Translational Profiling Research Articles

Origin of translational control by eIF2α phosphorylation: insights from genome-wide translational profiling studies in fission yeast.

During amino acid limitation, the protein kinase Gcn2 phosphorylates the α subunit of eIF2, thereby regulating mRNA translation. In yeast Saccharomyces cerevisiae and mammals, eIF2α phosphorylation regulates translation of related transcription factors Gcn4 and Atf4 through upstream open reading frames (uORFs) to activate transcription genome wide. However, mammals encode three more eIF2α kinases activated by distinct stimuli. Did the translational control system involving eIF2α phosphorylation evolve from so simple (as found in yeast S. cerevisiae) to complex (as found in humans)? Recent genome-wide translational profiling studies of amino acid starvation response in the fission yeast Schizosaccharomyces pombe provide an unexpected answer to this question.

Induction of autophagy mitigates TDP-43 pathology and translational repression of neurofilament mRNAs in mouse models of ALS/FTD

BackgroundTDP-43 proteinopathy is a pathological hallmark of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). So far, there is no therapy available for these neurodegenerative diseases. In addition, the impact of TDP-43 proteinopathy on neuronal translational profile also remains unknown.MethodsBiochemical, immunohistology and assay-based studies were done with cell cultures and transgenic mice models. We also used Ribotag with microarray and proteomic analysis to determine the neuronal translational profile in the mice model of ALS/FTD.ResultsHere, we report that oral administration of a novel analog (IMS-088) of withaferin-A, an antagonist of nuclear factor kappa-B (NF-ĸB) essential modulator (NEMO), induced autophagy and reduced TDP-43 proteinopathy in the brain and spinal cord of transgenic mice expressing human TDP-43 mutants, models of ALS/FTD. Treatment with IMS-088 ameliorated cognitive impairment, reduced gliosis in the brain of ALS/FTD mouse models. With the Ribotrap method, we investigated the impact of TDP-43 proteinopathy and IMS-088 treatment on the translation profile of neurons of one-year old hTDP-43A315T mice. TDP-43 proteinopathy caused translational dysregulation of specific mRNAs including translational suppression of neurofilament mRNAs resulting in 3 to 4-fold decrease in levels type IV neurofilament proteins. Oral administration of IMS-088 rescued the translational defects associated with TDP-43 proteinopathy and restored the synthesis of neurofilament proteins, which are essential for axon integrity and synaptic function.ConclusionsOur study revealed that induction of autophagy reduces TDP-43 pathology and ameliorates the translational defect seen in mice models of ALS/FTD. Based on these results, we suggest IMS-088 and perhaps other inducers of autophagy should be considered as potential therapeutics for neurodegenerative disorders with TDP-43 proteinopathies.

The Earliest Spanish Dickens? The 1844 Alborada Translation of Pickwick's "Madman's Manuscript"

:This paper takes up a hitherto unnoticed 1844 Spanish translation of "A Madman's Manuscript," an interpolated narrative appearing in chapter 11 of The Pickwick Papers. I argue that the translation was likely the work of Guillermo Macpherson (1824–98), latterly Spain's leading Shakespeare translator; and that the translation must be direct, as the source text had been omitted from versions in French, the most likely mediating language. In analyzing the translation, I find evidence both of inexperience and of renderings consistent with the translator's profile as a heritage speaker. In contextualizing the translation, I document the expatriate and binational Anglophone readerships around Cádiz, where the translation was issued, and examine how English print, in British or continental editions, reached Spanish markets in Dickens's lifetime. I argue that English print reached Cádiz in primarily British editions, by sea, making Andalusia's Englishknowing readers a suggestive case of the diasporic history of print.

Global translational landscape of the Candida albicans morphological transition.

