Eukaryotic Elongation Research Articles

Single-Molecule Studies of Cognate and Near-Cognate Elongation in an in vitro Eukaryotic Translation System.

The ribosome plays a central role in translation of the genetic code into amino acid sequences during synthesis of polypeptides. During each cycle of peptide elongation, the ribosome must discriminate between correct and incorrect aminoacyl-tRNAs according to the codon present in its A-site. Ribosomes rely on a complex sequence of proofreading mechanisms to minimize erroneous selection of incorrect aminoacyl-tRNAs that would lead to mistakes in translation. These mechanisms have been studied extensively in prokaryotic organisms, but eukaryotic elongation is less well understood. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) with an in vitro eukaryotic translation system to investigate tRNA selection and subsequent steps during peptide elongation. We compared accommodation of a tryptophan-aminoacyl-tRNA into the ribosomal A-site containing either a cognate or near-cognate codon and unexpectedly found that, following an initial slow sampling event, subsequent near-cognate sampling events proceeded more rapidly than the initial event. Further, we found a strong negative correlation between the concentration of near-cognate aminoacyl-tRNA and the efficiency of tRNA accommodation. These novel characteristics of near-cognate interaction with the eukaryotic ribosome suggest that rejection of a near-cognate tRNAs leads to formation of an altered ribosomal conformation that assists in rejecting subsequent incorrect tRNA interactions.

Abstract 11462: A Surprising Role of TNIK in Regulating Endothelial Interferon Signaling and Activation

Background: Endothelial cells (ECs) predominantly govern vascular tone and homeostasis. Activation of ECs is the central pathophysiological process in the initiation of atherosclerosis (AS). Interferon signaling (IFN Type I and II) is activated in ECs under inflammatory conditions such as systemic lupus erythematosus and is implicated in EC activation. Yet, the underlying mechanisms are still unknown. Through RNA sequencing and pathway analysis we have identified, TNIK, a Ste20 family of MAP kinases (MAP4K) to regulate IFN signaling in ECs. Our study is the first to report the role and function of TNIK in IFN and EC activation. Hypothesis: TNIK regulates IFNs through focal adhesion kinase (FAK) mediated STAT signaling in EC activation (Fig 1A). Methods: Cultured human aortic endothelial cells (HAECs) were treated with control or TNIK-targeted siRNA. Later cells were subjected to static, laminar or disturbed flow conditions. Total RNA was isolated and then RNA-sequencing was performed. The differential expression data were analyzed using CLC genomics and Ingenuity Pathway Analysis (IPA). Western blotting and qRT-PCR were performed to validate STAT activation and interferon-stimulated genes (ISGs) Results: We found >450 genes differentially expressed in siTNIK vs siCTRL treated ECs (fold change>2, FDR<0.05). IPA revealed that under static (ST) and disturbed flow (DF) conditions TNIK depletion led to inhibition IFN (-log P-value >11) and upregulation of eukaryotic transcription elongation factor signaling (eIF2, -log P-value >7) (Fig 1B, C). In addition, under DF, TNIK depletion led to downregulation of hypercytokinemia/hyperchemokinemia (-log P-value >8, Fig 1B). Validation through western blotting and qRT-PCR (Fig 1D, E) confirmed that TNIK regulates FAK and STAT phosphorylation and IFN stimulated gene targets. Conclusions: Our study elucidates the unique role of TNIK in regulating FAK-STAT mediated IFN signaling and subsequently EC activation.

