Translation Initiation Factor eIF4E Research Articles

Structural studies of the eIF4E-VPg complex reveal a direct competition for capped RNA: Implications for translation.

Viruses have transformed our understanding of mammalian RNA processing, including facilitating the discovery of the methyl-7-guanosine (m7G) cap on the 5' end of RNAs. The m7G cap is required for RNAs to bind the eukaryotic translation initiation factor eIF4E and associate with the translation machinery across plant and animal kingdoms. The potyvirus-derived viral genome-linked protein (VPg) is covalently bound to the 5' end of viral genomic RNA (gRNA) and associates with host eIF4E for successful infection. Divergent models to explain these observations proposed either an unknown mode of eIF4E engagement or a competition of VPg for the m7G cap-binding site. To dissect these possibilities, we resolved the structure of VPg, revealing a previously unknown 3-dimensional (3D) fold, and characterized the VPg-eIF4E complex using NMR and biophysical techniques. VPg directly bound the cap-binding site of eIF4E and competed for m7G cap analog binding. In human cells, VPg inhibited eIF4E-dependent RNA export, translation, and oncogenic transformation. Moreover, VPg formed trimeric complexes with eIF4E-eIF4G, eIF4E bound VPg-luciferase RNA conjugates, and these VPg-RNA conjugates were templates for translation. Informatic analyses revealed structural similarities between VPg and the human kinesin EG5. Consistently, EG5 directly bound eIF4E in a similar manner to VPg, demonstrating that this form of engagement is relevant beyond potyviruses. In all, we revealed an unprecedented modality for control and engagement of eIF4E and show that VPg-RNA conjugates functionally engage eIF4E. As such, potyvirus VPg provides a unique model system to interrogate eIF4E.

Functional characterization of the translation initiation factor eIF4E of Echinococcus granulosus.

The eukaryotic initiation factor 4E (eIF4E) specifically recognizes the 5' mRNA cap, a rate-limiting step in the translation initiation process. Although the 7-methylguanosine cap (MMGcap) is the most common 5' cap structure in eukaryotes, the trans-splicing process that occurs in several organism groups, including nematodes and flatworms, leads to the addition of a trimethylguanosine cap (TMGcap) to some RNA transcripts. In some helminths, eIF4E can have a dual capacity to bind both MMGcap and TMGcap. In the present work, we evaluated the distribution of eIF4E protein sequences in platyhelminths and we showed that only one gene coding for eIF4E is present in most parasitic flatworms. Based on this result, we cloned the Echinococcus granulosus cDNA sequence encoding eIF4E in Escherichia coli, expressed the recombinant eIF4E as a fusion protein to GST, and tested its ability to capture mRNAs through the 5' cap using pull-down assay and qPCR. Our results indicate that the recombinant eIF4E was able to bind preferentially 5'-capped mRNAs compared with rRNAs from total RNA preparations of E. granulosus. By qPCR, we observed an enrichment in MMG-capped mRNA compared with TMG-capped mRNAs among Eg-eIF4E-GST pull-down RNAs. Eg-eIF4E structural model using the Schistosoma mansoni eIF4E as template showed to be well preserved with only a few differences between chemically similar amino acid residues at the binding sites. These data showed that E. granulosus eIF4E can be used as a potential tool to study full-length 5'-capped mRNA, besides being a potential drug target against parasitic flatworms.

Abstract 1302: Targeting hormone receptor-dependent cancers with potent, selective and orally-available small molecule inhibitors of eIF4E

Abstract The PI3K/mTOR pathway is commonly dysregulated in many hormone receptor-dependent tumors and plays a key role in promoting tumor growth and mediating drug resistance. In particular, PI3K and mTORC1/2 inhibitors have been intensively studied in the treatment of hormone receptor-dependent cancers and have shown benefit in some clinical settings. However, issues such as dose-limiting toxicities and emergent resistance limit the broader utility of these inhibitors. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for both the PI3K/mTOR and MAPK signaling pathways. We have designed a series of potent, selective and orally-available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind to eIF4E either as its free form or with eIF4E-4EBP and eIF4F complexes within tumor cells and downregulate hormone receptor-dependent signaling. Ribosomal profiling of eIF4E inhibitor-treated tumor cells identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also a unique set of translationally regulated target mRNAs. Consistent with this observation, our eIF4E inhibitors show some similarities yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiological assays. Finally, significant anti-tumor efficacy was observed with eIF4E inhibition in vitro and in vivo. Taken together, these results highlight the potential for targeting eIF4E as a novel therapeutic strategy to treat hormone-receptor dependent cancers. Citation Format: Gary G. Chiang, Gregory S. Parker, Ivy N. Hung, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Cody Parker, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urkalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Kevin R. Webster. Targeting hormone receptor-dependent cancers with potent, selective and orally-available small molecule inhibitors of eIF4E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1302.

