Translation Initiation Factor eIF4G Research Articles

Linc00941 fuels ribogenesis and protein synthesis by supporting robust cMYC translation in malignant pleural mesothelioma

Malignant pleural mesothelioma is a rare and lethal cancer caused by exposure to asbestos. The highly inflammatory environment caused by fibers accumulation forces cells to undergo profound adaptation to gain survival advantages. Prioritizing the synthesis of essential transcripts is an efficient mechanism coordinated by multiple molecules, including long non-coding RNAs. Enhancing the knowledge about these mechanisms is an essential weapon in combating mesothelioma. Linc00941 correlates to bad prognosis in various cancers, but it is reported to partake in distinct and apparently irreconcilable processes. In this work, we report that linc00941 supports the survival and aggressiveness of mesothelioma cells by influencing protein synthesis and ribosome biogenesis. Linc00941 binds to the translation initiation factor eIF4G, promoting the selective protein synthesis of cMYC, which, in turn, enhances the expression of key genes involved in translation. We analyzed a retrospective cohort of 97 mesothelioma patients' samples from our institution, revealing that linc00941 expression strongly correlates with reduced survival probability. This discovery clarifies linc00941's role in mesothelioma and proposes a unified mechanism of action for this lncRNA involving the selective translation of essential oncogenes, reconciling the discrepancies about its function.

EIF4G as a switch for heat shock mRNA translation

The heat shock response is crucial for cell survival. In this issue of Molecular Cell, Desroches Altamirano etal.1 demonstrate that a temperature-induced conformational change in the translation initiation factor eIF4G is a key mechanism regulating translation during the heat shock response.

Eukaryotic translation initiation factor EIF4G1 p.Ser637Cys mutation in a family with Parkinson's disease with antecedent essential tremor.

Essential tremor (ET) and Parkinson's disease (PD) are common chronic movement disorders that can cause a substantial degree of disability. However, the etiology underlying these two conditions remains poorly understood. In the present study, Whole-exome sequencing of peripheral blood samples from the proband and Sanger sequencing of the other 18 family members, and pedigree analysis of four generations of 29 individuals with both ET and PD in a nonconsanguineous Chinese family were performed. Specifically, family members who had available medical information, including historical documentation and physical examination records, were included. A novel c.1909A>T (p.Ser637Cys) missense mutation was identified in the eukaryotic translation initiation factor 4γ1 (EIF4G1) gene as the candidate likely responsible for both conditions. In total, 9 family members exhibited tremor of the bilateral upper limbs and/or head starting from ages of ≥40 years, 3 of whom began showing evidence of PD in their 70s. Eukaryotic initiation factor 4 (eIF4)G1, a component of the translation initiation complex eIF4F, serves as a scaffold protein that interacts with many initiation factors and then binds to the 40S ribosomal subunit. The EIF4G1 (p.Ser637Cys) might inhibit the recruitment of the mRNA to the ribosome. In conclusion, the results from the present study suggested that EIF4G1 may be responsible for the hereditary PD with 'antecedent ET' reported in the family assessed.

Translational enhancement of target endogenous mRNA in mammalian cells using programmable RNA-binding pentatricopeptide repeat proteins

Programmable protein scaffolds are invaluable in the development of genome engineering tools. The pentatricopeptide repeat (PPR) protein is an attractive platform for RNA manipulation because of its programmable RNA-binding selectivity, which is determined by the combination of amino acid species at three specific sites in the PPR motif. Translation is a key RNA regulatory step that determines the final gene expression level and is involved in various human diseases. In this study, designer PPR protein was used to develop a translational enhancement technique by fusion with the translation initiation factor eIF4G. The results showed that the PPR-eIF4G fusion protein could activate the translation of endogenous c-Myc and p53 mRNAs and control cell fate, indicating that PPR-based translational enhancement is a versatile technique applicable to various endogenous mRNAs in mammalian cells. In addition, the translational enhancement was dependent on both the target position and presence of eIF4G, suggesting the presence of an unknown translation activation mechanism.