Candida albicans, a major human fungal pathogen associated with high mortality and/or morbidity rates in a wide variety of immunocompromised individuals, undergoes a reversible morphological transition from yeast to filamentous cells that is required for virulence. While previous studies have identified and characterized global transcriptional mechanisms important for driving this transition, as well as other virulence properties, in C. albicans and other pathogens, considerably little is known about the role of genome-wide translational mechanisms. Using ribosome profiling, we report the first global translational profile associated with C. albicans morphogenesis. Strikingly, many genes involved in pathogenesis, filamentation, and the response to stress show reduced translational efficiency (TE). Several of these genes are known to be strongly induced at the transcriptional level, suggesting that a translational fine-tuning mechanism is in place. We also identify potential upstream open reading frames (uORFs), associated with genes involved in pathogenesis, and novel ORFs, several of which show altered TE during filamentation. Using a novel bioinformatics method for global analysis of ribosome pausing that will be applicable to a wide variety of genetic systems, we demonstrate an enrichment of ribosome pausing sites in C. albicans genes associated with protein synthesis and cell wall functions. Altogether, our results suggest that the C. albicans morphological transition, and most likely additional virulence processes in fungal pathogens, is associated with widespread global alterations in TE that do not simply reflect changes in transcript levels. These alterations affect the expression of many genes associated with processes essential for virulence and pathogenesis.

Disruption of the architecture of the junctional sarcoplasmic reticulum in recessive catecholaminergic polymorphic ventricular tachycardia is caused by the er-shaping proteins reep5 and climp63

Abstract Background The recessive variant of Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), a highly lethal inherited arrhythmic disease, is caused by loss of functions mutations in the genes encoding cardiac Calsequestrin (CASQ2) and Triadin (TRD). Disease-mediated decrease in expression of either CASQ2 and TRD in cardiomyocytes, beside affecting calcium handling, profoundly affects the architecture of the junctional SR (jSR) cisternae that appears enlarged and fragmented thus potentially modifying calcium-induced calcium-release. Purpose The present study explores the involvement of SR structural proteins in a recessive CPVT mouse model. We focused on the role of REEP5 and CLIMP63 in shaping the SR in cardiomyocytes in CASQ2-KO mice. The two proteins exert opposing actions: while REEP5 promotes the membrane curvature that helps forming tubules, CLIMP63 promotes the formation of flat cisternal “sheets”. Methods In cardiac tissue of WT and CASQ2-KO mice we compared the transcriptional, translational and post-translational profile of genes encoding for proteins that regulate the ER-architecture. We studied protein interaction and localization by co-immunoprecipitation and immunofluorescence. We processed protein extracts to evaluate the extent of palmitoylation of CLIMP63. Results Our data demonstrate transcriptional repression of REEP5 (p<0.05), and upregulation of CLIMP63 at the transcriptional and translational level (p<0.05) in the heart of CASQ2-KO mice compared to controls. We also investigated the interplay between RyR2 and CLIMP63 by co-immunoprecipitation and documented that in the heart of CASQ2-KO mice this interaction is doubled as compared to WT. Interestingly, we observed that in cardiomyocytes of CASQ2-KO mice the co-localization between RyR2 and CLIMP63 is not affected by the absence of CASQ2. Since it has been shown that palmitoylation is a post-translational modification that regulated the turnover and the retention of CLIMP63 in the endoplasmic reticulum, we therefore hypothesized that the increased abundance of CLIMP63 and its association with RyR2 could be mediated by an increased level in palmitoylation. In agreement with our hypothesis we observed that the amount of protein that is affinity-purified through palmitoylated cysteines is increased in CASQ2-KO mice compared to WT suggesting that this modification, by slowing the turnover of the protein, mediates its accumulation and leads to expansion of SR cisternae. Conclusion Our data, represent the first evidence that post translational modifications of CLIMP63 contribute to the loss of SR homeostatic environment and SR integrity in CPVT mice by breaking the balance between REEP5 and CLIMP63 and therefore reducing the formation of curved tubular membranes in favour of the flat sheet morphology. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Research and University - Dipartimenti di eccellenza 2018-2022 grant to the Molecular Medicine Department (University of Pavia)

Zinc offers splenic protection through suppressing PERK/IRE1-driven apoptosis pathway in common carp (Cyprinus carpio) under arsenic stress

