of non-canonical protein functions can identify novel tissue homeostasis pathways. Herein, we describe a role for the Bcl-2 family member BAD in postnatal mammary gland morphogenesis. In Bad knock-in mice, where BAD cannot undergo phosphorylation at 3 key serine residues, pubertal gland development is delayed due to aberrant tubulogenesis of the ductal epithelium. Proteomic and RPPA analyses identify that BAD regulates focal adhesions and the mRNA translation repressor, 4E-BP1. These results suggest that BAD modulates localized translation that drives focal adhesion maturation and cell motility. Consistent with this, cells within Bad organoids contain unstable protrusions with decreased compartmentalized mRNA translation and focal adhesions, and exhibit reduced cell migration and tubulogenesis. Critically, protrusion stability is rescued by 4E-BP1 depletion. Together our results confirm an unexpected role of BAD in controlling localized translation and cell migration during mammary gland development.
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http://dx.doi.org/10.1038/s41467-021-23269-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8134504PMC
May 2021

4E-BP2-dependent translation in cerebellar Purkinje cells controls spatial memory but not autism-like behaviors.

Cell Rep 2021 04;35(4):109036

Department of Anesthesia and Faculty of Dentistry, McGill University, Montreal, QC H3G 1Y6, Canada; Alan Edwards Centre for Research on Pain, McGill University, Montreal, QC H3A 0G1, Canada. Electronic address:

Recent studies have demonstrated that selective activation of mammalian target of rapamycin complex 1 (mTORC1) in the cerebellum by deletion of the mTORC1 upstream repressors TSC1 or phosphatase and tensin homolog (PTEN) in Purkinje cells (PCs) causes autism-like features and cognitive deficits. However, the molecular mechanisms by which overactivated mTORC1 in the cerebellum engenders these behaviors remain unknown. The eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) is a central translational repressor downstream of mTORC1. Here, we show that mice with selective ablation of 4E-BP2 in PCs display a reduced number of PCs, increased regularity of PC action potential firing, and deficits in motor learning. Surprisingly, although spatial memory is impaired in these mice, they exhibit normal social interaction and show no deficits in repetitive behavior. Our data suggest that, downstream of mTORC1/4E-BP2, there are distinct cerebellar mechanisms independently controlling social behavior and memory formation.
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http://dx.doi.org/10.1016/j.celrep.2021.109036DOI Listing
April 2021

4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold.

Proc Natl Acad Sci U S A 2021 04;118(15)

Department of Anesthesia and Faculty of Dentistry, McGill University, Montreal, QC H3A 2B4, Canada;

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as , , , , and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)-induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human mutation that enhances the activity of the PI3K-AKT pathway ( ) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.
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http://dx.doi.org/10.1073/pnas.2025522118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053921PMC
April 2021

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

Nucleic Acids Res 2021 05;49(9):4803-4815

Goodman Cancer Research Center, McGill University, Montréal H3G 1Y6, Canada.

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.
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http://dx.doi.org/10.1093/nar/gkab162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136787PMC
May 2021

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses.

J Clin Invest 2021 04;131(8)

Lady Davis Institute, Jewish General Hospital, Montréal, Quebec, Canada.

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.
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http://dx.doi.org/10.1172/JCI140752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262472PMC
April 2021

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

Mol Cell 2021 03 12;81(6):1187-1199.e5. Epub 2021 Feb 12.

Goodman Cancer Centre, Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada. Electronic address:

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-β production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-β induction, prompting a negative feedback regulatory mechanism that represses IFN-β 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.
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http://dx.doi.org/10.1016/j.molcel.2021.01.030DOI Listing
March 2021

Lysergic acid diethylamide (LSD) promotes social behavior through mTORC1 in the excitatory neurotransmission.