RRM1 and PAB domains of translation initiation factor eIF4G (Tif4631p) play a crucial role in the nuclear degradation of export-defective mRNAs in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the CBC-Tif4631p-dependent exosomal targeting (CTEXT) complex consisting of Cbc1/2p, Tif4631p and Upf3p promotes the exosomal degradation of aberrantly long 3'-extended, export-defective transcripts and a small group of normal (termed 'special') mRNAs. We carried out a systematic analysis of all previously characterized functional domains of the major CTEXT component Tif4631p by deleting each of them and interrogating their involvement in the nuclear surveillance of abnormally long 3'-extended and export-defective messages. Our analyses show that the N-terminal RNA recognition motif 1 (RRM1) and poly(A)-binding protein (PAB) domains of Tif4631p, spanning amino acid residues, 1-82 and 188-299 in its primary structure, respectively, play a crucial role in degrading these aberrant messages. Furthermore, the physical association of the nuclear exosome with the altered/variant CTEXT complex harboring any of the mutant Tif4631p proteins lacking either the RRM1 or PAB domain becomes abolished. This finding indicates that the association between CTEXT and the exosome is accomplished via interaction between these Tif4631p domains with the major exosome component, Rrp6p. Abolition of interaction between altered CTEXT (harboring any of the RRM1/PAB-deleted versions of Tif4631p) and the exosome further leads to the impaired recruitment of the RNA targets to the Rrp6p subunit of the exosome carried out by the RRM1/PAB domains of Tif4631p. When analyzing the Tif4631p-interacting proteins, we identified a DEAD-box RNA helicase (Dbp2p), as an interacting partner that turned out to be a previously unknown component of CTEXT. The present study provides a more complete description of the CTEXT complex and offers insight into the functional relationship of this complex with the nuclear exosome.

The mRNA translation initiation factor eIF4G1 controls mitochondrial oxidative phosphorylation, axonal morphogenesis, and memory

mRNA translation initiation plays a critical role in learning and memory. The eIF4F complex, composed of the cap-binding protein eIF4E, ATP-dependent RNA helicase eIF4A, and scaffolding protein eIF4G, is a pivotal factor in the mRNA translation initiation process. eIF4G1, the major paralogue of the three eIF4G family members, is indispensable for development, but its function in learning and memory is unknown. To study the role of eIF4G1 in cognition, we used an eIF4G1 haploinsufficient (eIF4G1-1D) mouse model. The axonal arborization of eIF4G1-1D primary hippocampal neurons was significantly disrupted, and the mice displayed impairment in hippocampus-dependent learning and memory. Translatome analysis showed that the translation of mRNAs encoding proteins of the mitochondrial oxidative phosphorylation (OXPHOS) system was decreased in the eIF4G1-1D brain, and OXPHOS was decreased in eIF4G1-silenced cells. Thus, eIF4G1-mediated mRNA translation is crucial for optimal cognitive function, which is dependent on OXPHOS and neuronal morphogenesis.

Effect of Gene Silencing of Translation Initiation Factors eIF(iso)4G and eIF(iso)4E on Sour Cherry Rootstock Resistance to Sharka Disease.

Sharka disease, caused by the Plum pox virus (PPV), is one of the most harmful, quarantine viral diseases that affect stone fruit crops. The absence of natural resistance to the virus in stone fruits has become a decisive factor for the use of genetic transformation methods to obtain stable forms. The eIF(iso)4G and eIF(iso)4E genes encode translation initiation factors used in the PPV life cycle. In the presented study, the effect of silencing these genes using the RNA interference method on the resistance of sour cherry rootstock 146-2 plants (Prunus pumila L. x Prunus tomentosa Thunb) to the sharka disease was studied. Two vectors have been created for the genetic transformation of plants, with self-complementary sequences of the eIF(iso)4G and eIF(iso)4E gene fragments. The hairpin expression cassette contains a strong promoter of the peach ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) gene, as well as an intron and terminator of the same gene. We used the pMF1 vector containing recombinase R and a codA-nptII gene which makes it possible to obtain intragenic marker-free plants. A successful genetic transformation was carried out by the AGL0 strain of A. tumefaciens. Whole leaves of shoots cultivated in vitro were used as a source of explants. Eight independent transgenic lines of rootstock 146-2 were obtained in experiments (sixlines with a hairpin to the eIF(iso)4G gene and two lines with a hairpin to the eIF(iso)4E gene). Their status was confirmed by the PCR and Southern blotting. The obtained plants were acclimatized in a greenhouse. The silencing of the eIF(iso)4G and eIF(iso)4E genes in transgenic plants was confirmed by the quantitative PCR. The presence of specific small interfering (si) RNAs was confirmed by the method of Northern blotting. Plants of all transgenic rootstock lines were infected with PPV by the method of grafting with infected buds. Resistance to the PPV infection of the obtained transgenic plants was carried out by using an enzyme immunoassay. The ELISA results showed that silencing the eIF(iso)4G gene did not lead to increased resistance while silencing the eIF(iso)4E factor gene led to increased resistance to the PPV, and the one line's plants showed no signs of infection for two years after infecting. The work demonstrates a (promising) approach in which the creation of stone cultures resistant to the plum pox virus can be achieved by suppressing the genes of translation initiation factors in clonal rootstocks.