Arsenic (As) occurs naturally and concentrations in water bodies can reach high levels, leading to accumulation in vital organs like the spleen. Being an important organ in immune response and blood development processes, toxic effects of As on the spleen could compromise immunity and cause associated disorders in affected individuals. Splenic detoxification is key to improving the chances of survival but relatively little is known about the mechanisms involved. Essential trace elements like zinc have shown immune-modulatory effects humans and livestock. This study aimed to investigate the mechanisms involved in As-induced splenic toxicity in the common carp (Cyprinus carpio), and the protective effects of zinc (Zn). Our findings suggest that environmental exposure to As caused severe histological injuries and Ca2+ accumulation in the spleen of common carp. Additionally, transcriptional and translational profiles of endoplasmic reticulum stress, apoptosis and autophagy-related genes of the spleen showed upward trends under As toxicity. Treatment with Zn appears to offer protection against As-induced splenic injury in common carp and the pathologic changes above were alleviated. Our results provide additional insight into the mechanism of As toxicity in common carp while elucidating the role of Zn, a natural immune-modulator, as a potential antidote against As poisoning.

Genome-wide translational profiling of amygdala Crh-expressing neurons reveals role for CREB in fear extinction learning

Fear and extinction learning are adaptive processes caused by molecular changes in specific neural circuits. Neurons expressing the corticotropin-releasing hormone gene (Crh) in central amygdala (CeA) are implicated in threat regulation, yet little is known of cell type-specific gene pathways mediating adaptive learning. We translationally profiled the transcriptome of CeA Crh-expressing cells (Crh neurons) after fear conditioning or extinction in mice using translating ribosome affinity purification (TRAP) and RNAseq. Differential gene expression and co-expression network analyses identified diverse networks activated or inhibited by fear vs extinction. Upstream regulator analysis demonstrated that extinction associates with reduced CREB expression, and viral vector-induced increased CREB expression in Crh neurons increased fear expression and inhibited extinction. These findings suggest that CREB, within CeA Crh neurons, may function as a molecular switch that regulates expression of fear and its extinction. Cell-type specific translational analyses may suggest targets useful for understanding and treating stress-related psychiatric illness.

Therapeutic miRNA-Enriched Extracellular Vesicles: Current Approaches and Future Prospects.

Extracellular vesicles (EVs) are 50–300 nm vesicles secreted by eukaryotic cells. They can carry cargo (including miRNA) from the donor cell to the recipient cell. miRNAs in EVs can change the translational profile of the recipient cell and modulate cellular morphology. This endogenous mechanism has attracted the attention of the drug-delivery community in the last few years. EVs can be enriched with exogenous therapeutic miRNAs and used for treatment of diseases by targeting pathological recipient cells. However, there are some obstacles that need to be addressed before introducing therapeutic miRNA-enriched EVs in clinics. Here, we focused on the progress in the field of therapeutic miRNA enriched EVs, highlighted important areas where research is needed, and discussed the potential to use them as therapeutic miRNA carriers in the future.

MicroRNA exchange via extracellular vesicles in cancer.

Cells utilize different means of inter‐cellular communication to function properly. Here, we review the crosstalk between cancer cells and their surrounding environment through microRNA (miRNA)‐containing extracellular vesicles (EVs). The current findings suggest that the export of miRNAs and uptake of miRNA‐containing EVs might be an active process. As post‐transcriptional regulators of gene expression, cancer‐derived miRNAs that are taken up by normal cells can change the translational profile of the recipient cell towards a transformed proteome. Stromal cells can also deliver miRNAs via EVs to cancer cells to support tumour growth and cancer progression. Therefore, gaining a better understanding of EV‐mediated inter‐cellular communication in the tumour microenvironment might lead to the development of novel diagnostic and therapeutic strategies.