Characterization of Hydration Properties in Structural Ensembles of Biomolecules.

Solute-solvent interactions are critical for biomolecular stability and recognition. Explicit solvent molecular dynamics (MD) simulations are routinely used to probe such interactions. However, detailed analyses and interpretation of the hydration patterns seen in MD simulations can be both complex and time-consuming. A variety of approaches/tools to compute and interrogate hydration properties in structural ensembles of proteins, nucleic acids, or in general any molecule are available and are complemented here with a new and free software package ("JAL"). Central to "JAL" is an intuitive atom centric approach of computing hydration properties. In addition to the standard metrics commonly used to understand hydration, "JAL" introduces two nonstandard utilities: a program to rapidly compute buried waters in an MD trajectory and a new method to compute multiwater bridges around a solute. We demonstrate the utility of the package by probing the hydration characteristics of the tumor suppressor protein p53 and the translation initiation factor eif4E. "JAL" is hosted online and can be accessed for free at http://mspc.bii.a-star.edu.sg/minhn/jal.html .

Biochemical and Structural Insights into the Eukaryotic Translation Initiation Factor eIF4E.

A major question in cell and cancer biology is concerned with understanding the flow of information from gene to protein. Indeed, many studies indicate that the proteome can be decoupled from the transcriptome. A major source of this decoupling is post-transcriptional regulation. The eukaryotic translation initiation factor eIF4E serves as an excellent example of a protein that can modulate the proteome at the post-transcriptional level. eIF4E is elevated in many cancers thus highlighting the relevance of this mode of control to biology. In this review, we provide a brief overview of various functions of eIF4E in RNA metabolism e.g. in nuclear-cytoplasmic RNA export, translation, RNA stability and/or sequestration. We focus on the modalities of eIF4E regulation at the biochemical and particularly structural level. In this instance, we describe not only the importance for the m7Gcap eIF4E interaction but also of recently discovered non-traditional RNA-eIF4E interactions as well as cap-independent activities of eIF4E. Further, we describe several distinct structural modalities used by the cell and some viruses to regulate or co-opt eIF4E, substantially extending the types of proteins that can regulate eIF4E from the traditional eIF4E-binding proteins (e.g. 4E-BP1 and eIF4G). Finally, we provide an overview of the results of targeting eIF4E activity in the clinic.

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins.

The mRNA 5′ cap consists of N7-methylguanosine bound by a 5′,5′-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap–protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap–protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays.

Hydrodynamic Behavior of the Intrinsically Disordered Potyvirus Protein VPg, of the Translation Initiation Factor eIF4E and of their Binary Complex.

Protein intrinsic disorder is involved in many biological processes and good experimental models are valuable to investigate its functions. The potyvirus genome-linked protein, VPg, displays many features of an intrinsically disordered protein. The virus cycle requires the formation of a complex between VPg and eIF4E, one of the host translation initiation factors. An in-depth characterization of the hydrodynamic properties of VPg, eIF4E, and of their binary complex VPg-eIF4E was carried out. Two complementary experimental approaches, size-exclusion chromatography and fluorescence anisotropy, which is more resolving and revealed especially suitable when protein concentration is the limiting factor, allowed to estimate monomers compaction upon complex formation. VPg possesses a high degree of hydration which is in agreement with its classification as a partially folded protein in between a molten and pre-molten globule. The natively disordered first 46 amino acids of eIF4E contribute to modulate the protein hydrodynamic properties. The addition of an N-ter His tag decreased the conformational entropy of this intrinsically disordered region. A comparative study between the two tagged and untagged proteins revealed the His tag contribution to proteins hydrodynamic behavior.