Abstract B55: MYCN promotes m6a-dependent translation of eEF2K mRNA during adaptation to ER stress

Abstract MYCN-amplified neuroblastoma is one of the most common and malignant pediatric tumors with low survival rates. Attempts to develop specific inhibitors to target MYCN have not been successful, prioritizing research into MYCN downstream targets. It was previously shown that eukaryotic translation elongation factor 2 (eEF2K) is upregulated in MYCN-amplified neuroblastoma and facilitates its adaptation to the nutrient deprivation (ND). However, the mechanism of eEF2K overexpression and its role in such adaptation is unknown. Using SUnSET assays, we showed that MYCN overexpression increases protein synthesis rates, demonstrating that MYCN-amplified cells depend on protein biosynthesis to cover the needs of fast proliferation. Enhanced protein biosynthesis increases the susceptibility for protein aggregates and subsequent unfolded protein response (UPR) activation and ER stress. ER stress also occurs under ND due to accumulation of unglycosylated proteins in the ER. To check how MYCN-amplified tumors might adapt to ER stress, we performed gene set enrichment analyses (GSEA) of genes that are overexpressed in MYCN amplified neuroblastoma and found “UPR” in the top 10 gene sets. As eEF2K regulates protein synthesis rates by slowing down the elongation step of mRNA translation, we examined whether eEF2K is part of the ER stress adaptation mechanism in MYCN-amplified cells. We found that during ND, eEF2K KO cells are more sensitive to thapsigargin (Tg) and tunicamycin (Tm), ER stress-inducing compounds, and show higher levels of ATF4, a major ER stress marker. Notably, low doses of cycloheximide, which blocks translation elongation, rescued the phenotype in eEF2K KO cells, confirming that the observed effect of eEF2K KO is linked to translation elongation. Finally, GSEA of RNAseq data obtained from TET21N WT vs. eEF2K KO after ND showed upregulation of genes responsible for the UPR and ER stress in eEF2K KO cells. Therefore, eEF2K appears to be linked to ER stress adaptation. To find potential mechanisms of MYCN-dependent regulation of eEF2K expression, we performed bioinformatic analyses of the eEF2K mRNA and found potential binding sites for METTL3, an enzyme that functions in the methylation of adenosine residues in mRNAs, in the eEF2K 3´-UTR. METTL3 knockdown resulted in a marked decrease of eEF2K protein without significant changes at the mRNA level. We applied polysome fractionation to study the effect of m6a modification on eEF2K mRNA translation. We discovered that METTL3 KD brings eEF2K mRNA to lighter polysome fractions compared with WT cells. As YTHDF1 is the only known reader of m6a modification that increases translation, we performed YTHDF1 knockdown and observed a decrease in eEF2K protein levels. Our findings indicate that MYCN amplified neuroblastoma cells increase translation of eEF2K mRNAs via a METTL3-YTHDF1 axis, and that this potentially functions as part of an adaptation mechanism under increased protein synthesis and subsequent ER stress. Citation Format: Andrii Vislovukh, Cristopher Hughes, Michael Lizardo, Poul H. Sorensen. MYCN promotes m6a-dependent translation of eEF2K mRNA during adaptation to ER stress [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B55.

CagA orchestrates eEF1A1 and PKC\u03b4 to induce interleukin-6 expression in Helicobacter pylori-infected gastric epithelial cells

BackgroundHelicobacter pylori colonises the stomach of approximately 50% of the global population. Cytotoxin-associated gene A protein (CagA) is one of the important virulent factors responsible for the increased inflammation and increases the risk of developing peptic ulcers and gastric carcinoma. The cytokine interleukin-6 (IL-6) has particularly important roles in the malignant transformation of gastric and intestinal epithelial cells as it is upregulated in H. pylori-infected gastric mucosa. In this study, we investigated the underlying mechanisms of CagA-induced IL-6 up-regulation during H. pylori infection. AGS cells, a human gastric adenocarcinoma cell line, lacking eEF1A1 were infected with CagA+H. pylori (NCTC11637), CagA−H. pylori (NCTC11637ΔcagA), or transduced by Ad-cagA/Ad-GFP. The expression and production of IL-6 were measured by quantitative real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The interactions among CagA, eukaryotic translation elongation factor 1-alpha 1 (eEF1A1), protein kinase Cδ (PKCδ), and signal transducer and activator of transcription 3 (STAT3) were determined by western blot or co-immunoprecipitation.ResultsDuring H. pylori infection, CagA-M (residues 256‒871aa) was found to interact with eEF1A1-I (residues 1‒240aa). NCTC11637 increased the expression of IL-6 in AGS cells compared with NCTC11637ΔcagA whereas knockdown of eEF1A1 in AGS cells completely abrogated these effects. Moreover, the CagA-eEF1A1 complex promoted the expression of IL-6 in AGS cells. CagA and eEF1A1 cooperated to mediate the expression of IL-6 by affecting the activity of p-STATS727 in the nucleus. Further, CagA-eEF1A1 affected the activity of STAT3 by recruiting PKCδ. However, blocking PKCδ inhibited the phosphorylation of STAT3S727 and induction of IL-6 by CagA.ConclusionsCagA promotes the expression of IL-6 in AGS cells by recruiting PKCδ through eEF1A1 in the cytoplasm to increase the phosphorylation of STAT3S727 in the nucleus. These findings provide new insights into the function of CagA-eEF1A1 interaction in gastric adenocarcinoma.