Abstract A26: mTOR inhibition promotes differentiation of human regulatory T cells via privileged mRNA translation

Abstract Regulatory T cells (Tregs) maintain immune system homeostasis in the organism. In the tumor microenvironment, however, Tregs can hinder the antitumor immune response. Treg maturation in peripheral sites (pTregs) requires inhibition of protein kinase mTORC1, which paradoxically blocks mRNA translation. mTORC1 phosphorylates (inactivates) 4E-BP1, a negative regulator of the translation initiation factor eIF4E. When mTORC1 is active, 4E-BP1 is hyperphosphorylated and releases eIF4E, which can then bind the mRNA cap structure and promote eIF4F complex assembly and ribosome recruitment. Inhibition of mTORC1 results in hypo-phosphorylation (activation) of 4E-BP1, which then binds and sequesters eIF4E, preventing its interaction with the cap and ribosome association to the mRNA. Using human CD4+ T cells differentiated in vitro, we show that mTORC1 inhibition strongly impairs protein synthesis, yet with exposure to TGFbeta is essential to mediate iTreg differentiation and immune suppression activity. We found that TGFbeta reprograms the T-cell transcriptome, while mTORC1 inhibition reprograms the translatome (genome-wide translation signature), mediating Treg differentiation. Genome-wide transcription and translation profiling identified TGFbeta-upregulated mRNAs resistant to mTORC1 inhibition, in part due to their 5'-untranslated regions, that induce Treg differentiation and immune-suppression activity. These include canonical pTreg fate-determining mRNAs FOXP3, CTLA-4, CD101 and CD103, among others. Inhibition of mTORC1 alone, or TGFbeta alone, induces iTregs with poor immune-suppression activity. Treg differentiation is therefore mediated by an mTORC1-independent privileged mRNA translation mechanism that converts activated CD4+ T cells to Tregs. These findings are quite relevant in the tumor microenvironment, where mTORC1-inhibiting conditions such as hypoxia and deprivation of nutrients and energy are present. Investigation of molecular targets acting in this mTORC1/eIF4E-independent translation of Treg mRNAs is ongoing. These targets could be drugged to selectively inhibit Treg differentiation in the tumor microenvironment, thus allowing effector cells to mount the response against cancer cells. Citation Format: Viviana Volta, Amanda Ernlund, Abhilash Gadi, Amanda Valeta-Magara, Columba de la Parra, Preeyam Patel, Robert J. Schneider. mTOR inhibition promotes differentiation of human regulatory T cells via privileged mRNA translation [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A26.

Abstract IA11: Translating the cancer genome one codon at a time and its therapeutic implications

Abstract Our research is centered on understanding translational control of gene expression in both normal health and disease in a cell- and tissue-specific manner, with a particular focus on cancer biology. Our research combines mouse genetics with genome-wide translational profiling, in-depth molecular biology, and pharmacology to systematically define the points of regulation, in cis and trans, by which the genome is selectively decoded into proteins. We have uncovered that a common denominator of multiple oncogenic pathways is their ability to directly control the core translation machinery of a cell, resulting in the rapid rewiring of mRNA translation programs that promote distinct hallmarks of cancer development such as cell growth, metabolism, and increased motility. For example, our most recent findings delineate the in vivo requirements for a distinct threshold of the major cap-binding protein, eIF4E, a key translation factor downstream of mTOR signaling, in normal organismal development compared to that required for translating the cancer genome. We show that cancer cells require increased eIF4E activity for their survival as distinct subsets of mRNAs that regulate the cancer cell oxidative response are marked by the presence of a novel eIF4E-dependent cis-acting translational control motif present in their 5'UTRs. I will also discuss the generation of the first comprehensive systems-level analysis of the “cancer translatome” during each phase of cancer development in vivo that highlights a dichotomy in transcriptional vs. translational control of gene expression guiding key, select steps in cancer development and evolution. These studies have uncovered a previously unappreciated translation program guiding tumor cell evasion of immune checkpoints. The immediate impact of our research has been the design of a new generation of compounds to target the aberrant translation machinery in cancer cells, which are currently in clinical trials and may reflect a new frontier in cancer therapy. Citation Format: Davide Ruggero. Translating the cancer genome one codon at a time and its therapeutic implications [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA11.