Proc Natl Acad Sci U S A 2021 02;118(5)

Neurobiological Psychiatry Unit, Department of Psychiatry, McGill University, Montreal, QC, Canada, H3A 1A1;

Clinical studies have reported that the psychedelic lysergic acid diethylamide (LSD) enhances empathy and social behavior (SB) in humans, but its mechanism of action remains elusive. Using a multidisciplinary approach including in vivo electrophysiology, optogenetics, behavioral paradigms, and molecular biology, the effects of LSD on SB and glutamatergic neurotransmission in the medial prefrontal cortex (mPFC) were studied in male mice. Acute LSD (30 μg/kg) injection failed to increase SB. However, repeated LSD (30 μg/kg, once a day, for 7 days) administration promotes SB, without eliciting antidepressant/anxiolytic-like effects. Optogenetic inhibition of mPFC excitatory neurons dramatically inhibits social interaction and nullifies the prosocial effect of LSD. LSD potentiates the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and 5-HT, but not -methyl-D-aspartate (NMDA) and 5-HT, synaptic responses in the mPFC and increases the phosphorylation of the serine-threonine protein kinases Akt and mTOR. In conditional knockout mice lacking Raptor (one of the structural components of the mTORC1 complex) in excitatory glutamatergic neurons ( ), the prosocial effects of LSD and the potentiation of 5-HT/AMPA synaptic responses were nullified, demonstrating that LSD requires the integrity of mTORC1 in excitatory neurons to promote SB. Conversely, in knockout mice lacking Raptor in GABAergic neurons of the mPFC ( ), LSD promotes SB. These results indicate that LSD selectively enhances SB by potentiating mPFC excitatory transmission through 5-HT/AMPA receptors and mTOR signaling. The activation of 5-HT/AMPA/mTORC1 in the mPFC by psychedelic drugs should be explored for the treatment of mental diseases with SB impairments such as autism spectrum disorder and social anxiety disorder.
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http://dx.doi.org/10.1073/pnas.2020705118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865169PMC
February 2021

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

Mol Cell 2021 01 18;81(2):398-407.e4. Epub 2020 Dec 18.

Goodman Cancer Research Centre, McGill University, Montreal, QC H3A 1A3, Canada; Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2020.11.036DOI Listing
January 2021

Antidepressant actions of ketamine engage cell-specific translation via eIF4E.

Nature 2021 02 16;590(7845):315-319. Epub 2020 Dec 16.

Department of Biochemistry and Goodman Cancer Centre, McGill University, Montreal, Quebec, Canada.

Effective pharmacotherapy for major depressive disorder remains a major challenge, as more than 30% of patients are resistant to the first line of treatment (selective serotonin reuptake inhibitors). Sub-anaesthetic doses of ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist, provide rapid and long-lasting antidepressant effects in these patients, but the molecular mechanism of these effects remains unclear. Ketamine has been proposed to exert its antidepressant effects through its metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK). The antidepressant effects of ketamine and (2R,6R)-HNK in rodents require activation of the mTORC1 kinase. mTORC1 controls various neuronal functions, particularly through cap-dependent initiation of mRNA translation via the phosphorylation and inactivation of eukaryotic initiation factor 4E-binding proteins (4E-BPs). Here we show that 4E-BP1 and 4E-BP2 are key effectors of the antidepressant activity of ketamine and (2R,6R)-HNK, and that ketamine-induced hippocampal synaptic plasticity depends on 4E-BP2 and, to a lesser extent, 4E-BP1. It has been hypothesized that ketamine activates mTORC1-4E-BP signalling in pyramidal excitatory cells of the cortex. To test this hypothesis, we studied the behavioural response to ketamine and (2R,6R)-HNK in mice lacking 4E-BPs in either excitatory or inhibitory neurons. The antidepressant activity of the drugs is mediated by 4E-BP2 in excitatory neurons, and 4E-BP1 and 4E-BP2 in inhibitory neurons. Notably, genetic deletion of 4E-BP2 in inhibitory neurons induced a reduction in baseline immobility in the forced swim test, mimicking an antidepressant effect. Deletion of 4E-BP2 specifically in inhibitory neurons also prevented the ketamine-induced increase in hippocampal excitatory neurotransmission, and this effect concurred with the inability of ketamine to induce a long-lasting decrease in inhibitory neurotransmission. Overall, our data show that 4E-BPs are central to the antidepressant activity of ketamine.
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http://dx.doi.org/10.1038/s41586-020-03047-0DOI Listing
February 2021

MNK Inhibition Sensitizes -Mutant Colorectal Cancer to mTORC1 Inhibition by Reducing eIF4E Phosphorylation and c-MYC Expression.

