Loss of Elp3 blocks intestinal tuft cell differentiation via an mTORC1-Atf4 axis.

Intestinal tuft cells are critical for anti-helminth parasite immunity because they produce IL-25, which triggers IL-13 secretion by activated group 2 innate lymphoid cells (ILC2s) to expand both goblet and tuft cells. We show that epithelial Elp3, a tRNA-modifying enzyme, promotes tuft cell differentiation and is consequently critical for IL-25 production, ILC2 activation, goblet cell expansion and control of Nippostrongylus brasiliensis helminth infection in mice. Elp3 is essential for the generation of intestinal immature tuft cells and for the IL-13-dependent induction of glycolytic enzymes such as Hexokinase 1 and Aldolase A. Importantly, loss of epithelial Elp3 in the intestine blocks the codon-dependent translation of the Gator1 subunit Nprl2, an mTORC1 inhibitor, which consequently enhances mTORC1 activation and stabilizes Atf4 in progenitor cells. Likewise, Atf4 overexpression in mouse intestinal epithelium blocks tuft cell differentiation in response to intestinal helminth infection. Collectively, our data define Atf4 as a negative regulator of tuft cells and provide insights into promotion of intestinal type 2 immune response to parasites through tRNA modifications.

Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.

Elongator controls the migration and differentiation of cortical neurons through acetylation of alpha-tubulin.

The generation of cortical projection neurons relies on the coordination of radial migration with branching. Here, we report that the multisubunit histone acetyltransferase Elongator complex, which contributes to transcript elongation, also regulates the maturation of projection neurons. Indeed, silencing of its scaffold (Elp1) or catalytic subunit (Elp3) cell-autonomously delays the migration and impairs the branching of projection neurons. Strikingly, neurons defective in Elongator show reduced levels of acetylated alpha-tubulin. Reduction of alpha-tubulin acetylation via expression of a nonacetylatable alpha-tubulin mutant leads to comparable defects in cortical neurons and suggests that alpha-tubulin is a target of Elp3. This is further supported by the demonstration that Elp3 promotes acetylation and counteracts HDAC6-mediated deacetylation of this substrate in vitro. Our results uncover alpha-tubulin as a target of the Elongator complex and suggest that a tight regulation of its acetylation underlies the maturation of cortical projection neurons.

Codon-specific translation reprogramming promotes resistance to targeted therapy.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

Loss of the Transfer RNA Wobble Uridine-Modifying Enzyme Elp3 Delays T Cell Cycle Entry and Impairs T Follicular Helper Cell Responses through Deregulation of Atf4.

The activation of T cells is accompanied by intensive posttranscriptional remodeling of their proteome. We observed that protein expression of enzymes that modify wobble uridine in specific tRNAs, namely elongator subunit 3 (Elp3) and cytosolic thiouridylase (Ctu)2, increased in the course of T cell activation. To investigate the role of these tRNA epitranscriptomic modifiers in T cell biology, we generated mice deficient for Elp3 in T cells. We show that deletion of Elp3 has discrete effects on T cells. In vitro, Elp3-deficient naive CD4+ T cells polarize normally but are delayed in entering the first cell cycle following activation. In vivo, different models of immunization revealed that Elp3-deficient T cells display reduced expansion, resulting in functional impairment of T follicular helper (TFH) responses, but not of other CD4+ effector T cell responses. Transcriptomic analyses identified a progressive overactivation of the stress-responsive transcription factor Atf4 in Elp3-deficient T cells. Overexpression of Atf4 in wild-type T cells phenocopies the effect of Elp3 loss on T cell cycle entry and TFH cell responses. Reciprocally, partial silencing of Atf4 or deletion of its downstream effector transcription factor Chop rescues TFH responses of Elp3-deficient T cells. Together, our results reveal that specific epitranscriptomic tRNA modifications contribute to T cell cycle entry and promote optimal TFH responses.

Proteostasis is essential during cochlear development for neuron survival and hair cell polarity.