Chicken eEF1α is a Critical Factor for the Polymerase Complex Activity of Very Virulent Infectious Bursal Disease Virus.

Infectious bursal disease (IBD) is an immunosuppressive, highly contagious, and lethal disease of young chickens caused by IBD virus (IBDV). It results in huge economic loss to the poultry industry worldwide. Infection caused by very virulent IBDV (vvIBDV) strains results in high mortality in young chicken flocks. However, the replication characteristics of vvIBDV are not well studied. Publications have shown that virus protein 3 (VP3) binds to VP1 and viral double-stranded RNA, and together they form a ribonucleoprotein complex that plays a key role in virus replication. In this study, vvIBDV VP3 was used to identify host proteins potentially involved in modulating vvIBDV replication. Chicken eukaryotic translation elongation factor 1α (cheEF1α) was chosen to further investigate effects on vvIBDV replication. By small interfering RNA-mediated cheEF1α knockdown, we demonstrated the possibility of significantly reducing viral polymerase activity, with a subsequent reduction in virus yields. Conversely, over-expression of cheEF1α significantly increased viral polymerase activity and virus replication. Further study confirmed that cheEF1α interacted only with vvIBDV VP3 but not with attenuated IBDV (aIBDV) VP3. Furthermore, the amino acids at the N- and C-termini were important in the interaction between vvIBDV VP3 and cheEF1α. Domain III was essential for interactions between cheEF1α and vvIBDV VP3. In summary, cheEF1α enhances vvIBDV replication by promoting the activity of virus polymerase. Our study indicates cheEF1α is a potential target for limiting vvIBDV infection.

BC094916 suppressed SP 2/0 xenograft tumor by down-regulating Creb1 and Bcl2 transcription

BackgroundBoth multiple myeloma (MM) and systemic lupus erythematosus (SLE) are associated with abnormal production of plasma cells, although their pathological mechanism of each disease is different. The main characteristic of both diseases is uncontrolled differentiation of B cells into plasmablast/plasma cells. Despite continuous research on prognostic factors and the introduction of new agents for MM and SLE, treatments still do not exist for controlling plasmablast/plasma cells. Thus, it is necessary to identify novel therapeutic targets of plasmablast/plasma cells. Because of its plasmablast-like characteristics, the mus musculus myeloma SP 2/0 cell line was used in this study to test the effect of a novel therapeutic agent (BC094916 overexpression) on plasmablast/plasma cells.MethodsWe first determined gene expression profiles of plasma cells using Affymetrix microarrays and RNA-sequencing. The effect of BC094916 on SP 2/0 cell proliferation, cell cycle, and apoptosis was determined by CCK8 and fluorescence-activated cell sorting. The SP 2/0 xenograft mouse model was used to assess the impact of BC094916 on tumor progression. The luciferase reporter system was used to evaluate the effect of BC094916 on Creb1 and Bcl2 transcription.ResultsWe found that BC094916 mRNA was decreased in plasma cells. The mouse myeloma cell line SP 2/0 expressed low levels of BC094916 mRNA, whereas BC094916 overexpression suppressed SP 2/0 cell proliferation by inducing apoptosis. BC094916 overexpression suppressed tumor progression in the SP 2/0 xenograft mouse model. We also found that BC094916 mediate apoptosis by suppressing transcription of the Creb1 and Bcl2 genes, which promote the transcription of eukaryotic translation initiation and elongation factor genes.ConclusionsBC094916 overexpression suppressed Creb1 and Bcl2 transcription to induce cell apoptosis, which suppressed SP 2/0 proliferation and xenograft tumor progression. Thus, BC094916 overexpression may be a potential therapeutic agent for treatment of MM and autoimmune diseases such as SLE.