Cancer Discov 2021 05 16;11(5):1228-1247. Epub 2020 Dec 16.

Lifelong Health, South Australian Health and Medical Research Institute, North Terrace, Adelaide, South Australia, Australia.

-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for ∼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in -mutant colorectal cancer. Using -mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRAS models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple -mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in -mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611341PMC
May 2021

The multifaceted eukaryotic cap structure.

Wiley Interdiscip Rev RNA 2021 03 9;12(2):e1636. Epub 2020 Dec 9.

Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

The 5' cap structure is added onto RNA polymerase II transcripts soon after initiation of transcription and modulates several post-transcriptional regulatory events involved in RNA maturation. It is also required for stimulating translation initiation of many cytoplasmic mRNAs and serves to protect mRNAs from degradation. These functional properties of the cap are mediated by several cap binding proteins (CBPs) involved in nuclear and cytoplasmic gene expression steps. The role that CBPs play in gene regulation, as well as the biophysical nature by which they recognize the cap, is quite intricate. Differences in mechanisms of capping as well as nuances in cap recognition speak to the potential of targeting these processes for drug development. In this review, we focus on recent findings concerning the cap epitranscriptome, our understanding of cap binding by different CBPs, and explore therapeutic targeting of CBP-cap interaction. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > Capping and 5' End Modifications Translation > Translation Mechanisms.
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http://dx.doi.org/10.1002/wrna.1636DOI Listing
March 2021

The eIF4E homolog 4EHP (eIF4E2) regulates hippocampal long-term depression and impacts social behavior.

Mol Autism 2020 11 23;11(1):92. Epub 2020 Nov 23.

Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, QC, H3G 1Y6, Canada.

Background: The regulation of protein synthesis is a critical step in gene expression, and its dysfunction is implicated in autism spectrum disorder (ASD). The eIF4E homologous protein (4EHP, also termed eIF4E2) binds to the mRNA 5' cap to repress translation. The stability of 4EHP is maintained through physical interaction with GRB10 interacting GYF protein 2 (GIGYF2). Gene-disruptive mutations in GIGYF2 are linked to ASD, but causality is lacking. We hypothesized that GIGYF2 mutations cause ASD by disrupting 4EHP function.

Methods: Since homozygous deletion of either gene is lethal, we generated a cell-type-specific knockout model where Eif4e2 (the gene encoding 4EHP) is deleted in excitatory neurons of the forebrain (4EHP-eKO). In this model, we investigated ASD-associated synaptic plasticity dysfunction, ASD-like behaviors, and global translational control. We also utilized mice lacking one copy of Gigyf2, Eif4e2 or co-deletion of one copy of each gene to further investigate ASD-like behaviors.

Results: 4EHP is expressed in excitatory neurons and synaptosomes, and its amount increases during development. 4EHP-eKO mice display exaggerated mGluR-LTD, a phenotype frequently observed in mouse models of ASD. Consistent with synaptic plasticity dysfunction, the mice displayed social behavior impairments without being confounded by deficits in olfaction, anxiety, locomotion, or motor ability. Repetitive behaviors and vocal communication were not affected by loss of 4EHP in excitatory neurons. Heterozygous deletion of either Gigyf2, Eif4e2, or both genes in mice did not result in ASD-like behaviors (i.e. decreases in social behavior or increases in marble burying). Interestingly, exaggerated mGluR-LTD and impaired social behaviors were not attributed to changes in hippocampal global protein synthesis, which suggests that 4EHP and GIGYF2 regulate the translation of specific mRNAs to mediate these effects.