Protein homeostasis is essential to cell function, and a compromised ability to reduce the load of misfolded and aggregated proteins is linked to numerous age-related diseases, including hearing loss. Here, we show that altered proteostasis consequent to Elongator complex deficiency also impacts the proper development of the cochlea and results in deafness. In the absence of the catalytic subunit Elp3, differentiating spiral ganglion neurons display large aggresome-like structures and undergo apoptosis before birth. The cochlear mechanosensory cells are able to survive proteostasis disruption but suffer defects in polarity and stereociliary bundle morphogenesis. We demonstrate that protein aggregates accumulate at the apical surface of hair cells, where they cause a local slowdown of microtubular trafficking, altering the distribution of intrinsic polarity proteins and affecting kinocilium position and length. Alleviation of protein misfolding using the chemical chaperone 4-phenylbutyric acid during embryonic development ameliorates hair cell polarity in Elp3-deficient animals. Our study highlights the importance of developmental proteostasis in the cochlea and unveils an unexpected link between proteome integrity and polarized organization of cellular components.

Elongator subunit 3 (ELP3) modifies ALS through tRNA modification.

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder of which the progression is influenced by several disease-modifying factors. Here, we investigated ELP3, a subunit of the elongator complex that modifies tRNA wobble uridines, as one of such ALS disease modifiers. ELP3 attenuated the axonopathy of a mutant SOD1, as well as of a mutant C9orf72 ALS zebrafish model. Furthermore, the expression of ELP3 in the SOD1G93A mouse extended the survival and attenuated the denervation in this model. Depletion of ELP3 in vitro reduced the modified tRNA wobble uridine mcm5s2U and increased abundance of insoluble mutant SOD1, which was reverted by exogenous ELP3 expression. Interestingly, the expression of ELP3 in the motor cortex of ALS patients was reduced and correlated with mcm5s2U levels. Our results demonstrate that ELP3 is a modifier of ALS and suggest a link between tRNA modification and neurodegeneration.

DBIRD complex integrates alternative mRNA splicing with RNA polymerase II transcript elongation.

Alternative messenger RNA splicing is the main reason that vast mammalian proteomic complexity can be achieved with a limited number of genes. Splicing is physically and functionally coupled to transcription, and is greatly affected by the rate of transcript elongation. As the nascent pre-mRNA emerges from transcribing RNA polymerase II (RNAPII), it is assembled into a messenger ribonucleoprotein (mRNP) particle; this is the functional form of the nascent pre-mRNA and determines the fate of the mature transcript. However, factors that connect the transcribing polymerase with the mRNP particle and help to integrate transcript elongation with mRNA splicing remain unclear. Here we characterize the human interactome of chromatin-associated mRNP particles. This led us to identify deleted in breast cancer 1 (DBC1) and ZNF326 (which we call ZNF-protein interacting with nuclear mRNPs and DBC1 (ZIRD)) as subunits of a novel protein complex--named DBIRD--that binds directly to RNAPII. DBIRD regulates alternative splicing of a large set of exons embedded in (A + T)-rich DNA, and is present at the affected exons. RNA-interference-mediated DBIRD depletion results in region-specific decreases in transcript elongation, particularly across areas encompassing affected exons. Together, these data indicate that the DBIRD complex acts at the interface between mRNP particles and RNAPII, integrating transcript elongation with the regulation of alternative splicing.

KIAA1199: A novel regulator of MEK/ERK-induced Schwann cell dedifferentiation.

The molecular mechanisms that regulate Schwann cell (SC) plasticity and the role of the Nrg1/ErbB-induced MEK1/ERK1/2 signalling pathway in SC dedifferentiation or in myelination remain unclear. It is currently believed that different levels of MEK1/ERK1/2 activation define the state of SC differentiation. Thus, the identification of new regulators of MEK1/ERK1/2 signalling could help to decipher the context-specific aspects driving the effects of this pathway on SC plasticity. In this perspective, we have investigated the potential role of KIAA1199, a protein that promotes ErbB and MEK1/ERK1/2 signalling in cancer cells, in SC plasticity. We depleted KIAA1199 in the SC-derived MSC80 cell line with RNA-interference-based strategy and also generated Tamoxifen-inducible and conditional mouse models in which KIAA1199 is inactivated through homologous recombination, using the Cre-lox technology. We show that the invalidation of KIAA1199 in SC decreases the expression of cJun and other negative regulators of myelination and elevates Krox20, driving them towards a pro-myelinating phenotype. We further show that in dedifferentiation conditions, SC invalidated for KIAA1199 exhibit lower myelin clearance as well as increased myelination capacity. Finally, the Nrg1-induced activation of the MEK/ERK/1/2 pathway is severely reduced when KIAA1199 is absent, indicating that KIAA1199 promotes Nrg1-dependent MEK1 and ERK1/2 activation in SCs. In conclusion, this work identifies KIAA1199 as a novel regulator of MEK/ERK-induced SC dedifferentiation and contributes to a better understanding of the molecular control of SC dedifferentiation.