Generation and Comprehensive Analysis of Host Cell Interactome of the PA Protein of the Highly Pathogenic H5N1 Avian Influenza Virus in Mammalian Cells.

Accumulating data have identified the important roles of PA protein in replication and pathogenicity of influenza A virus (IAV). Identification of host factors that interact with the PA protein may accelerate our understanding of IAV pathogenesis. In this study, using immunoprecipitation assay combined with liquid chromatography-tandem mass spectrometry, we identified 278 human cellular proteins that might interact with PA of H5N1 IAV. Gene Ontology annotation revealed that the identified proteins are highly associated with viral translation and replication. Further KEGG pathway analysis of the interactome profile highlighted cellular pathways associated with translation, infectious disease, and signal transduction. In addition, Diseases and Functions analysis suggested that these cellular proteins are highly related with Organismal Injury and Abnormalities and Cell Death and Survival. Moreover, two cellular proteins (nucleolin and eukaryotic translation elongation factor 1-alpha 1) identified both in this study and others were further validated to interact with PA using co-immunoprecipitation and co-localization assays. Therefore, this study presented the interactome data of H5N1 IAV PA protein in human cells which may provide novel cellular target proteins for elucidating the potential molecular functions of PA in regulating the lifecycle of IAV in human cells.

Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A.

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.

Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa.

The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT–PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation.

Abstract 4472: Plitidepsin shows antitumor activity in patient-derived tumor xenografts and hematologic malignancies

Abstract Plitidepsin is a new marine-derived anti-tumor agent, originally isolated from the tunicate Aplidium albicans that is now fully obtained by total chemical synthesis. The compound is currently being evaluated in a pivotal phase III trial in patients with refractory/relapsed multiple myeloma. Previous in vitro studies in 26 hematological cell lines demonstrated that plitidepsin was more active in multiple myeloma and non-Hodgkin lymphoma compared to CML, AML and ALL derived cell lines as determined in a 4 day proliferation assay in suspension cultures. We have recently shown that plitidepsin interacts selectively with eEF1A2 (eukaryotic translation elongation factor 1 alpha 2) in tumor cells, which results in apoptotic cell death. To identify target tumor types for future clinical studies, the inhibition of colony formation of plitidepsin was assessed in vitro in 116 established patient derived xenografts tumor models from 23 different tumor types, including solid tumors and hematological malignancies such as myeloma, leukemia and lymphoma. The anti-tumor activity of plitidepsin varied depending on the concentration used, achieving a mean IC70 (inhibitory concentration of 70%) value of 3 nM (range between 0.03 nM and >1 μM), which can be also achieved in patients treated using the clinical recommended dose or less. 39 out of 116 tumors showed above average sensitivity (IC70 < 1nM) and IC70 values in these tumors were about 9-fold lower than the mean of all tumors as tested. Sensitive tumor types were seen among different tumor types, such as hematological malignancies, pleuramesothelioma, melanoma, small cell and non-small cell lung cancer (both adeno and large cell), as well as renal cancer. In conclusion plitidepsin showed a broad differential antitumor activity in vitro in hematological malignancies and in solid tumors. Bioinformatic analyses are currently underway to identify biomarkers and/or gene signatures that can predict sensitivity or resistance to plitidepsin for selecting patients being responsive to the drug. Citation Format: Armin Maier, Gerhard Kelter, Vincent Vuaroquaux, Pablo M. Avilés Marin, Carmen Cuevas, Carlos M. Galmarini, Heinz H. Fiebig. Plitidepsin shows antitumor activity in patient-derived tumor xenografts and hematologic malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4472. doi:10.1158/1538-7445.AM2015-4472

A bumpy road for RNA polymerase II.