Limitations: This study did not identify which genes are translationally regulated by 4EHP and GIGYF2. Identification of mistranslated genes in 4EHP-eKO mice might provide a mechanistic explanation for the observed impairment in social behavior and exaggerated LTD. Future experiments employing affinity purification of translating ribosomes and mRNA sequencing in 4EHP-eKO mice will address this relevant issue.

Conclusions: Together these results demonstrate an important role of 4EHP in regulating hippocampal plasticity and ASD-associated social behaviors, consistent with the link between mutations in GIGYF2 and ASD.
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http://dx.doi.org/10.1186/s13229-020-00394-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682028PMC
November 2020

Unorthodox Mechanisms to Initiate Translation Open Novel Paths for Gene Expression.

J Mol Biol 2020 12 7;432(24):166702. Epub 2020 Nov 7.

Department of Biology, McGill University, Montreal, Québec, Canada; Department of Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands.

Translation in eukaryotes is dependent on the activity of translation initiation factor (eIF) 4G family of proteins, a scaffold protein that, during the initiation step, coordinates the activity of other eIFs to recruit the 40S ribosomal subunit to the mRNA. Three decades of research on protein synthesis and its regulation has provided a wealth of evidence supporting the crucial role of cap-dependent translation initiation, which involves eIF4G. However, the recent discovery of a surprising variety of alternative mechanisms to initiate translation in the absence of eIF4G has stirred the orthodox view of how protein synthesis is performed. These mechanisms involve novel interactions among known eIFs, or between known eIFs and other proteins not previously linked to translation. Thus, a new picture is emerging in which the unorthodox translation initiation complexes contribute to the diversity of mechanisms that regulate gene expression in eukaryotes.
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http://dx.doi.org/10.1016/j.jmb.2020.10.035DOI Listing
December 2020

eIF4E S209 phosphorylation licenses myc- and stress-driven oncogenesis.

Elife 2020 11 2;9. Epub 2020 Nov 2.

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, United States.

To better understand a role of eIF4E S209 in oncogenic translation, we generated heterozygous knockin (4EKI) HCT 116 human colorectal cancer (CRC) cells. 4EKI had little impact on total eIF4E levels, cap binding or global translation, but markedly reduced HCT 116 cell growth in spheroids and mice, and CRC organoid growth. 4EKI strongly inhibited Myc and ATF4 translation, the integrated stress response (ISR)-dependent glutamine metabolic signature, AKT activation and proliferation in vivo. 4EKI inhibited polyposis in mice by suppressing Myc protein and AKT activation. Furthermore, p-eIF4E was highly elevated in CRC precursor lesions in mouse and human. p-eIF4E cooperated with mutant to promote Myc and ISR-dependent glutamine addiction in various CRC cell lines, characterized by increased cell death, transcriptomic heterogeneity and immune suppression upon deprivation. These findings demonstrate a critical role of eIF4E S209-dependent translation in Myc and stress-driven oncogenesis and as a potential therapeutic vulnerability.
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http://dx.doi.org/10.7554/eLife.60151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665890PMC
November 2020

eIF2α controls memory consolidation via excitatory and somatostatin neurons.

Nature 2020 10 7;586(7829):412-416. Epub 2020 Oct 7.

Department of Biochemistry, McGill University, Montréal, Québec, Canada.

An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF2. Phosphorylation of the α-subunit of eIF2 (p-eIF2α), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds. By contrast, inhibiting the ISR by mutating the eIF2α phosphorylation site, genetically and pharmacologically inhibiting the ISR kinases, or mimicking reduced p-eIF2α with the ISR inhibitor ISRIB, enhances long-term memory in health and disease. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2α phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2α in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2α-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons.
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http://dx.doi.org/10.1038/s41586-020-2805-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874887PMC
October 2020

Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5' leader regions.