A role for APPL1 in TLR3/4-dependent TBK1 and IKKε activation in macrophages.

Endosomes have important roles in intracellular signal transduction as a sorting platform. Signaling cascades from TLR engagement to IRF3-dependent gene transcription rely on endosomes, yet the proteins that specifically recruit IRF3-activating molecules to them are poorly defined. We show that adaptor protein containing a pleckstrin-homology domain, a phosphotyrosine-binding domain, and a leucine zipper motif (APPL)1, an early endosomal protein, is required for both TRIF- and retinoic acid-inducible gene 1-dependent signaling cascades to induce IRF3 activation. APPL1, but not early endosome Ag 1, deficiency impairs IRF3 target gene expression upon engagement of both TLR3 and TLR4 pathways, as well as in H1N1-infected macrophages. The IRF3-phosphorylating kinases TBK1 and IKKε are recruited to APPL1 endosomes in LPS-stimulated macrophages. Interestingly, APPL1 undergoes proteasome-mediated degradation through ERK1/2 to turn off signaling. APPL1 degradation is blocked when signaling through the endosome is inhibited by chloroquine or dynasore. Therefore, APPL1 endosomes are critical for IRF3-dependent gene expression in response to some viral and bacterial infections in macrophages. Those signaling pathways involve the signal-induced degradation of APPL1 to prevent aberrant IRF3-dependent gene expression linked to immune diseases.

Targeting osteopontin suppresses glioblastoma stem-like cell character and tumorigenicity in vivo.

Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence.

Molecular mechanism of IKK catalytic dimer docking to NF-κB substrates.

The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKß and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBß) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/ß dimerization in substrate motif recognition.

Valine aminoacyl-tRNA synthetase promotes therapy resistance in melanoma.

Transfer RNA dynamics contribute to cancer development through regulation of codon-specific messenger RNA translation. Specific aminoacyl-tRNA synthetases can either promote or suppress tumourigenesis. Here we show that valine aminoacyl-tRNA synthetase (VARS) is a key player in the codon-biased translation reprogramming induced by resistance to targeted (MAPK) therapy in melanoma. The proteome rewiring in patient-derived MAPK therapy-resistant melanoma is biased towards the usage of valine and coincides with the upregulation of valine cognate tRNAs and of VARS expression and activity. Strikingly, VARS knockdown re-sensitizes MAPK-therapy-resistant patient-derived melanoma in vitro and in vivo. Mechanistically, VARS regulates the messenger RNA translation of valine-enriched transcripts, among which hydroxyacyl-CoA dehydrogenase mRNA encodes for a key enzyme in fatty acid oxidation. Resistant melanoma cultures rely on fatty acid oxidation and hydroxyacyl-CoA dehydrogenase for their survival upon MAPK treatment. Together, our data demonstrate that VARS may represent an attractive therapeutic target for the treatment of therapy-resistant melanoma.

DERP6 (ELP5) and C3ORF75 (ELP6) regulate tumorigenicity and migration of melanoma cells as subunits of Elongator.

The Elongator complex is composed of 6 subunits (Elp1-Elp6) and promotes RNAPII transcript elongation through histone acetylation in the nucleus as well as tRNA modification in the cytoplasm. This acetyltransferase complex directly or indirectly regulates numerous biological processes ranging from exocytosis and resistance to heat shock in yeast to cell migration and neuronal differentiation in higher eukaryotes. The identity of human ELP1 through ELP4 has been reported but human ELP5 and ELP6 have remained uncharacterized. Here, we report that DERP6 (ELP5) and C3ORF75 (ELP6) encode these subunits of human Elongator. We further investigated the importance and function of these two subunits by a combination of biochemical analysis and cellular assays. Our results show that DERP6/ELP5 is required for the integrity of Elongator and directly connects ELP3 to ELP4. Importantly, the migration and tumorigenicity of melanoma-derived cells are significantly decreased upon Elongator depletion through ELP1 or ELP3. Strikingly, DERP6/ELP5 and C3ORF75/ELP6-depleted melanoma cells have similar defects, further supporting the idea that DERP6/ELP5 and C3ORF75/ELP6 are essential for Elongator function. Together, our data identify DERP6/ELP5 and C3ORF75/ELP6 as key players for migration, invasion and tumorigenicity of melanoma cells, as integral subunits of Elongator.

Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly activated?