The identification of a second regulatory checkpoint controlling RNA polymerase II elongation near the poly(A) site of protein-coding genes reveals an additional level of complexity in the modulation of eukaryotic transcriptional elongation and termination.

A putative role of the Sup35p C-terminal domain in the cytoskeleton organization during yeast mitosis

Sup35 protein (Sup35p), or eukaryotic peptide chain release factor GTP binding subunit (eRF3), is a well-known yeast prion responsible for the characteristic [PSI(+)] trait. N- and M-domains of this protein have been the foci of intensive research due to their importance for the prion formation. Sup35p C-terminal domain (Sup35pC) is essential for translation termination and cell viability. Deletion of Sup35pC was shown to lead to malformation of cells during mitosis. In this study we confirm that Sup35pC domain possesses high sequence and structural similarity to the eukaryotic translation elongation factor 1-α (eEF1A) from yeast and show that its sequence is conserved across different species including human. Because cell malformation during mitosis could be due to the deregulation of cytoskeleton formation, and since a Sup35 paralog eEF1A is known to act as an actin modulating protein, we focused on establishing of the relationships between the Sup35pC and modulation of the cytoskeleton formation. We found 104 co-partners between Sup35pC and EF1A of S. cerevisiae, and 18 partners of human ERF3A. Based on the analysis of known and modeled structures of some effectors and partners we found possible protein-protein interactions. Based on our study, we propose that Sup35pC may serve as actin modulator during mitosis.

Abstract 195: TIP30 Interferes With Protein Synthesis And Inhibits Cardiac Hypertrophy

Background: Pathological cardiac hypertrophy is a crucial risk factor for the development of heart failure. It is characterized by a massive increase of contractile and structural proteins within cardiomyocytes, mainly as a result of enhanced protein synthesis. The regulatory mechanisms of increased RNA translation during hypertrophy, however, remain largely unknown. Here, we examined the cardiac function of TIP30, which has previously emerged as a tumor suppressor gene with reduced expression in malignant tumours. Results: We identified myocardial TIP30 mRNA as dramatically downregulated (>80%) in a mouse model of dilative cardiomyopathy (MLP knock-out mice) as well as in human failing hearts. A protein pull-down screen with a GST-TIP30 fusion protein and proteomic candidate identification revealed the eukaryotic translation elongation factor 1A1 (eEF1A1) as binding partner of TIP30, which was independently verified by GST-pulldown and co-immunoprecipitation (IP). Interestingly, this interaction between TIP30 and eEF1A1 was not detectable during pro-hypertrophic (phenylephrine, PE) stimulation in neonatal rat cardiac myocytes (NRCM). Adenoviral (Ad-) TIP30 overexpression in NRCM during PE stimulation led to a reduced hypertrophic response compared to Ad-control infected cells (cell size in % of control: Ad-TIP30 135 ± 4 vs. Ad-control 200 ± 3 p<0.05). At the same time, a dramatic decrease in protein:DNA ratio was observed. siRNA downregulation of eEF1A1 ablated the anti-hypertrophic effects of TIP30. Measurement of translational activity by determination of the polysome/monosome ratio revealed a blunted translation rate after TIP30 overexpression. In line with our in vitro data, myocardial overexpression of TIP30 by an adeno-associated virus (AAV9-TIP30) significantly reduced cardiac hypertrophy during two weeks of Angiotensin (Ang)/PE infusion in vivo. In turn, Ang/PE infusion as well as aortic constriction (TAC) led to an increased hypertrophic response in TIP30 knock-out versus wild-type mice. Conclusion: TIP30 inhibits hypertrophy and translational activity most likely through inhibition of eEF1A1 in cardiomyocytes. TIP30 is downregulated in human failing hearts and could thereby contribute to aggravated hypertrophy.