Nucleic Acids Res 2020 09;48(17):9521-9537

Department of Biochemistry, McGill University, Montreal H3G 1Y6, Canada.

Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5' leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5' leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.
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http://dx.doi.org/10.1093/nar/gkaa662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515738PMC
September 2020

Metformin inhibits RAN translation through PKR pathway and mitigates disease in ALS/FTD mice.

Proc Natl Acad Sci U S A 2020 08 20;117(31):18591-18599. Epub 2020 Jul 20.

Center for NeuroGenetics, College of Medicine, University of Florida, Gainesville, FL 32610;

Repeat associated non-AUG (RAN) translation is found in a growing number of microsatellite expansion diseases, but the mechanisms remain unclear. We show that RAN translation is highly regulated by the double-stranded RNA-dependent protein kinase (PKR). In cells, structured CAG, CCUG, CAGG, and GC expansion RNAs activate PKR, which leads to increased levels of multiple RAN proteins. Blocking PKR using PKR-K296R, the TAR RNA binding protein or PKR-KO cells, reduces RAN protein levels. p-PKR is elevated in ALS/FTD human and mouse brains, and inhibiting PKR in BAC transgenic mice using AAV-PKR-K296R or the Food and Drug Administration (FDA)-approved drug metformin, decreases RAN proteins, and improves behavior and pathology. In summary, targeting PKR, including by use of metformin, is a promising therapeutic approach for ALS/FTD and other expansion diseases.
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http://dx.doi.org/10.1073/pnas.2005748117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414156PMC
August 2020

Aster-C coordinates with COP I vesicles to regulate lysosomal trafficking and activation of mTORC1.

EMBO Rep 2020 09 9;21(9):e49898. Epub 2020 Jul 9.

Department of Pharmacology, Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

Nutrient sensing by the mTOR complex 1 (mTORC1) requires its translocation to the lysosomal membrane. Upon amino acids removal, mTORC1 becomes cytosolic and inactive, yet its precise subcellular localization and the mechanism of inhibition remain elusive. Here, we identified Aster-C as a negative regulator of mTORC1 signaling. Aster-C earmarked a special rough ER subdomain where it sequestered mTOR together with the GATOR2 complex to prevent mTORC1 activation during nutrient starvation. Amino acids stimulated rapid disassociation of mTORC1 from Aster-C concurrently with assembly of COP I vesicles which escorted mTORC1 to the lysosomal membrane. Consequently, ablation of Aster-C led to spontaneous activation of mTORC1 and dissociation of TSC2 from lysosomes, whereas inhibition of COP I vesicle biogenesis or actin dynamics prevented mTORC1 activation. Together, these findings identified Aster-C as a missing link between lysosomal trafficking and mTORC1 activation by revealing an unexpected role of COP I vesicles in mTORC1 signaling.
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http://dx.doi.org/10.15252/embr.201949898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507422PMC
September 2020

Elevated V-ATPase Activity Following PTEN Loss Is Required for Enhanced Oncogenic Signaling in Breast Cancer.

Mol Cancer Res 2020 10 25;18(10):1477-1490. Epub 2020 Jun 25.

Goodman Cancer Research Centre, McGill University, Montreal, Quebec, Canada.