Mutation of FMDV Lpro H138 residue drives viral attenuation in cell culture and in vivo in swine

The foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) is a papain like protease that cleaves the viral polyprotein and several host factors affecting host cell translation and induction of innate immunity. Introduction of Lpro mutations ablating catalytic activity is not tolerated by the virus, however, complete coding sequence deletion or introduction of targeted amino acid substitutions can render viable progeny. In proof-of-concept studies, we have previously identified and characterized FMDV Lpro mutants that are attenuated in cell culture and in animals, while retaining their capacity for inducing a strong adaptive immunity. By using molecular modeling, we have now identified a His residue (H138), that resides outside the substrate binding and catalytic domain, and is highly conserved across serotypes. Mutation of H138 renders possible FMDV variants of reduced virulence in vitro and in vivo. Kinetics studies showed that FMDV A12-LH138L mutant replicates similarly to FMDV A12-wild type (WT) virus in cells that do not offer immune selective pressure, but attenuation is observed upon infection of primary or low passage porcine epithelial cells. Western blot analysis on protein extracts from these cells, revealed that while processing of translation initiation factor eIF-4G was slightly delayed, no degradation of innate sensors or effector molecules such as NF-κB or G3BP2 was observed, and higher levels of interferon (IFN) and IFN-stimulated genes (ISGs) were induced after infection with A12-LH138L as compared to WT FMDV. Consistent with the results in porcine cells, inoculation of swine with this mutant resulted in a mild, or in some cases, no clinical disease but induction of a strong serological adaptive immune response. These results further support previous evidence that Lpro is a reliable target to derive numerous viable FMDV strains that alone or in combination could be exploited for the development of novel FMD vaccine platforms.

Abstract P2026: S100a1 Protects Cardiomyocytes From Hypertrophic Growth By Controlling The De-novo Synthesis Of Contractile And Mitochondrial Protein Programs

Background and Hypothesis: Loss of S100A1 expression in heart failure contributes to adverse cardiomyocyte (CM) remodeling via an unknown molecular mechanism. Conversely, the antihypertrophic effect of viral-based restoration of S100A1 expression in diseased hearts is not understood yet. We hypothesized that S100A1 may directly interfere with mRNA translation in CMs. Methods and Results: An unbiased mass spectrometry/affinity purification approach using human recombinant S100A1 protein as a bait and murine S100A1 -/- knock-out CM homogenates as prey identified the translation initiation factors EIF4G, EIF2b, RPS6 and PABP as targets for Ca 2+ -bound (holo) S100A1 protein, amongst other translational regulators. Computational modeling revealed top-scoring binding modes for holo-S100A1’s C-terminus in close proximity to RPS6’s ser-235/236 phosphorylation site harboring domain, and PLA detected S100A1/RPS6 complexes in neonatal ventricular cardiomyocytes (NVCMs). siRNA silencing of S100A1 in NVCMs caused a significant increase in cell size (+44%*) and fetal gene expression (e.g. MYH7 4.2-fold*) with a prompt enhancement of the de-novo protein synthesis rate (+89%*), as assessed by IF, RT-PCR and puromycin incorp. up to 48h (n=3-5, *P<0.05 vs contr). Although phospho-specific IB revealed an increase in RPS6-ser235/236 phosphorylation (+38%, n=10, P<0.05) due to S100A1 knock-down, the activity of the upstream Akt/mTORC2 pathway remained unaltered. A nascent proteome analysis unveiled the significant increase and decrease in the de-novo translation of 554 and 17 proteins (FC>1.5), respectively, in S100A1-silenced NVCMs vs contr. with e.g. MYH7, ALC-1, ACTN2 or Nppb in the top 10 newly synthesized proteins. Heart muscle contraction and metabolism-related GO-terms (e.g. myofibril assembly or FA oxidation and OXPHOS) characterized the top 100 upregulated proteins. Conclusion: This is the first study providing evidence for an impact of S100A1 on the mRNA translation machinery in CMs by inhibiting translation of contractile and mitochondrial protein programs required for hypertrophic growth. S100A1 may mechanistically interfere with molecular factors of the translation initiation machinery, which warrants further investigation.