O-GlcNAcylation alters the selection of mRNAs for translation and promotes 4E-BP1–dependent mitochondrial dysfunction in the retina

Diabetes promotes the posttranslational modification of proteins by O-linked addition of GlcNAc (O-GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is O-GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O-GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O-GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs (i.e. mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, ∼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of O-GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O-GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O-GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O-GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation.

Nuclear eIF4E Stimulates 3′-End Cleavage of Target RNAs

SUMMARYThe eukaryotic translation initiation factor eIF4E is nuclear and cytoplasmic where it plays roles in export and translation of specific transcripts, respectively. When we were studying its mRNA export activity, we unexpectedly discovered that eIF4E drives the protein expression of elements of the 3′-end core cleavage complex involved in cleavage and polyadenylation (CPA), including CPSF3, the enzyme responsible for cleavage, as well as its co-factors CPSF1, CPSF2, CPSF4, Symplekin, WDR33, and FIP1L1. Using multiple strategies, we demonstrate that eIF4E stimulates 3′-end cleavage of selected RNAs. eIF4E physically interacts with CPSF3, CPSF1, and uncleaved target RNA, suggesting it acts directly and indirectly on the pathway. Through these effects, eIF4E can generate better substrates for its mRNA export and translation activities. Thus, we identified an unanticipated function for eIF4E in 3′-end processing of specific target RNAs, and this function could potentially affect the expression of a broad range of oncoproteins.

The 3' Untranslated Region of a Plant Viral RNA Directs Efficient Cap-Independent Translation in Plant and Mammalian Systems.

Many plant viral RNA genomes lack a 5′ cap, and instead are translated via a cap-independent translation element (CITE) in the 3′ untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation initiation factor eIF4E to facilitate translation. eIF4E is structurally conserved between plants and animals, so we tested cap-independent translation efficiency of PTEs of nine plant viruses in plant and mammalian systems. The PTE from thin paspalum asymptomatic virus (TPAV) facilitated efficient cap-independent translation in wheat germ extract, rabbit reticulocyte lysate, HeLa cell lysate, and in oat and mammalian (BHK) cells. Human eIF4E bound the TPAV PTE but not a PTE that did not stimulate cap-independent translation in mammalian extracts or cells. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting revealed that both human and wheat eIF4E protected the conserved guanosine (G)-rich domain in the TPAV PTE pseudoknot. The central G plays a key role, as it was found to be required for translation and protection from SHAPE modification by eIF4E. These results provide insight on how plant viruses gain access to the host’s translational machinery, an essential step in infection, and raise the possibility that similar PTE-like mechanisms may exist in mRNAs of mammals or their viruses.

Phosphoglycerate dehydrogenase promotes pancreatic cancer development by interacting with eIF4A1 and eIF4E

BackgroundPancreatic cancer is one of the most malignant cancers. The overall 5-year survival rate of its patients is 8%, the lowest among major cancer types. It is very urgent to study the development mechanisms of this cancer and provide potential targets for therapeutics design. Glucose, one of the most essential nutrients, is highly exploited for aerobic glycolysis in tumor cells to provide building blocks. However, the glucose consumption manner in pancreatic cancer cells is unclear. And the mechanism of the substantial metabolic pathway promoting pancreatic cancer development is also unrevealed.Methods13C6 glucose was used to trace the glucose carbon flux and detected by mass spectrum. The expressions of PHGDH were determined in cells and pancreatic adenocarcinomas. Knockdown and overexpression were performed to investigate the roles of PHGDH on pancreatic cancer cell proliferation, colony formation and tumor growth. The mechanisms of PHGDH promoting pancreatic cancer development were studied by identifying the interacting proteins and detecting the regulatory functions on translation initiations.ResultsPancreatic cancer cells PANC-1 consumed large amounts of glucose in the serine and glycine de novo synthesis. Phosphoglycerate dehydrogenase (PHGDH) highly expressed and controlled this pathway. Knockdown of PHGDH significantly attenuated the tumor growth and prolonged the survival of tumor bearing mice. The pancreatic adenocarcinoma patients with low PHGDH expression had better overall survival. Mechanistically, knockdown of PHGDH inhibited cell proliferation and tumorigenesis through disrupting the cell-cell tight junctions and the related proteins expression. Besides catalyzing serine synthesis to activate AKT pathway, PHGDH was found to interact with the translation initiation factors eIF4A1 and eIF4E and facilitated the assembly of the complex eIF4F on 5’ mRNA structure to promote the relevant proteins expression.ConclusionBesides catalyzing serine synthesis, PHGDH promotes pancreatic cancer development through enhancing the translation initiations by interacting with eIF4A1 and eIF4E. Inhibiting the interactions of PHGDH/eIF4A1 and PHGDH/eIF4E will provide potential targets for anti-tumor therapeutics development.