Transcriptome-Wide Analysis of Interplay between mRNA Stability, Translation and Small RNAs in Response to Neuronal Membrane Depolarization

Experience-dependent changes to neural circuitry are shaped by spatially-restricted activity-dependent mRNA translation. Although the complexity of mRNA translation in neuronal cells is widely appreciated, translational profiles associated with neuronal excitation remain largely uncharacterized, and the associated regulatory mechanisms are poorly understood. Here, we employed ribosome profiling, mRNA sequencing and small RNA sequencing to profile transcriptome-wide changes in mRNA translation after whole cell depolarization of differentiated neuroblast cultures, and investigate the contribution of sequence-specific regulatory mechanisms. Immediately after depolarization, a functional partition between transcriptional and translational responses was uncovered, in which many mRNAs were subjected to significant changes in abundance or ribosomal occupancy, but not both. After an extended (2 h) post-stimulus rest phase, however, these changes became synchronized, suggesting that there are different layers of post-transcriptional regulation which are temporally separated but become coordinated over time. Globally, changes in mRNA abundance and translation were found to be associated with a number of intrinsic mRNA features, including mRNA length, GC% and secondary structures; however, the effect of these factors differed between both post-depolarization time-points. Furthermore, small RNA sequencing revealed that miRNAs and tRNA-derived small RNA fragments were subjected to peak changes in expression immediately after stimulation, during which these molecules were predominantly associated with fluctuations in mRNA abundance, consistent with known regulatory mechanisms. These data suggest that excitation-associated neuronal translation is subjected to extensive temporal coordination, with substantial contributions from a number of sequence-dependent regulatory mechanisms.

Ribosomal profiling during prion disease uncovers progressive translational derangement in glia but not in neurons.

Prion diseases are caused by PrPSc, a self-replicating pathologically misfolded protein that exerts toxicity predominantly in the brain. The administration of PrPSc causes a robust, reproducible and specific disease manifestation. Here, we have applied a combination of translating ribosome affinity purification and ribosome profiling to identify biologically relevant prion-induced changes during disease progression in a cell-type-specific and genome-wide manner. Terminally diseased mice with severe neurological symptoms showed extensive alterations in astrocytes and microglia. Surprisingly, we detected only minor changes in the translational profiles of neurons. Prion-induced alterations in glia overlapped with those identified in other neurodegenerative diseases, suggesting that similar events occur in a broad spectrum of pathologies. Our results suggest that aberrant translation within glia may suffice to cause severe neurological symptoms and may even be the primary driver of prion disease.

Theory of arbitrary rigid object motion autofocus for non‐uniform target rotation and translation

Moving targets in synthetic aperture radar (SAR) imagery often yield signatures that are smeared beyond recognition. One mitigation strategy is to attempt to refocus such smears as if the corresponding targets were stationary. This problem is especially difficult for cases in which the target executes time-varying rotation rates for roll, pitch and yaw, as well as for simultaneous non-linear target translational motion. The current analysis develops an arbitrary rigid object motion autofocus (AROMA) for performing automatic refocus of such targets having non-uniform rotation and translation profiles. AROMA uses a maximum likelihood signal-theoretic analysis of the physical rotation angles and translation distances arising from the unknown rigid object motions in order to generate refocused target imagery. The efficacy of this methodology is demonstrated via the injection of a simulated rotating and translating target comprised of multiple scattering centres into a cluttered background of measured Ku-band SAR data. Furthermore, the application of AROMA to an input defocused smear corresponding to a ‘target-of-opportunity’ vehicle in measured Ka-band SAR imagery yields significant improvement in the target focus, thus further validating the underlying theoretical analysis developed herein.

Glycolytic and immunological alterations in human U937 monocytes in response to H1N1 infection.

