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.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL.

Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

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.

Translational offsetting as a mode of estrogen receptor α-dependent regulation of gene expression.

Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as "translational offsetting" of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5'UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34-modifying enzymes and thereby controls levels of U34-modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα-dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.

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.

ITGBL1 is a new immunomodulator that favors development of melanoma tumors by inhibiting natural killer cells cytotoxicity.

Resistances to immunotherapies remains a major hurdle towards a cure for melanoma in numerous patients. An increase in the mesenchymal phenotype and a loss of differentiation have been clearly associated with resistance to targeted therapies. Similar phenotypes have been more recently also linked to resistance to immune checkpoint therapies. We demonstrated here that the loss of MIcrophthalmia associated Transcription Factor (MITF), a pivotal player in melanocyte differentiation, favors the escape of melanoma cells from the immune system. We identified Integrin beta-like protein 1 (ITGBL1), a secreted protein, upregulated in anti-PD1 resistant patients and in MITFlow melanoma cells, as the key immunomodulator. ITGBL1 inhibited immune cell cytotoxicity against melanoma cells by inhibiting NK cells cytotoxicity and counteracting beneficial effects of anti-PD1 treatment, both in vitro and in vivo. Mechanistically, MITF inhibited RUNX2, an activator of ITGBL1 transcription. Interestingly, VitaminD3, an inhibitor of RUNX2, improved melanoma cells to death by immune cells. In conclusion, our data suggest that inhibition of ITGBL1 might improve melanoma response to immunotherapies.

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.

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.

Reversing the relative time courses of the peptide bond reaction with oligopeptides of different lengths and charged amino acid distributions in the ribosome exit tunnel.

The kinetics of the protein elongation cycle by the ribosome depends on intertwined factors. One of these factors is the electrostatic interaction of the nascent protein with the ribosome exit tunnel. In this computational biology theoretical study, we focus on the rate of the peptide bond formation and its dependence on the ribosome exit tunnel electrostatic potential profile. We quantitatively predict how oligopeptides of variable lengths can affect the peptide bond formation rate. We applied the Michaelis-Menten model as previously extended to incorporate the mechano-biochemical effects of forces on the rate of reaction at the catalytic site of the ribosome. For a given pair of carboxy-terminal amino acid substrate at the P- and an aminoacyl-tRNA at the A-sites, the relative time courses of the peptide bond formation reaction can be reversed depending on the oligopeptide sequence embedded in the tunnel and their variable lengths from the P-site. The reversal is predicted to occur from a shift in positions of charged amino acids upstream in the oligopeptidyl-tRNA at the P-site. The position shift must be adjusted by clever design of the oligopeptide probes using the electrostatic potential profile along the exit tunnel axial path. These predicted quantitative results bring strong evidence of the importance and relative contribution of the electrostatic interaction of the ribosome exit tunnel with the nascent peptide chain during elongation.

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.

A simple geometrical model of the electrostatic environment around the catalytic center of the ribosome and its significance for the elongation cycle kinetics.

The central function of the large subunit of the ribosome is to catalyze peptide bond formation. This biochemical reaction is conducted at the peptidyl transferase center (PTC). Experimental evidence shows that the catalytic activity is affected by the electrostatic environment around the peptidyl transferase center. Here, we set up a minimal geometrical model fitting the available x-ray solved structures of the ribonucleic cavity around the catalytic center of the large subunit of the ribosome. The purpose of this phenomenological model is to estimate quantitatively the electrostatic potential and electric field that are experienced during the peptidyl transfer reaction. At least two reasons motivate the need for developing this quantification. First, we inquire whether the electric field in this particular catalytic environment, made only of nucleic acids, is of the same order of magnitude as the one prevailing in catalytic centers of the proteic enzymes counterparts. Second, the protein synthesis rate is dependent on the nature of the amino acid sequentially incorporated in the nascent chain. The activation energy of the catalytic reaction and its detailed kinetics are shown to be dependent on the mechanical work exerted on the amino acids by the electric field, especially when one of the four charged amino acid residues (R, K, E, D) has previously been incorporated at the carboxy-terminal end of the peptidyl-tRNA. Physical values of the electric field provide quantitative knowledge of mechanical work, activation energy and rate of the peptide bond formation catalyzed by the ribosome. We show that our theoretical calculations are consistent with two independent sets of previously published experimental results. Experimental results for E.coli in the minimal case of the dipeptide bond formation when puromycin is used as the final amino acid acceptor strongly support our theoretically derived reaction time courses. Experimental Ribo-Seq results on E. coli and S. cerevisiae comparing the residence time distribution of ribosomes upon specific codons are also well accounted for by our theoretical calculations. The statistical queueing time theory was used to model the ribosome residence time per codon during nascent protein elongation and applied for the interpretation of the Ribo-Seq data. The hypo-exponential distribution fits the residence time observed distribution of the ribosome on a codon. An educated deconvolution of this distribution is used to estimate the rates of each elongation step in a codon specific manner. Our interpretation of all these results sheds light on the functional role of the electrostatic profile around the PTC and its impact on the ribosome elongation cycle.