Abstract A222: Exploiting mTORC1-independent protein synthesis in Tregs to boost antitumor immune response

Abstract Regulatory T-cells (Tregs) maintain immune system homeostasis in the organism. In the tumor micro-environment, however, Tregs can hinder 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 induces 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, Columba De la Parra, Abilash Gadi, Amanda Valeta-Magara, Robert J. Schneider. Exploiting mTORC1-independent protein synthesis in Tregs to boost antitumor immune response [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A222.

Chemical shift assignment of the viral protein genome-linked (VPg) from potato virus Y.

The dysregulation of translation contributes to many pathogenic conditions in humans. Discovering new translational mechanisms is important to understanding the diversity of this process and its potential mechanisms. Such mechanisms can be initially observed in viruses. With this in mind, we studied the viral protein genome-linked VPg factor from the largest genus of plant viruses. Studies in plants show that VPg binds to the eukaryotic translation initiation factor eIF4E for translation of viral RNAs. VPg contains no known eIF4E binding motifs and no sequence homology to any known proteins. Thus, as a first step in understanding the structural basis of this interaction, we carried out NMR assignments of the VPg from the potato virus Y potyvirus protein.

Roles of eIF4E-binding protein Caf20 in Ste12 translation and P-body formation in yeast

Translation initiation factor eIF4E forms eIF4E-eIF4G complex at the 5' cap of mRNA. This interaction can be inhibited by the family of 4E-binding proteins (4E-BP). In yeast Saccharomyces cerevisiae, two 4E-BPs, Caf20 and Eap1, compete with eIF4G for binding to eIF4E via the shared conserved interaction motif. In order to investigate the roles of Caf20 in gene-specific translational regulation and the formation of mRNA granules (P-bodies), we introduced substitution mutations, caf20-Y4A or caf20-L9A, in the eIF4E-binding motif for CAF20. Overexpression of the wild-type CAF20 showed an increased protein level of Ste12 transcription factor as well as highly developed P-body formation. However, 4E-binding site mutations of CAF20 led to a reduced number of P-body foci and decreased levels of Ste12 protein. The phenotypes of the caf20 deletion mutation were also analyzed, and we suggest that Caf20 plays a critical role in Ste12 protein expression and in the control of P-body formation.

Mutation of a Nicotiana tabacum L. eukaryotic translation-initiation factor gene reduces susceptibility to a resistance-breaking strain of Potato virus Y.

Eukaryotic translation-initiation factors eIF4E and eIF(iso)4E in plants play key roles in infection by potyviruses and other plant RNA viruses. Mutations in the genes encoding these factors reduce susceptibility to the viruses, and are the basis of several recessive virus resistance genes widely used in plant breeding. Because virus variants occasionally break such resistance, the molecular basis for this process must be elucidated. Although deletion mutants of eIF4E1-S of tobacco (Nicotiana tabacum L.) resist Potato virus Y (PVY; the type member of the genus Potyvirus), resistance-breaking strains of PVY threaten tobacco production worldwide. Here, we used RNA interference technology to knock down tobacco eIF4E2-S and eIF4E2-T genes or eIF(iso)4E-S and eIF(iso)4E-T genes. Transgenic plants with reduced transcript levels of both eIF(iso)4E-S and eIF(iso)4E-T showed reduced susceptibility to a resistance-breaking PVY strain with a K105E mutation in the viral genome-associated protein (VPg). By screening a population of chemically induced mutants of eIF(iso)4E-S and eIF(iso)4E-T, we showed that plants with a nonsense mutation in eIF(iso)4E-T, but not eIF(iso)4E-S, showed reduced susceptibility to the resistance-breaking PVY strain. In a yeast two-hybrid assay, VPg of the resistance-breaking strain, but not wild-type PVY, physically interacted with the eIF(iso)4E-T protein. Thus, eIF4E1-S is required for infection by PVY, but eIF(iso)4E-T is required for infection by the resistance-breaking strain. Our study provides the first evidence for the involvement of a host eukaryotic translation-initiation factor in the infection cycle of a resistance-breaking virus strain. The eIF(iso)4E-T mutants will be useful in tobacco breeding to introduce resistance against resistance-breaking PVY strains.