First person – Yun-Ting Tseng

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Yun-Ting Tseng is first author on ‘ Translation initiation factor eIF4G1 modulates assembly of the polypeptide exit tunnel region in yeast ribosome biogenesis’, published in JCS. Yun-Ting is a PhD student in the lab of Kai-Yin Lo at the Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University, working within the field of ribosome biogenesis, exploring how ribosomes are assembled and matured correctly.

Translation initiation factor eIF4G1 modulates assembly of the polypeptide exit tunnel region in yeast ribosome biogenesis

eIF4G is an important eukaryotic translation initiation factor. In this study, eIF4G1, one of the eIF4G isoforms, was shown to directly participate in biogenesis of the large (60S) ribosomal subunit in Saccharomyces cerevisiae cells. Mutation of eIF4G1 decreased the amount 60S ribosomal subunits significantly. The C-terminal fragment of eIF4G1 could complement the function in 60S biogenesis. Analyses of its purified complex with mass spectrometry indicated that eIF4G1 associated with the pre-60S form directly. Strong genetic and direct protein-protein interactions were observed between eIF4G1 and Ssf1 protein. Upon deletion of eIF4G1, Ssf1, Rrp15, Rrp14 and Mak16 were abnormally retained on the pre-60S complex. This purturbed the loading of Arx1 and eL31 at the polypeptide exit tunnel (PET) site and the transition to a Nog2 complex. Our data indicate that eIF4G1 is important in facilitating PET maturation and 27S processing correctly. This article has an associated First Person interview with the first author of the paper.

Proteomic elucidation of the targets and primary functions of the picornavirus 2A protease

Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2Apro and 3Cpro, which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2Apro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2Apro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2Apro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions.

Ribosomal leaky scanning through a translated uORF requires eIF4G2.

eIF4G2 (DAP5 or Nat1) is a homologue of the canonical translation initiation factor eIF4G1 in higher eukaryotes but its function remains poorly understood. Unlike eIF4G1, eIF4G2 does not interact with the cap-binding protein eIF4E and is believed to drive translation under stress when eIF4E activity is impaired. Here, we show that eIF4G2 operates under normal conditions as well and promotes scanning downstream of the eIF4G1-mediated 40S recruitment and cap-proximal scanning. Specifically, eIF4G2 facilitates leaky scanning for a subset of mRNAs. Apparently, eIF4G2 replaces eIF4G1 during scanning of 5′ UTR and the necessity for eIF4G2 only arises when eIF4G1 dissociates from the scanning complex. In particular, this event can occur when the leaky scanning complexes interfere with initiating or elongating 80S ribosomes within a translated uORF. This mechanism is therefore crucial for higher eukaryotes which are known to have long 5′ UTRs with highly frequent uORFs. We suggest that uORFs are not the only obstacle on the way of scanning complexes towards the main start codon, because certain eIF4G2 mRNA targets lack uORF(s). Thus, higher eukaryotes possess two distinct scanning complexes: the principal one that binds mRNA and initiates scanning, and the accessory one that rescues scanning when the former fails.

Binding of eukaryotic initiation factor 3 (eIF3) to Barley Yellow Dwarf Virus (BYDV) untranslated regions (UTRs)