Identification and characterization of the interaction between viroplasm-associated proteins from two different plant-infecting reoviruses and eEF-1A of rice

A rice protein homologous to eukaryotic translation elongation factor 1A (eEF-1A) was found to interact with the Pns6 of rice ragged stunt virus (RRSV), the type member of the genus Oryzavirus, family Reoviridae, in yeast two-hybrid screening. The interaction between the rice protein, designated OseEF-1A, and RRSV Pns6 was confirmed by bimolecular fluorescence complementation. Besides Pns6, OseEF-1A also interacted with the viroplasm matrix protein, Pns10, of RRSV. When expressed together, OseEF-1A co-localized with RRSV Pns10 in epidermal cells of Nicotiana benthamiana. Pns6 of southern rice black-streaked dwarf virus (SRBSDV), a newly reported member of the genus Fijivirus, family Reoviridae, was the only non-structural SRBSDV protein studied here that also interacted with OseEF-1A. Like Pns6 of rice black-streaked dwarf virus (RBSDV), SRBSDV Pns6 interacted with itself and co-localized with Pns9-1 in N. benthamiana. In the presence of Pns6, OseEF-1A co-localized with Pns9-1, the putative viroplasm matrix protein of SRBSDV.

Abstract 4300: The EEF1A2-PI3K-AKT-mTOR axis supports the protumorigenic function of MDM4 in human hepatocellular carcinoma.

Abstract Background & Aims: The Mouse Double Minute homolog 4 (MDM4) is one of the main negative p53 regulators in mammalian cells and mutational inactivation of p53 is a rare event in Western hepatocellular carcinomas (HCCs). 1q gains promote the upregulation of MDM4 in HCC, leading to a negative regulation of p53 activity. However, additional mechanisms might be involved that explain the increased MDM4 activity observed in HCCs with balanced MDM4 gene locus. Here we aimed at the identification of post-transcriptional mechanisms involved in the upregulation of MDM4 in HCC. Methods: To investigate a potential role of PI3K-AKT-mTOR signaling on MDM4 activity, HCC cell lines were treated with small molecule inhibitors and siRNAs and expression changes as well as functional analyses were recorded. Expression changes of central pathway components were investigated in human liver samples (normal liver, peritumorous liver tissue, and HCC). An AKT transgenic mouse model was used to show the involvement of AKT pathway in the regulation of MDM4 in vivo. Results: The inhibition of both PI3K-AKT and mTOR signaling pathways resulted in reduced MDM4 protein levels in HCC cell lines, which was associated with the transcriptional activation of p53-target genes. Biochemical assays revealed that both AKT-mediated phosphorylation and ubiquitin-specific protease 2a (USP2a)-mediated deubiquitination protected MDM4 from proteasomal in human HCC cell lines. In addition, AKT transgenic mice showed increased MDM4 protein levels indicating that AKT signaling is involved in the stabilization of MDM4 protein in vivo. Furthermore, the Eukaryotic translational Elongation Factor 1 alpha 2 (EEF1A2), which is frequently upregulated in human HCC, sustained the PI3K-AKT-mTOR cascade both in vitro and in vivo. In human HCCs, a strong positive correlation between the overexpression of EEF1A2, pAKT, USP2a, and MDM4 was observed, which was associated with shorter survival of HCC patients. Conclusions: Our data demonstrate that the EEF1A2/PI3K/AKT/mTOR cascade inactivates wild-type p53 in human HCC through the stabilization of the MDM4 protooncogene via a post-transcriptional mechanism involving an AKT-mediated phosphorylation of MDM4 and USP2a-mediated de-ubiquitination. Since the sustained activation of the EEF1A2/PI3K/AKT/mTOR/MDM4 axis has impact on the survival probability of HCC patients, it may thus represent a promising therapeutic target. Citation Format: Rossella Pellegrino, Diego F. Calvisi, Olaf Neumann, Xin Chen, Chunmei Wang, Bernhard Radlwimmer, Sara Ladu, Frank Dombrowski, Matthias Evert, Peter Schirmacher, Thomas Longerich. The EEF1A2-PI3K-AKT-mTOR axis supports the protumorigenic function of MDM4 in human hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4300. doi:10.1158/1538-7445.AM2013-4300