PTEN loss-of-function contributes to hyperactivation of the PI3K pathway and to drug resistance in breast cancer. Unchecked PI3K pathway signaling increases activation of the mechanistic target of rapamycin complex 1 (mTORC1), which promotes tumorigenicity. Several studies have suggested that vacuolar (H)-ATPase (V-ATPase) complex activity is regulated by PI3K signaling. In this study, we showed that loss of PTEN elevated V-ATPase activity. Enhanced V-ATPase activity was mediated by increased expression of the ATPase H+ transporting accessory protein 2 (), also known as the prorenin receptor (PRR). PRR is cleaved into a secreted extracellular fragment (sPRR) and an intracellular fragment (M8.9) that remains associated with the V-ATPase complex. Reduced PTEN expression increased V-ATPase complex activity in a PRR-dependent manner. Breast cancer cell lines with reduced PTEN expression demonstrated increased PRR expression. Similarly, PRR expression became elevated upon PTEN deletion in a mouse model of breast cancer. Interestingly, concentration of sPRR was elevated in the plasma of patients with breast cancer and correlated with tumor burden in HER2-enriched cancers. Moreover, PRR was essential for proper HER2 receptor expression, localization, and signaling. PRR knockdown attenuated HER2 signaling and resulted in reduced Akt and ERK 1/2 phosphorylation, and in lower mTORC1 activity. Overall, our study demonstrates a mechanism by which PTEN loss in breast cancer can potentiate multiple signaling pathways through upregulation of the V-ATPase complex. IMPLICATIONS: Our study contributed to the understanding of the role of the V-ATPase complex in breast cancer cell tumorigenesis and provided a potential biomarker in breast cancer.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0088DOI Listing
October 2020

Non-cooperative 4E-BP2 folding with exchange between eIF4E-binding and binding-incompatible states tunes cap-dependent translation inhibition.

Nat Commun 2020 06 19;11(1):3146. Epub 2020 Jun 19.

Program in Molecular Medicine, The Hospital for Sick Children, Toronto, ON, M5G 0A4, Canada.

Phosphorylation of intrinsically disordered eIF4E binding proteins (4E-BPs) regulates cap-dependent translation by weakening their ability to compete with eIF4G for eIF4E binding within the translation initiation complex. We previously showed that phosphorylation of T37 and T46 in 4E-BP2 induces folding of a four-stranded beta-fold domain, partially sequestering the canonical eIF4E-binding helix. The C-terminal intrinsically disordered region (C-IDR), remaining disordered after phosphorylation, contains the secondary eIF4E-binding site and three other phospho-sites, whose mechanisms in inhibiting binding are not understood. Here we report that the domain is non-cooperatively folded, with exchange between beta strands and helical conformations. C-IDR phosphorylation shifts the conformational equilibrium, controlling access to eIF4E binding sites. The hairpin turns formed by pT37/pT46 are remarkably stable and function as transplantable units for phospho-regulation of stability. These results demonstrate how non-cooperative folding and conformational exchange leads to graded inhibition of 4E-BP2:eIF4E binding, shifting 4E-BP2 into an eIF4E binding-incompatible conformation and regulating translation initiation.
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http://dx.doi.org/10.1038/s41467-020-16783-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305185PMC
June 2020

Dysregulated translational control in brain disorders: from genes to behavior.

Curr Opin Genet Dev 2020 12 11;65:34-41. Epub 2020 Jun 11.

Department of Biochemistry, McGill University, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, QC H3G 1Y6, Canada; Goodman Cancer Research Centre, 1160 Pine Avenue West, Montreal, QC H3A 1A3, Canada. Electronic address:

Control of protein synthesis (mRNA translation) is essential for proper brain development and function. Perturbations to the mechanisms governing mRNA translation have repeatedly been shown to constitute a neurodegenerative, neuropsychiatric, and neurodevelopmental disorder risk factor. Developing effective therapeutics for brain disorders will require a better understanding of the molecular mechanisms underlying the control of protein synthesis in brain function. Studies using transgenic animal models have been invaluable towards this end, providing exciting new insights into the genetic basis of brain disorders with hopeful prospects for new and effective treatment options.
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http://dx.doi.org/10.1016/j.gde.2020.05.005DOI Listing
December 2020

Rheb1-Independent Activation of mTORC1 in Mammary Tumors Occurs through Activating Mutations in mTOR.

