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1
Smolle, Maria Anna, Piotr Czapiewski, Sylwia Lapińska-Szumczyk, Hanna Majewska, Anna Supernat, Anna Zaczek, Wojciech Biernat, Nicole Golob-Schwarzl, and Johannes Haybaeck. "The Prognostic Significance of Eukaryotic Translation Initiation Factors (eIFs) in Endometrial Cancer." International Journal of Molecular Sciences 20, no. 24 (December 6, 2019): 6169. http://dx.doi.org/10.3390/ijms20246169.
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Whilst the role of eukaryotic translation initiation factors (eIFs) has already been investigated in several human cancers, their role in endometrial cancer (EC) is relatively unknown. In the present retrospective study, 279 patients with EC (1180 samples) were included (mean age: 63.0 years, mean follow-up: 6.1 years). Samples were analysed for expression of 7 eIFs subunits (eIF2α, eIF3c, eIF3h, eIF4e, eIF4g, eIF5, eIF6) through immunohistochemistry and western blotting. Fifteen samples of healthy endometrium served as controls. Density and intensity were assessed and mean combined scores (CS) calculated for each patient. Upon immunohistochemistry, median eIF5 CS were significantly higher in EC as compared with non-neoplastic tissue (NNT, p < 0.001), whilst median eIF6 CS were significantly lower in EC (p < 0.001). Moreover, eIF5 (p = 0.002), eIF6 (p = 0.032) and eIF4g CS (p = 0.014) were significantly different when comparing NNT with EC grading types. Median eIF4g CS was higher in type II EC (p = 0.034). Upon western blot analysis, eIF4g (p < 0.001), peIF2α (p < 0.001) and eIF3h (p < 0.05) were significantly overexpressed in EC, while expression of eIF3c was significantly reduced in EC as compared with NNT (p < 0.001). The remaining eIFs were non-significant. Besides tumour stage (p < 0.001) and patient’s age (p < 0.001), high eIF4g CS-levels were independently associated with poor prognosis (HR: 1.604, 95%CI: 1.037–2.483, p = 0.034). The other eIFs had no prognostic significance. Notably, the independent prognostic significance of eIF4g was lost when adding tumour type. Considering the difficulties in differentiating EC type I and II, eIF4g may serve as a novel prognostic marker indicating patient outcome.
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Wang, Xiaoyan, Guangchao Xu, Fangyingnan Zhang, Yating Wei, Jiawen Deng, Lan Mu, Jinqing He, et al. "eIF6 modulates skin wound healing by upregulating keratin 6B." Stem Cells Translational Medicine 13, no. 11 (October 15, 2024): 1101–12. http://dx.doi.org/10.1093/stcltm/szae064.
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Abstract Eukaryotic translation initiation factor 6 (eIF6) plays a crucial role in 60S ribosome biogenesis and protein translation, as well as in hypertrophic scar formation, but its potential role in epithelialization is still poorly understood. Herein, we found that eIF6 negatively correlated with the wound healing process. Mice with genetically knockdown eIF6 (eIF6+/−) showed faster re-epithelization as shown by the longer tongue of the newly formed epidermis. Furthermore, eIF6 ablation accelerated the wound healing process by targeting basal keratinocytes in the eIF6 keratinocyte-conditional knockout (eIF6f/+; Krt5-Cre+) mice. Mechanistically, keratin 6B, an important wound-activated protein, was significantly upregulated in eIF6f/+; Krt5-Cre+ mice skin as proved by RNA-seq, western immunoblots, and immunofluorescence staining. Moreover, an elevated level of KRT6B and accelerated proliferative capacity were also observed in stable knockdown eIF6 HaCaT cells. Taken together, eIF6 downregulation could accelerate epithelialization by upregulating KRT6B expression and promoting keratinocyte proliferation. Our results for the first time indicate that eIF6 might be a novel target to regulate re-epithelialization.