Isoforms of elongation factor eEF1A may be differently regulated at post-transcriptional level in breast cancer progression

Eukaryotic translation elongation factor 1A exists as two 98 % homologous isoforms: eEF1A1 (A1) and eEF1A2 (A2) which are tissue and development specific. Despite high homology in an open reading frame (ORF) region, mRNAs coding for eEF1A1 and eEF1A2 are different in their untranslated regions (UTR), suggesting a possibility of their dissimilar post-transcriptional regulation. Aim. To analyze the existence of cis-acting motifs in the UTRs of EEF1A1/A2 mRNAs, to confirm the possibility of post-transcriptional control of eEF1A1 and eEF1A2 expression. Methods. An ensemble of bioinformatic methods was applied to predict regulatory motifs in the UTRs of EEF1A1/A2 mRNAs. Dual-luciferase reporter assay was employed to detect post-transcriptional regulation of eEF1A1/A2 expression. Results. Numerous regulatory motifs in the UTR of EEF1A1/A2 mRNAs were found bioinformatically. The experimental evidence was obtained for the existence of negative regulation of EEF1A1 and positive regulation of EEF1A2 mRNA in the model of breast cancer development. Conclusions. EEF1A1 and EEF1A2 mRNAs contain distinct motifs in the UTRs and are differently regulated in cancer suggesting the possibility of their control by different cellular signals.

Single-Molecule Fluorescence Observations of Eukaryotic Translation

For the two last decades, structural and single-molecule investigations of the prokaryotic ribosome have provided a better understanding of the highly dynamic nature of translation. Eukaryotic ribosome has received far less attention, most of the conclusions being extrapolated from prokaryotic data. Although both ribosomes share functional and structural features, eukaryotic ribosome contains many additional domains of unknown function. Applying single-molecule techniques to eukaryote ribosomes would clarify the function of these additional domains.In order to study structural rearrangement of the ribosome during eukaryotic elongation, we bypass canonical initiation using an initiation factor free Internal Ribosome Entry Site (IRES) enabling elongation in the desired reading frame. Ribosomal subunits, purified from Rabbit Reticulocyte Lysate (RRL), are first bound to the IRES. Ribosome-mRNA complexes are then fixed on a microscope coverslip via the 5'end of the mRNA. All factors necessary for elongation are provided by injection of RRL. using single molecule Total Internal Reflection Fluorescence (TIRF) microscopy, we monitor elongation thanks to a fluorescently labelled oligonucleotide hybridized on the mRNA. The oligonucleotide is detached by the helicase activity of a translating ribosome. The evanescent wave generated by the TIRF apparatus allows solely the detection of molecules close to the surface. In this configuration, probe detachments induce the loss of fluorescence signals which are used to measure the distribution of translation speed. A thorough care is given to photobleaching during data analysis. Controls using antibiotics further show that the loss of fluorescence is a signature of a single ribosome activity.

Abstract LB-85: Ectopic expression of eukaryotic translation elongation factor eEF1A2 is a hallmark for human lung cancer and is associated with poor prognosis