Viruses remain a major threat to human health and the global food sources. Viruses are obligate parasites that use their host's cellular machinery to translate their proteins, usually employing unique non-canonical translation mechanisms to outcompete the host cell mRNA for this machinery. BYDV is a positive-strand RNA virus that lacks the canonical 5ʹ 7-methylguanosine cap and a poly-A tail in its mRNA, instead utilizing a CITE (cap-independent translation element) in the 3ʹ UTR, termed as the 3ʹ BTE (BYDV like CITE). The BYDV 3ʹ BTE is 105-nt long and has a cruciform secondary structure with three stem loops SL-I, SL-II, SL-III and a base stem (SL-IV). BTE has been shown to use the host cell's eukaryotic translation initiation factor (eIF) 4G, eIF4A, eIF4B and ATP (an active helicase complex) to recruit the 40S ribosomal subunit. This 3ʹ BTE-recruited translation initiation complex must transfer to the 5ʹ end of the mRNA for scanning, AUG detection, and translation elongation to proceed. The 5ʹ UTR of BYDV is also structured, consisting of four stem loops, (SL-A, B, C and D), and the 3ʹ to 5ʹ transfer process is aided by a relatively weak, long-distance, ‘kissing-loop’, base pairing interaction between stem-loops in the 5ʹ and 3ʹ ends of BYDV. Recently, we showed that eIF3 plays a crucial role in this transfer by simultaneously binding to both the BYDV 5ʹ and 3ʹ UTRs. These binding interactions were found to be helicase dependent, and the UTRs did not compete for eIF3 binding, suggesting the presence of a transient complex. eIF3 has been shown to interact with both the 5ʹ IRES (Internal ribosome entry site) and 3ʹ UTR of Hepatitis C Virus (HCV) simultaneously. In HCV-like IRESs, the eIF3 and the IRES binding site on the 40S subunit has been shown to overlap, requiring structural rearrangements and dislocation of eIF3 for IRES to bind. In BYDV, we expect similar rearrangements in the UTRs for eIF3 to successfully transfer the 40S complex from the 3ʹ to the 5ʹ end. To understand this macromolecular complex consisting of eIF3, the helicase complex, and the BYDV UTRs, we used a fusion RNA oligo comprising the BYDV 5ʹ UTR and 3ʹ BTE linked by a 12-base RNA linker and performed fluorescence anisotropy-based binding studies to eIF3. Interestingly, the helicase complex was not required for eIF3 binding to the Fusion oligo. SHAPE footprinting analyses performed on the fusion oligo showed that the presence of all helicase factors shifts the eIF3 binding site on the RNA closer to the kissing loop interaction between the UTRs. These data suggest that the helicase complex is not required for binding if the UTRs are in close proximity, but it does aid in rearranging the UTR RNAs to accommodate optimum eIF3 binding for eventual transfer of the 40S complex from the 3ʹ to the 5ʹ end of BYDV.

Articles of Significant Interest in This Issue

Opium poppy mosaic virus (OPMV) is a single-stranded plus-sense RNA virus that does not encode a capsid protein and requires a helper virus. Ilyas et al. (e02109-20) found that OPMV generates two similarly sized subgenomic RNAs (sgRNAs), one of which is synthesized by the replicase and is the template for translation of viral movement proteins. The other is generated by an exoribonuclease-resistant sequence, a mechanism that normally produces small, noncoding sgRNAs. However, this sgRNA codes for a 30-kDa protein with a functional role in OPMV accumulation. The authors also determined that OPMV uses a Barley yellow dwarf virus-like translation element (BTE) as one of its 3′ cap-independent translation enhancers. While BTEs were thought to have little in common beyond a 17-nucleotide conserved sequence, analysis of the 14 known BTE structures showed that BTEs contain additional, strongly conserved sequences and structural features that include residues known to interact with translation initiation factor eIF4G.

Critical Roles of Translation Initiation and RNA Uridylation in Endogenous Retroviral Expression and Neural Differentiation in Pluripotent Stem Cells

Previous studies have suggested that the loss of the translation initiation factor eIF4G1 homolog NAT1 induces excessive self-renewability of naive pluripotent stem cells (PSCs); yet the role of NAT1 in the self-renewal and differentiation of primed PSCs is still unclear. Here, we generate a conditional knockout of NAT1 in primed PSCs and use the cells for the functional analyses of NAT1. Our results show that NAT1 is required for the self-renewal and neural differentiation of primed PSCs. In contrast, NAT1 deficiency in naive pluripotency attenuates the differentiation to all cell types. We also find that NAT1 is involved in efficient protein expression of an RNA uridyltransferase, TUT7. TUT7 is involved in the neural differentiation of primed PSCs via the regulation of human endogenous retrovirus accumulation. These data demonstrate the essential roles of NAT1 and TUT7 in the precise transition of stem cell fate.