We monitored changes that took place in glycolytic enzymes, the pyruvate end product of glycolysis, tumor necrosis factor α (TNFα), and toll-like receptors (TLRs) both at the transcriptional and translational levels upon direct interaction between PR8-H1N1 and the human monocytes U937 in vitro system. U937 were first treated with H1N1 infectious viral particles or phorbol-12-myristate-13-acetate (PMA) or left untreated and later infected with the H1N1 virus. Levels of phosphofructokinase 1 (PFK1) and pyruvate were biochemically quantified. In addition, levels of TNFα, TLR3, and TLR7 were measured by ELISA. The transcriptional profiles of PFKs, inflammatory cytokines, TLR3 and TLR7 were relatively quantified by qRT-PCR. The results generally revealed significant changes in both the transcriptional and translational profiles of the studied biochemical and immunological parameters upon influenza infection in a time-dependent manner. In conclusion, H1N1 infection triggers transcriptional and translational changes in immortalized human monocytes, which might serve as markers for infection subject for further validation for their specificities.

MTOR Modulates Intercellular Signals for Enlargement and Infiltration in Glioblastoma Multiforme

Simple SummaryGlioblastoma multiforme (GBM) is the most aggressive and lethal primary brain tumor. Emerging evidence indicate the multi-faceted role of extracellular vesicles (EVs) in GBM growth and proliferation. In fact, GBM-derived EVs can alter the phenotype of GBM-associated parenchymal cells; thus, promoting tumor growth, angiogenesis, and immune evasion. Remarkably, among several pathways that are frequently deregulated in GBM, mammalian Target of Rapamycin (mTOR) up-regulation, and subsequent autophagy (ATG) depression are considered hallmarks of GBM. In fact, mTOR-dependent ATG inhibition strongly correlates with the presence of EVs, which in turn promotes glioblastoma cancer stem cells (GSCs) self-renewal, proliferation, and infiltration. ATG and exosome release are reciprocally regulated. In detail, a failure in ATG enhances exosomal release. Therefore, strategies aimed at targeting on mTOR-dependent extracellular vesicles could be a promising approach for GBM prevention and treatment.Recently, exosomal release has been related to the acquisition of a malignant phenotype in glioblastoma cancer stem cells (GSCs). Remarkably, intriguing reports demonstrate that GSC-derived extracellular vesicles (EVs) contribute to glioblastoma multiforme (GBM) tumorigenesis via multiple pathways by regulating tumor growth, infiltration, and immune invasion. In fact, GSCs release tumor-promoting macrovesicles that can disseminate as paracrine factors to induce phenotypic alterations in glioma-associated parenchymal cells. In this way, GBM can actively recruit different stromal cells, which, in turn, may participate in tumor microenvironment (TME) remodeling and, thus, alter tumor progression. Vice versa, parenchymal cells can transfer their protein and genetic contents to GSCs by EVs; thus, promoting GSCs tumorigenicity. Moreover, GBM was shown to hijack EV-mediated cell-to-cell communication for self-maintenance. The present review examines the role of the mammalian Target of Rapamycin (mTOR) pathway in altering EVs/exosome-based cell-to-cell communication, thus modulating GBM infiltration and volume growth. In fact, exosomes have been implicated in GSC niche maintenance trough the modulation of GSCs stem cell-like properties, thus, affecting GBM infiltration and relapse. The present manuscript will focus on how EVs, and mostly exosomes, may act on GSCs and neighbor non tumorigenic stromal cells to modify their expression and translational profile, while making the TME surrounding the GSC niche more favorable for GBM growth and infiltration. Novel insights into the mTOR-dependent mechanisms regulating EV-mediated intercellular communication within GBM TME hold promising directions for future therapeutic applications.