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Jivotovskaya, Antonina V., Leoš Valášek, Alan G. Hinnebusch, and Klaus H. Nielsen. "Eukaryotic Translation Initiation Factor 3 (eIF3) and eIF2 Can Promote mRNA Binding to 40S Subunits Independently of eIF4G in Yeast." Molecular and Cellular Biology 26, no. 4 (February 15, 2006): 1355–72. http://dx.doi.org/10.1128/mcb.26.4.1355-1372.2006.
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ABSTRACT Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAi Met ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo.
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Harish, Kavya Meena, Poonam Roshan, Aparna Biswas, Stella Anagnos, Riley Luebbers, and Sofia Origanti. "Abstract 7078: Targeting eIF6 to modulate ribosomal subunit association dynamics in cancers." Cancer Research 84, no. 6_Supplement (March 22, 2024): 7078. http://dx.doi.org/10.1158/1538-7445.am2024-7078.
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Abstract Deregulation of ribosomal subunit levels and accessibility are hallmarks of cancer. Eukaryotic translation initiation factor 6 (eIF6) is a key regulator of ribosomal subunit association that is over expressed in many cancers including lung, colon, ovarian and breast cancers, and deregulation of eIF6 is correlated with a poor cancer prognosis. Association of eIF6 with 60S is critical for 60S assembly. However, eIF6 must be released from 60S prior to translation to permit inter subunit interactions between 60S and 40S and to facilitate the formation of translationally proficient 80S monosome. Release of eIF6 is deregulated in the ribosomopathy- Shwachman Diamond Syndrome that is predisposed to leukemias. Also, loss of eIF6 markedly delays tumorigenesis without affecting normal growth, which presents eIF6 as a viable therapeutic target. A potential therapeutic strategy is to target the eIF6 and 60S ribosomal interaction interface. Through biophysical analyses we had identified residues that are critical for the interaction between eIF6 and 60S ribosomal subunit. We show that targeting these key residues in the eIF6-60S interaction interface markedly delays colonic cancer growth and inhibits protein synthesis. Furthermore, we show that the levels of other eIFs and the release factors: SBDS, and EFL1-GTPase are unaltered in the mutant cells suggesting that the translational inhibition is primarily attributed to the deregulation of eIF6. Interestingly, targeting eIF6 leads to an upregulation of p53 independent of the DNA damage response, suggesting that ribosomal stress contributes to the stabilization of p53. Previously, it was shown that translation of Cdc42 was upregulated by eIF6 to promote invasiveness. However, Cdc42 levels were unaltered in the mutant cells. Future studies will determine the mRNA substrates that are translationally deregulated in the mutants. We will also determine the effect of disrupting eIF6 function in inhibiting tumor growth and progression in vivo. Category: MCB, Subcategory: MCB07-Gene regulation sub classification: Translational Control Citation Format: Kavya Meena Harish, Poonam Roshan, Aparna Biswas, Stella Anagnos, Riley Luebbers, Sofia Origanti. Targeting eIF6 to modulate ribosomal subunit association dynamics in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7078.
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Golob-Schwarzl, Nicole, Christina Wodlej, Florian Kleinegger, Margit Gogg-Kamerer, Anna Maria Birkl-Toeglhofer, Johannes Petzold, Ariane Aigelsreiter, Michael Thalhammer, Young Nyun Park, and Johannes Haybaeck. "Eukaryotic translation initiation factor 6 overexpression plays a major role in the translational control of gallbladder cancer." Journal of Cancer Research and Clinical Oncology 145, no. 11 (October 4, 2019): 2699–711. http://dx.doi.org/10.1007/s00432-019-03030-x.