PABPN1, a Target of p63, Modulates Keratinocyte Differentiation through Regulation of p63α mRNA Translation

p63 is expressed from two promoters and produces two N-terminal isoforms, TAp63 and ΔNp63. Alternative splicing creates three C-terminal isoforms p63α, p63β, and p63δ, whereas alternative polyadenylation (APA) in coding sequence creates two more C-terminal isoforms p63γ and p63ε. Although several transcription factors have been identified to differentially regulate the N-terminal p63 isoforms, it is unclear how the C-terminal p63 isoforms are regulated. Thus, we determined whether PABPN1, a key regulator of APA, may differentially regulate the C-terminal p63 isoforms. We found that PABPN1 deficiency increases p63γ mRNA through APA in coding sequence. We also found that PABPN1 is necessary for p63α translation by modulating the binding of translation initiation factors eIF4E and eIF4G to p63α mRNA. Moreover, we found that the p53 family, especially p63α, regulates PABPN1 transcription, suggesting that the mutual regulation between p63 and PABPN1 forms a feedback loop. Furthermore, we found that PABPN1 deficiency inhibits keratinocyte cell growth, which can be rescued by ectopic ΔNp63α. Finally, we found that PABPN1 controls the terminal differentiation of HaCaT keratinocytes by modulating ΔNp63α expression. Taken together, our findings suggest that PABPN1 is a key regulator of the C-terminal p63 isoforms through APA in coding sequence and mRNA translation and that the p63-PABPN1 loop modulates p63 activity and the APA landscape.

Abstract 4142: Physical and functional interactions between MNK and mTOR signaling regulate the activation and differentiation of T cells

Abstract The role of the PI3K/mTOR and MAPK signaling pathways in regulating T cell activation and differentiation is well established. It has been shown that mTOR acts as a sensor of the T cell metabolic state, coordinating diverse inputs to determine the balance between effector versus memory cell fates. Similarly, the MAPK interacting kinases, MNK1 and MNK2, are key downstream effector kinases that mediate post-transcriptional gene regulation of critical mediators of the T cell response, including immune checkpoint proteins and cytokines, via the phosphorylation of specific RNA binding proteins such as eIF4E and hnRNPA1. eFT508 is a potent and selective inhibitor of both MNK1 and MNK2, which enhances anti-tumor immune responses by decreasing the expression of immune response modulators including PD-1, PD-L1, LAG3, TIM3 and IL-10. Furthermore, eFT508 treatment boosts memory T cell populations while increasing the effectiveness of cytotoxic T lymphocytes. Phosphoproteomic analysis of the effects of eFT508 on early T cell activation identified novel eFT508 sensitive phosphopeptides, including a subset that overlaps with mTOR-dependent phosphorylation sites. In vitro biochemical analysis has shown that eFT508 is not a mTOR kinase inhibitor and that most of these sites are not direct MNK substrates. These results suggest inhibition of MNK by eFT508 can have significant effects on mTOR signaling. Consistent with these findings, we observe a physical association between MNK and mTOR that is disrupted by either eFT508 or the allosteric mTOR inhibitor rapamycin. Our phosphoproteomic analysis also identified novel MNK-dependent phosphorylation sites within the translation initiation factor eIF4G1 that regulate the ability of mTOR to recognize eIF4G1 as a substrate. In addition, phosphorylation of the mTOR target 4EBP1 is decreased upon treatment of T cells with eFT508, although 4EBP1 itself is not a direct substrate of MNK in vitro. These data are consistent with a role for MNK in regulating mRNA translation, not only through phosphorylation of eIF4E, but also by modulating the activity of mTOR toward specific substrates. Given the role of mTOR in controlling memory T cell differentiation, our results provide a potential mechanistic basis to explain the impact of MNK inhibition on T cell differentiation. Ongoing studies are further characterizing the consequences of eFT508 regulation on MNK/mTOR signaling and T cell differentiation. This work significantly expands our current understanding of eFT508’s mechanism of action as well as its ability to promote a prolonged anti-tumor immune response. Citation Format: Craig R. Stumpf, Vikas K. Goel, Rajesh K. Sharma, Gary G. Chiang, Peggy A. Thompson, Kevin R. Webster. Physical and functional interactions between MNK and mTOR signaling regulate the activation and differentiation of T cells [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 4142.

RGG-motif self-association regulates eIF4G-binding translation repressor protein Scd6

ABSTRACTRegulation of mRNA translation plays a key role in the control of gene expression. Scd6, a conserved RGG-motif containing protein represses translation by binding to translation initiation factor eIF4G1. Here we report that Scd6 binds itself in RGG-motif dependent manner and self-association regulates its repression activity. Scd6 self-interaction competes with eIF4G1 binding and methylation of Scd6 RGG-motif by Hmt1 negatively affects self-association. Results pertaining to Sbp1 indicate that self-association could be a general feature of RGG-motif containing translation repressor proteins. Taken together, our study reveals a mechanism of regulation of eIF4G-binding RGG-motif translation repressors.

Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo.

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.