Abstract Background: The elongation factor eEF1A is one of the most abundantly expressed cellular proteins. It exists in two forms, eEF1A2, expressed in neurons and striated muscle, and eEF1A1, expressed in all other cell types. No normal adult cell expresses both forms. We investigated their role in lung cancer. Methods: We studied over 500 NSCLC and SCLC lung cancers and over 170 cell lines, as well as corresponding non- malignant lung tissues and immortalized respiratory epithelial lines. Methods included microarray, qPCR, immunostaining and western blots for eEF1A1 and eEF1A2, genomic sequencing and SNP analyses. Results: All normal and malignant cells highly expressed eEF1A1. EEF1A2 was expressed in 70-80% of all forms of lung cancers and 95% of cell lines, while all non-malignant tissues were negative. Expression of eEF1A2 in neuroendocrine tumors was particular high. The ectopic expression of eEF1A2 was not the consequence of mutations or copy number gains. Use of demethylating agents and HDAC inhibitors resulted in expression of eEF1A2 in respiratory epithelial cells, indicating that epigenetic mechanisms prevented expression in non-malignant cells. Transient knockdown of the eEF1A2 expression inhibited proliferation and colony formation of lung cancer cell lines. Furthermore, the expression of eEF1A2 in resected NSCLC cancers was associated with poor survival. Conclusions: Highly specific ectopic expression of eEF1A2 gene is frequent in all types of lung cancer, plays an important role in tumor pathogenesis and is a potential diagnostic and prognostic factor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-85. doi:1538-7445.AM2012-LB-85

The effect of silent homo sapiens eukaryotic translation elongation factor 1 alpha 2 gene on the growth of pancreatic cancer xenograft in nude mice

Objective To explore the effect and the possible mechanisms of silent homo sapiens eukaryotic translation elongation factor 1 alpha 2(EEF1A2)gene on the growth of pancreatic cancer cell in vivo.Methods The pancreatic cancer xenograft models in mice were established.The mice were equally divided into control group,negative control group and EEF1A2 group,which were injected with PBS,negative control siRNA and EEF1A2 siRNA into xenograft tumors respectively.The size and weight of tumors in each group were measured.The expression of EEF1A2 and PCNA in tumor tissue of each group was detected by immunohistochemistry.The cell apoptosis rate in tumor tissue of each group was determined by TUNEL.Results In xenograft nude mice models,since the 17th day of injection,the growth of tumor size in EEF1A2 group was obviously slower than that of negative control group and control group(all P＜0.05).By the end of the treatment,the tumors were cut off and weighted.The weight of tumors in EEF1A2 group(0.27g± 0.06g)were significantly lower than those of control group and negative control group(0.39g± 0.08g and 0.43g± 0.07g,P＜0.05).EEF1A2 mostly expressed in cytoplasm of pancreatic cancer cell.In negative control group and control group,the positive cells distributed densely and the positive rate was(72.58 ± 25.47)％ and (76.75±23.19)％ respectively.The distribution of positive cells in EEF1A2 group was scattered and the positive rate was(34.78±21.36)％,the difference was statisically significant(P＜0.01).The expression of PCNA at protein level in EEF1A2 group was significantly lower those that of control group and negative control group(P＜ 0.01).The result of TUNEL test indicated that the cell apoptosis rate in EEF1A2 group was higher than those of control group and negative control group (P＜0.01).Conclusions The EEF1A2 gene can be effectively silented in vivo,which significantly inhibits the growth of pancreatic cancer cell.It may be related with inhibition of cell proliferation and promotion cell apoptosis. Key words: Petide elongation factor 1; Pancreatic neoplasms; Mice, nude; Disease models,animal; RNA interference; Apoptosis

Protein expression profile in the testis of rats chronically exposed to low-dose 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin

The chronic effects low-dose 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined on protein expression profiles in rat testis, sperm, and serum gonadal hormones. A total of 32 male rats were randomly divided into three TCDD-exposed groups, administered either 140, 350, or 875 ng TCDD/kg/week for 29 weeks, respectively, and one control group receiving only corn oil. The proteins from rat testis were separated and analyzed by two-dimensional gel electrophoresis and mass spectrometry. TCDD induced significant decreases in sperm counts and serum gonadal hormone levels compared with controls. TCDD altered testicular protein expression levels. Several interesting volume-altered proteins that were related to the reproductive toxicities or other toxicities of TCDD were identified. Among these proteins, PERF15 was the only down-regulated protein; sperm protein SSP411, ubiquitin carboxyl-terminal hydrolase L-3, and eukaryotic translation elongation factor 1 gamma were up-regulated by TCDD. The differentially expressed proteins and other data provide further insight into the mechanisms of reproductive toxicity mediated by low-dose TCDD exposure.