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Abstract Background Gallbladder cancer (GBC) is a rare neoplasia of the biliary tract with high mortality rates and poor prognosis. Signs and symptoms of GBC are not specific and often arise at late stage of disease. For this reason, diagnosis is typically made when the cancer is already in advanced stages, and prognosis for survival is less than 5 years in 90% of cases. Biomarkers to monitor disease progression and novel therapeutic alternative targets for these tumors are strongly required. Commonly, dysregulated protein synthesis contributes to carcinogenesis and cancer progression. In this case, protein synthesis directs translation of specific mRNAs, and, in turn, promotes cell survival, invasion, angiogenesis, and metastasis of tumors. In eukaryotes, protein synthesis is regulated at its initiation, which is a rate-limiting step involving eukaryotic translation initiation factors (eIFs). We hypothesize that eIFs represent crossroads in the development of GBC, and might serve as potential biomarkers. The study focus was the role of eIF6 (an anti-association factor for the ribosomal subunits) in GBC. Methods In human GBC samples, the expression of eIF6 was analyzed biochemically at the protein (immunohistochemistry, immunoblot analyses) and mRNA levels (qRT-PCR). Results High levels of eIF6 correlated with shorter overall survival in biliary tract cancer (BTC) patients (n = 28). Immunohistochemical data from tissue microarrays (n = 114) demonstrated significantly higher expression levels of eIF6 in GBC compared to non-neoplastic tissue. Higher eIF6 expression on protein (immunoblot) and mRNA (qRT-PCR) level was confirmed by analyzing fresh frozen GBC patient samples (n = 14). Depletion of eIF6 (using specific siRNA-mediated knockdown) in Mz-ChA-2 and TFK-1 cell lines inhibited cell proliferation and induced apoptosis. Conclusion Our data indicates that eIF6 overexpression plays a major role in the translational control of GBC, and indicates its potential as a new biomarker and therapeutic target in GBC.
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Schatz, Christoph, Susanne Sprung, Volker Schartinger, Helena Codina-Martínez, Matt Lechner, Mario Hermsen, and Johannes Haybaeck. "Dysregulation of Translation Factors EIF2S1, EIF5A and EIF6 in Intestinal-Type Adenocarcinoma (ITAC)." Cancers 13, no. 22 (November 11, 2021): 5649. http://dx.doi.org/10.3390/cancers13225649.
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Intestinal-type adenocarcinoma (ITAC) is a rare cancer of the nasal cavity and paranasal sinuses that occurs sporadically or secondary to exposure to occupational hazards, such as wood dust and leather. Eukaryotic translation initiation factors have been described as promising targets for novel cancer treatments in many cancers, but hardly anything is known about these factors in ITAC. Here we performed in silico analyses, evaluated the protein levels of EIF2S1, EIF5A and EIF6 in tumour samples and non-neoplastic tissue controls obtained from 145 patients, and correlated these results with clinical outcome data, including tumour site, stage, adjuvant radiotherapy and survival. In silico analyses revealed significant upregulation of the translation factors EIF6 (ITGB4BP), EIF5, EIF2S1 and EIF2S2 (p < 0.05) with a higher arithmetic mean expression in ITAC compared to non-neoplastic tissue (NNT). Immunohistochemical analyses using antibodies against EIF2S1 and EIF6 confirmed a significantly different expression at the protein level (p < 0.05). In conclusion, this work identifies the eukaryotic translation initiation factors EIF2S1 and EIF6 to be significantly upregulated in ITAC. As these factors have been described as promising therapeutic targets in other cancers, this work identifies candidate therapeutic targets in this rare but often deadly cancer.
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Pesce, Elisa, Annarita Miluzio, Lorenzo Turcano, Claudia Minici, Delia Cirino, Piera Calamita, Nicola Manfrini, et al. "Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits." Cells 9, no. 1 (January 10, 2020): 172. http://dx.doi.org/10.3390/cells9010172.
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Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.
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Harish, Kavya Meena, Aparna Biswas, Poonam Roshan, and Sofia Origanti. "Abstract 3719: Targeting the eIF6 and 60S ribosomal subunit interaction interface in cancers." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3719. http://dx.doi.org/10.1158/1538-7445.am2023-3719.