Cracking the genetic code with neural networks.

The genetic code is textbook scientific knowledge that was soundly established without resorting to Artificial Intelligence (AI). The goal of our study was to check whether a neural network could re-discover, on its own, the mapping links between codons and amino acids and build the complete deciphering dictionary upon presentation of transcripts proteins data training pairs. We compared different Deep Learning neural network architectures and estimated quantitatively the size of the required human transcriptomic training set to achieve the best possible accuracy in the codon-to-amino-acid mapping. We also investigated the effect of a codon embedding layer assessing the semantic similarity between codons on the rate of increase of the training accuracy. We further investigated the benefit of quantifying and using the unbalanced representations of amino acids within real human proteins for a faster deciphering of rare amino acids codons. Deep neural networks require huge amount of data to train them. Deciphering the genetic code by a neural network is no exception. A test accuracy of 100% and the unequivocal deciphering of rare codons such as the tryptophan codon or the stop codons require a training dataset of the order of 4-22 millions cumulated pairs of codons with their associated amino acids presented to the neural network over around 7-40 training epochs, depending on the architecture and settings. We confirm that the wide generic capacities and modularity of deep neural networks allow them to be customized easily to learn the deciphering task of the genetic code efficiently.

Ribosome exit tunnel electrostatics.

The impact of ribosome exit tunnel electrostatics on the protein elongation rate or on forces acting upon the nascent polypeptide chain are currently not fully elucidated. In the past, researchers have measured the electrostatic potential inside the ribosome polypeptide exit tunnel at a limited number of spatial points, at least in rabbit reticulocytes. Here we present a basic electrostatic model of the exit tunnel of the ribosome, providing a quantitative physical description of the tunnel interaction with the nascent proteins at all centro-axial points inside the tunnel. We show that a strong electrostatic screening is due to water molecules (not mobile ions) attracted to the ribosomal nucleic acid phosphate moieties buried in the immediate vicinity of the tunnel wall. We also show how the tunnel wall components and local ribosomal protein protrusions impact on the electrostatic potential profile and impede charged amino acid residues from progressing through the tunnel, affecting the elongation rate in a range of -40% to +85% when compared to the average elongation rate. The time spent by the ribosome to decode the genetic encrypted message is constrained accordingly. We quantitatively derive, at single-residue resolution, the axial forces acting on the nascent peptide from its particular sequence embedded in the tunnel. The model sheds light on how the experimental data point measurements of the potential are linked to the local structural chemistry of the inner wall, shape, and size of the tunnel. The model consistently connects experimental observations coming from different fields in molecular biology, x-ray crystallography, physical chemistry, biomechanics, and synthetic and multiomics biology. Our model should be a valuable tool to gain insight into protein synthesis dynamics, translational control, and the role of the ribosome's mechanochemistry in the cotranslational protein folding.

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.

THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer.

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, we show that the ER membrane-associated protein THEM6 regulates intracellular levels of ether lipids and is essential to trigger the induction of the ER stress response (UPR). Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated activation of ATF4. Finally, we demonstrate that high THEM6 expression is associated with poor survival and correlates with high levels of UPR activation in PCa patients. Altogether, our results highlight THEM6 as a novel driver of therapy resistance in PCa as well as a promising target for the treatment of CRPC.

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.