Abstract 1955: Inhibition of eIF4A by eFT226 blocks KRAS mutant tumor growth

Abstract Mutations in KRAS are among the most common oncogenic lesions across a variety of human cancers. Activation of KRAS directs signaling via the MAPK and PI3K pathways to promote tumor growth. One outcome of enhanced KRAS signaling is the induction of mRNA translation by eIF4A, eIF4E, and eIF4G, which together comprise the eIF4F complex. eIF4A is an RNA helicase that functions to unwind elements in the 5'-untranslated region (UTR) of mRNAs to facilitate scanning of the 40S ribosomal subunit. eFT226 is a highly potent and selective inhibitor of eIF4A that functions by forming a ternary complex between eIF4A, eFT226 and specific polypurine motifs in the 5'-UTR of select mRNAs, thus blocking ribosome scanning and inhibiting mRNA translation. This polypurine sequence motif is highly enriched in the 5'-UTR of eFT226 target genes, many of which are known proto-oncogenes. Translational profiling revealed KRAS to be a target of eFT226 and 5'-UTR sequence analysis of KRAS mRNA identified the polypurine regulatory motif, which imparts sensitivity to eFT226 mediated inhibition of translation. In cell-based reporter assays used to monitor translation, mutation of the KRAS 5'-UTR polypurine motifs resulted in a 10-fold decrease in sensitivity to eFT226 relative to the wild-type sequence. In a cell panel screen for in vitro apoptosis induction by eFT226, the most sensitive models were enriched for cell lines driven by KRAS mutations. Consistent with these results, treatment with eFT226 decreased KRAS protein levels, repressed downstream MAPK signaling, inhibited cell proliferation, and induced apoptosis in a collection of non-small cell lung, colorectal and pancreatic KRAS mutant cancer cell lines. The ability of eFT226 to block tumor cell growth and induce apoptosis is independent of the specific KRAS mutation present (e.g. G12C/V, G13D, Q61H/L) suggesting that eFT226 could be broadly efficacious in treating tumors with activating KRAS mutations. Treatment of KRAS mutant solid tumor xenografts with eFT226 as a monotherapy significantly inhibited tumor growth. Together, these results highlight the ability of eFT226 to inhibit growth and promote apoptosis in KRAS mutant tumors and support the clinical development of eFT226 in KRAS driven tumors. A clinical trial evaluating eFT226 in patients with solid tumor malignancies has initiated. Citation Format: Craig R. Stumpf, Vikas K. Goel, Joan Chen, Jocelyn Staunton, Emily M. Santori, Maria Barrera, Haleigh Howard, Kevin R. Webster, Gary G. Chiang, Peggy A. Thompson. Inhibition of eIF4A by eFT226 blocks KRAS mutant tumor growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1955.

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

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.

Microbiota modulate sympathetic neurons via a gut–brain circuit

Gut-brain connections monitor the intestinal tissue and its microbial and dietary content1, regulating both intestinal physiological functions such as nutrient absorption and motility2,3, and brain–wired feeding behaviour2. It is therefore plausible that circuits exist to detect gut microbes and relay this information to central nervous system (CNS) areas that, in turn, regulate gut physiology4. We characterized the influence of the microbiota on enteric–associated neurons (EAN) by combining gnotobiotic mouse models with transcriptomics, circuit–tracing methods, and functional manipulations. We found that the gut microbiome modulates gut–extrinsic sympathetic neurons; while microbiota depletion led to increased cFos expression, colonization of germ-free mice with short-chain fatty acid–producing bacteria suppressed cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling, and anterograde tracing identified a subset of distal intestine-projecting vagal neurons positioned to play an afferent role in microbiota–mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identified brainstem sensory nuclei activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota–dependent control of gut extrinsic sympathetic activation through a gut-brain circuit.

MetaRibo-Seq measures translation in microbiomes

No method exists to measure large-scale translation of genes in uncultured organisms in microbiomes. To overcome this limitation, we develop MetaRibo-Seq, a method for simultaneous ribosome profiling of tens to hundreds of organisms in microbiome samples. MetaRibo-Seq was benchmarked against gold-standard Ribo-Seq in a mock microbial community and applied to five different human fecal samples. Unlike RNA-Seq, Ribo-Seq signal of a predicted gene suggests it encodes a translated protein. We demonstrate two applications of this technique: First, MetaRibo-Seq identifies small genes, whose identification until now has been challenging. For example, MetaRibo-Seq identifies 2,091 translated, previously unannotated small protein families from five fecal samples, more than doubling the number of small proteins predicted to exist in this niche. Second, the combined application of RNA-Seq and MetaRibo-Seq identifies differences in the translation of transcripts. In summary, MetaRibo-Seq enables comprehensive translational profiling in microbiomes and identifies previously unannotated small proteins.