Abstract 3855: Inhibition of MNK by eFT508 reprograms T-cell signaling to promote an antitumor immune response

Abstract Mitogen-activated protein (MAP) kinase signaling cascades play a vital role in T-cell activation upon antigen recognition. MNK1 and MNK2 are important downstream effector kinases in the MAPK pathway that largely function in regulating the expression of important signaling molecules, including cytokines and immune checkpoint receptors. MNKs are primarily thought to regulate the expression of select mRNAs, predominantly via post-transcriptional mechanisms involving the phosphorylation of the eukaryotic translation initiation factor eIF4E as well as the RNA binding proteins hnRNPA1 and PSF. eFT508 is a potent and highly selective inhibitor of MNK1 and MNK2 that has been shown to promote antitumor immunity by decreasing the expression of immunosuppressive molecules, such as immune checkpoint receptors. In order to identify key T-cell components that are regulated by MNK phosphorylation and may mediate the effects of eFT508 treatment, we performed an unbiased phosphoproteomic analysis of T cells during the early stages of T-cell receptor-mediated stimulation with and without eFT508 treatment. Primary human T cells were pretreated with eFT508 for two hours prior to stimulation with αCD3/αCD28 for an additional 30 minutes. Protein samples were then prepared for multiplexed phosphoproteomic analysis by mass spectrometry. Consistent with previous proteomic studies using stimulated T cells, a number of phosphopeptides associated with T-cell receptor signaling, among other cellular activities, were detected. Moreover, treatment with eFT508 specifically blocked the phosphorylation of distinct phosphosites on select proteins. The phosphoproteins modulated by eFT508 treatment are involved in important T-cell signaling pathways, cell proliferation and differentiation programs, stress responses, and post-transcriptional and translational gene regulation. Furthermore, there was enrichment for specific sequences surrounding the phosphorylation site in eFT508-sensitive peptides, highlighting a potential mechanism mediating MNK target recognition. Confirmation of MNK-mediated phosphorylation of novel substrates is being conducted in vitro by biochemical analysis of direct phosphorylation of potential substrates by MNK1 or MNK2 and in cellular lysates treated with eFT508 by Western blot analysis using phosphosite-specific antibodies. These findings have significantly expanded our understanding of cell signaling through MNK1 and MNK2 and will help to illuminate potential regulatory programs through which inhibition of MNKs by eFT508 can modulate antitumor immunity. Citation Format: Craig R. Stumpf, Joan Chen, Vikas K. Goel, Gregory S. Parker, Gary G. Chiang, Peggy A. Thompson, Kevin R. Webster. Inhibition of MNK by eFT508 reprograms T-cell signaling to promote an antitumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3855.

Recessive Resistance to Bean common mosaic virus Conferred by the bc-1 and bc-2 Genes in Common Bean (Phaseolus vulgaris) Affects Long-Distance Movement of the Virus.