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Abstract Translational control is integral to cancer initiation and progression. A subset of translation initiation factors are deregulated in cancers to facilitate their rampant growth and proliferation. Eukaryotic translation initiation factor 6 (eIF6) is one such factor that is over expressed in many cancers including lung, colon, ovarian and breast cancers, and deregulation of eIF6 is correlated with a poor cancer prognosis. Association of eIF6 with 60S is critical for 60S assembly. However, eIF6 must be released from 60S prior to translation to permit inter subunit interactions between 60S and 40S and to facilitate the formation of translationally proficient 80S complex. Release of eIF6 is deregulated in the ribosomopathy- Shwachman Diamond Syndrome that is predisposed to leukemias. Also, loss of eIF6 markedly delays tumorigenesis without affecting normal growth, which presents eIF6 as a viable therapeutic target. A potential therapeutic strategy is to target the eIF6 and 60S ribosomal interaction interface. Through extensive biophysical analyses, we had identified residues that are critical for the interaction between eIF6 and 60S ribosomal subunit. We show that targeting these key residues in the eIF6-60S interaction interface markedly delays colonic cancer growth and inhibits protein synthesis. Interestingly, targeting eIF6 leads to an upregulation of p53 independent of the DNA damage response, suggesting that ribosomal stress contributes to the stabilization of p53. Future studies will determine the mRNA targets that are translationally deregulated by inactivation of eIF6. We will also determine the effect of targeting eIF6 function in inhibiting tumor growth and progression in vivo. Citation Format: Kavya Meena Harish, Aparna Biswas, Poonam Roshan, Sofia Origanti. Targeting the eIF6 and 60S ribosomal subunit interaction interface in cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3719.
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Oyarbide, Usua, Valentino Bezzerri, Morgan Staton, Christian Boni, Arish Shah, Marco Cipolli, Eliezer Calo, and Seth J. Corey. "Eif6 Dosage Alleviates Activation of the Tp53 Pathway in Sbds-Deficient Cells: A Mechanism for Somatic Genetic Rescue in Shwachman-Diamond Syndrome." Blood 144, Supplement 1 (November 5, 2024): 4090. https://doi.org/10.1182/blood-2024-209812.
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Shwachman-Diamond syndrome (SDS) is characterized by skeletal abnormalities, pancreatic insufficiency, and neutropenia, with an increased risk of developing myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Nearly all cases of SDS are caused by biallelic mutations in the SBDS gene, which is critical for ribosome assembly. SBDS interacts with EFL1 to remove EIF6 from the 60S ribosomal subunit, facilitating ribosome formation in the cytoplasm. Research using mouse and zebrafish models has shown that Sbds deficiency activates the tumor suppressor protein Tp53, leading to developmental abnormalities and tissue-specific defects. TP53 biallelic mutations have been found in SDS patients with MDS/AML, suggesting a survival advantage for these mutations. SBDS deficiency is also linked to acquired deletions of chromosome 20q, where the EIF6 gene is located, or somatic mutations in EIF6. These changes, which have been termed somatic genetic rescue, are thought to compensate for the ribosome assembly defect in SDS and lower the risk of MDS/AML. Yet the mechanism of somatic genetic rescue has not been characterized. We created sbds-null zebrafish that exhibited Eif6 accumulation, changes in levels of ribosome proteins, and activation of Tp53 pathways. We have generated an eif6 knockout (KO) line which died earlier (~7-10 days post-fertilization) than the sbds KO line (~15 days post-fertilization). To determine the role of a Eif6 dose effect, we generated zebrafish mutants with low Eif6 protein expression (10% of the wildtype). Surprisingly, we observed that these eif6 hypomorph mutants survived to adulthood. Polysome profiling revealed significant reductions in the 80S monosomes and 40S ribosomal subunits in the eif6 KO at 5 dpf. However, the eif6 hypomorph mutants displayed polysome profiles similar to the eif6 wild type. We crossed the eif6 KO or hypomorph mutants with sbds-null fish and analyzed their phenotypic and molecular characteristics. In the sbds-null background, expression of eif6+/- significantly but partially extended their survival at 15 days post fertilization. The eif6 hypomorph mutants also increased larvae survival at 20 days post fertilization with Mendelian ratios. Low Eif6 levels did not rescue neutropenia in Sbds-deficient zebrafish. Notably, the eif6 hypomorph mutants in sbds-null background reduced the expression of Tp53-dependent targets (e.g., cdkn1a, bax, and puma). SDS patient-derived cell lines also showed accumulation of EIF6, TP53, and CDKN1A (p21). Knocking down EIF6 significantly decreased CDKN1A mRNA levels. These observations support the hypothesis that low levels of Eif6 mitigate Tp53 pathway activation and partially alleviate cellular stress in Sbds-deficient cells. These findings highlight the complex relationships between SBDS, EIF6, TP53, and stress responses in the pathogenesis of SDS. EIF6 dosage determines the degree of intracellular stress mediated by TP53, which may explain the somatic genetic rescue in SDS and the decreased risk for malignant transformation. Understanding these mechanisms will aid in developing therapeutic strategies for SDS. In addition, this partial dosage effect suggests additional stress pathways that are unlikely to be TP53-dependent. We are currently identifying TP53-independent mechanisms.