Cell Rep 2020 04;31(4):107571

Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; Faculty of Medicine, McGill University, Montreal, QC H3A 1A3, Canada; Rosalind & Morris Goodman Cancer Centre, McGill University, Montreal, QC H3A 1A3, Canada. Electronic address:

Mechanistic target of rapamycin complex 1 (mTORC1) is a master modulator of cellular growth, and its aberrant regulation is recurrently documented within breast cancer. While the small GTPase Rheb1 is the canonical activator of mTORC1, Rheb1-independent mechanisms of mTORC1 activation have also been reported but have not been fully understood. Employing multiple transgenic mouse models of breast cancer, we report that ablation of Rheb1 significantly impedes mammary tumorigenesis. In the absence of Rheb1, a block in tumor initiation can be overcome by multiple independent mutations in Mtor to allow Rheb1-independent reactivation of mTORC1. We further demonstrate that the mTOR kinase is indispensable for tumor initiation as the genetic ablation of mTOR abolishes mammary tumorigenesis. Collectively, our findings demonstrate that mTORC1 activation is indispensable for mammary tumor initiation and that tumors acquire alternative mechanisms of mTORC1 activation.
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http://dx.doi.org/10.1016/j.celrep.2020.107571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281837PMC
April 2020

The translational landscape of ground state pluripotency.

Nat Commun 2020 04 1;11(1):1617. Epub 2020 Apr 1.

Department of Molecular Biology, Faculty of Science, Radboud University, Radboud Institute for Molecular Life Sciences (RIMLS), 6525 GA, Nijmegen, The Netherlands.

Translational control plays a central role in regulation of gene expression and can lead to significant divergence between mRNA- and protein-abundance. Here, we used genome-wide approaches combined with time-course analysis to measure the mRNA-abundance, mRNA-translation rate and protein expression during the transition of naïve-to-primed mouse embryonic stem cells (ESCs). We find that the ground state ESCs cultured with GSK3-, MEK-inhibitors and LIF (2iL) display higher ribosome density on a selective set of mRNAs. This set of mRNAs undergo strong translational buffering to maintain stable protein expression levels in 2iL-ESCs. Importantly, we show that the global alteration of cellular proteome during the transition of naïve-to-primed pluripotency is largely accompanied by transcriptional rewiring. Thus, we provide a comprehensive and detailed overview of the global changes in gene expression in different states of ESCs and dissect the relative contributions of mRNA-transcription, translation and regulation of protein stability in controlling protein abundance.
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http://dx.doi.org/10.1038/s41467-020-15449-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113317PMC
April 2020

4E-BP-Dependent Translational Control of Mediates Adipose Tissue Macrophage Inflammatory Response.

J Immunol 2020 05 25;204(9):2392-2400. Epub 2020 Mar 25.

Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612;

Deregulation of mRNA translation engenders many human disorders, including obesity, neurodegenerative diseases, and cancer, and is associated with pathogen infections. The role of eIF4E-dependent translational control in macrophage inflammatory responses in vivo is largely unexplored. In this study, we investigated the involvement of the translation inhibitors eIF4E-binding proteins (4E-BPs) in the regulation of macrophage inflammatory responses in vitro and in vivo. We show that the lack of 4E-BPs exacerbates inflammatory polarization of bone marrow-derived macrophages and that 4E-BP-null adipose tissue macrophages display enhanced inflammatory gene expression following exposure to a high-fat diet (HFD). The exaggerated inflammatory response in HFD-fed 4E-BP-null mice coincides with significantly higher weight gain, higher mRNA translation, and increased expression of IRF8 in adipose tissue compared with wild-type mice. Thus, 4E-BP-dependent translational control limits, in part, the proinflammatory response during HFD. These data underscore the activity of the 4E-BP-IRF8 axis as a paramount regulatory mechanism of proinflammatory responses in adipose tissue macrophages.
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http://dx.doi.org/10.4049/jimmunol.1900538DOI Listing
May 2020

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Mol Cell 2020 03 29;77(6):1176-1192.e16. Epub 2020 Jan 29.

Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.
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http://dx.doi.org/10.1016/j.molcel.2020.01.006DOI Listing
March 2020
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