The IkappaB kinases (IKKs) IKK-alpha and IKK-beta, and the IKK-related kinases TBK1 and IKK-epsilon, have essential roles in innate immunity through signal-induced activation of NF-kappaB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-kappaB essential modulator coordinates some IKK complexes, whereas TANK, NF-kappaB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-epsilon complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-epsilon subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity.

The E3 ligase COP1 promotes ERα signaling and suppresses EMT in breast cancer.

ERα signaling drives proliferation, survival and cancer initiation in the mammary gland. Therefore, it is critical to elucidate mechanisms by which ERα expression is regulated. We show that the tumor suppressor E3 ligase COP1 promotes the degradative polyubiquitination of the microtubule-associated protein HPIP. As such, COP1 negatively regulates estrogen-dependent AKT activation in breast cancer cells. However, COP1 also induces ERα expression and ERα-dependent gene transcription, at least through c-Jun degradation. COP1 and ERα levels are positively correlated in clinical cases of breast cancer. COP1 also supports the metabolic reprogramming by estrogens, including glycolysis. On the other hand, COP1 suppresses EMT in breast cancer cells. COP1 deficiency also contributes to Tamoxifen resistance, at least through protective autophagy. Therefore, COP1 acts as an oncogenic E3 ligase by promoting ERα signaling but also acts as a tumor suppressor candidate by preventing EMT, which reflects a dual role of COP1 in breast cancer.

BCL-3 degradation involves its polyubiquitination through a FBW7-independent pathway and its binding to the proteasome subunit PSMB1.

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-kappaB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the mechanisms underlying its degradation remain poorly understood. Yeast two-hybrid analysis led to the identification of the proteasome subunit PSMB1 as a BCL-3-associated protein. The binding of BCL-3 to PSMB1 is required for its degradation through the proteasome. Indeed, PSMB1-depleted cells are defective in degrading polyubiquitinated BCL-3. The N-terminal part of BCL-3 includes lysines 13 and 26 required for the Lys(48)-linked polyubiquitination of BCL-3. Moreover, the E3 ligase FBW7, known to polyubiquitinate a variety of substrates phosphorylated by GSK3, is dispensable for BCL-3 degradation. Thus, our data defined a unique motif of BCL-3 that is needed for its recruitment to the proteasome and identified PSMB1 as a key protein required for the proteasome-mediated degradation of a nuclear and oncogenic IkappaB protein.

Involvement of placental growth factor in Wallerian degeneration.

Wallerian degeneration (WD) is an inflammatory process of nerve degeneration, which occurs more rapidly in the peripheral nervous system compared with the central nervous system, resulting, respectively in successful and aborted axon regeneration. In the peripheral nervous system, Schwann cells (SCs) and macrophages, under the control of a network of cytokines and chemokines, represent the main cell types involved in this process. Within this network, the role of placental growth factor (PlGF) remains totally unknown. However, properties like monocyte activation/attraction, ability to increase expression of pro-inflammatory molecules, as well as neuroprotective effects, make it a candidate likely implicated in this process. Also, nothing is described about the expression and localization of this molecule in the peripheral nervous system. To address these original questions, we decided to study PlGF expression under physiological and degenerative conditions and to explore its role in WD, using a model of sciatic nerve transection in wild-type and Pgf(-/-) mice. Our data show dynamic changes of PlGF expression, from periaxonal in normal nerve to SCs 24h postinjury, in parallel with a p65/NF-κB recruitment on Pgf promoter. After injury, SC proliferation is reduced by 30% in absence of PlGF. Macrophage invasion is significantly delayed in Pgf(-/-) mice compared with wild-type mice, which results in worse functional recovery. MCP-1 and proMMP-9 exhibit a 3-fold reduction of their relative expressions in Pgf(-/-) injured nerves, as demonstrated by cytokine array. In conclusion, this work originally describes PlGF as a novel member of the cytokine network of WD.

NF-κB, stem cells and breast cancer: the links get stronger.

Self-renewing breast cancer stem cells are key actors in perpetuating tumour existence and in treatment resistance and relapse. The molecular pathways required for their maintenance are starting to be elucidated. Among them is the transcription factor NF-κB, which is known to play critical roles in cell survival, inflammation and immunity. Recent studies indicate that mammary epithelial NF-κB regulates the self-renewal of breast cancer stem cells in a model of Her2-dependent tumourigenesis. We will describe here the NF-κB-activating pathways that are involved in this process and in which progenitor cells this transcription factor is actually activated.