Recessive resistance to Bean common mosaic virus (BCMV) in common bean (Phaseolus vulgaris) is governed by four genes that include one strain-nonspecific helper gene bc-u, and three strain-specific genes bc-1, bc-2, and bc-3. The bc-3 gene was identified as an eIF4E translation initiation factor gene mediating resistance through disruption of the interaction between this protein and the VPg protein of the virus. The mode of action of bc-1 and bc-2 in expression of BCMV resistance is unknown, although bc-1 gene was found to affect systemic spread of a related potyvirus, Bean common mosaic necrosis virus. To investigate the possible role of both bc-1 and bc-2 genes in replication, cell-to-cell, and long-distance movement of BCMV in P. vulgaris, we tested virus spread of eight BCMV isolates representing pathogroups I, IV, VI, VII, and VIII in a set of bean differentials expressing different combinations of six resistance alleles including bc-u, bc-1, bc-12, bc-2, bc-22, and bc-3. All studied BCMV isolates were able to replicate and spread in inoculated leaves of bean cultivars harboring bc-u, bc-1, bc-12, bc-2, and bc-22 alleles and their combinations, while no BCMV replication was found in inoculated leaves of cultivar IVT7214 carrying the bc-u, bc-2, and bc-3 genes, except for isolate 1755a, which was capable of overcoming the resistance conferred by bc-2 and bc-3. In contrast, the systemic spread of all BCMV isolates from pathogroups I, IV, VI, VII, and VIII was impaired in common bean cultivars carrying bc-1, bc-12, bc-2, and bc-22 alleles. The data suggest that bc-1 and bc-2 recessive resistance genes have no effect on the replication and cell-to-cell movement of BCMV, but affect systemic spread of BCMV in common bean. The BCMV resistance conferred by bc-1 and bc-2 and affecting systemic spread was found only partially effective when these two genes were expressed singly. The efficiency of the restriction of the systemic spread of the virus was greatly enhanced when the alleles of bc-1 and bc-2 genes were combined together.

Translatome Regulation in Neuronal Injury and Axon Regrowth.

Transcriptional events leading to outgrowth of neuronal axons have been intensively studied, but the role of translational regulation in this process is not well understood. Here, we use translatome analyses by ribosome pull-down and protein synthesis characterization by metabolic isotopic labeling to study nerve injury and axon outgrowth proteomes in rodent dorsal root ganglia (DRGs) and sensory neurons. We identify over 1600 gene products that are primarily translationally regulated in DRG neurons after nerve injury, many of which contain a 5’UTR cytosine-enriched regulator of translation (CERT) motif, implicating the translation initiation factor Eif4e in the injury response. We further identified approximately 200 proteins that undergo robust de novo synthesis in the initial stages of axon growth. ApoE is one of the highly synthesized proteins in neurons, and its receptor binding inhibition or knockout affects axon outgrowth. These findings provide a resource for future analyses of the role of translational regulation in neuronal injury responses and axon extension.

Rbm24, a target of p53, is necessary for proper expression of p53 and heart development.

Activation of p53-dependent apoptosis is critical for tumor suppression but aberrant activation of p53 also leads to developmental defects and heart failure. Here, we found that Rbm24 RNA-binding protein, a target of p53, regulates p53 mRNA translation. Mechanistically, we found that through binding to p53 mRNA and interaction with translation initiation factor eIF4E, Rbm24 prevents eIF4E from binding to p53 mRNA and inhibits the assembly of translation initiation complex. Importantly, we showed that mice deficient in Rbm24 die in utero due to the endocardial cushion defect in the heart at least in part due to aberrant activation of p53-dependent apoptosis. We also showed that the heart developmental defect in Rbm24-null mice can be partially rescued by p53 deficiency through decreased apoptosis in the heart. Together, we postulate that the p53-Rbm24 loop is critical for the heart development and may be explored for mitigating congenital heart diseases and heart failure.

Cytocapsular tubes conduct cell translocation

Cell locomotion is essential for multicellular organism embryo development, organ homeostasis, tissue regeneration, immune responses, and tumor metastasis. Here we report that single mammalian cells can generate two extracellular membranous compartments: cytocapsulae and cytocapsular tubes. Cells migrate in cytocapsulae and engender cytocapsular tubes, which exhibit pleiotropic biological functions and provide tubular routes for directed cell transportation. Ultrastructural analysis by electron microscope revealed that nanoprotrusions surround and anchor cytocapsular tubes in place. Multiple cytocapsular tubes interconnect and form networks supporting directed cell transportation in diverse directions. Enhanced translation initiation factor eIF4E up-regulates translation of transcripts encoding proteins important for organelle development. Thus, this study proposes a mechanism of directed cell translocation in cytocapsular tubes, which may facilitate the management of diseases, including tumor metastasis.