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De Marco, N., C. Campanella, and R. Carotenuto. "In X. laevis embryos high levels of the anti-apoptotic factor p27BBP/eIF6 are stage-dependently found in BrdU and TUNEL-reactive territories." Zygote 19, no. 2 (July 27, 2010): 157–63. http://dx.doi.org/10.1017/s0967199410000213.
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Summaryp27BBP/eIF6 (β4 binding protein/eukaryotic initiation factor 6) is a highly conserved protein necessary for cell life. In adult eIF6 mice, a 50% decrease in the protein levels in all tissues is accompanied by a reduction in cell proliferation only in the liver, fat cells and cultured fibroblasts. During X. laevis embryogenesis expression of p27BBP/eIF6 is abundant in high proliferative territories. However, in Xenopus cell proliferation appears unaffected following p27BBP/eIF6 over-expression or down-regulation. Indeed, p27BBP/eIF6 is an anti-apoptotic factor acting upstream of Bcl2 that reduces endogenous apoptosis. We studied p27BBP/eIF6 protein localization in wild type embryos and compared it to proliferation and apoptosis. At the beginning of embryogenesis, high levels of p27BBP/eIF6, proliferation and apoptosis overlap. In later development stages high proliferation levels are present in the same regions where higher p27BBP/eIF6 expression is observed, while apoptosis does not appear specifically concentrated in the same sites. The higher presence of p27BBP/eIF6 would appear related to an increased need of apoptosis control in the regions where cell death is essential for normal development.
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Jungers, Courtney F., Jonah M. Elliff, Daniela S. Masson-Meyers, Christopher J. Phiel, and Sofia Origanti. "Regulation of eukaryotic translation initiation factor 6 dynamics through multisite phosphorylation by GSK3." Journal of Biological Chemistry 295, no. 36 (July 23, 2020): 12796–813. http://dx.doi.org/10.1074/jbc.ra120.013324.
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Eukaryotic translation initiation factor 6 (eIF6) is essential for the synthesis of 60S ribosomal subunits and for regulating the association of 60S and 40S subunits. A mechanistic understanding of how eIF6 modulates translation in response to stress, specifically starvation-induced stress, is lacking. We here show a novel mode of eIF6 regulation by glycogen synthase kinase 3 (GSK3) that is predominantly active in response to serum starvation. Both GSK3α and GSK3β phosphorylate human eIF6. Multiple residues in the C terminus of eIF6 are phosphorylated by GSK3 in a sequential manner. In response to serum starvation, eIF6 accumulates in the cytoplasm, and this altered localization depends on phosphorylation by GSK3. Disruption of eIF6 phosphorylation exacerbates the translation inhibitory response to serum starvation and stalls cell growth. These results suggest that eIF6 regulation by GSK3 contributes to the attenuation of global protein synthesis that is critical for adaptation to starvation-induced stress.
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Miluzio, Annarita, Sara Ricciardi, Nicola Manfrini, Roberta Alfieri, Stefania Oliveto, Daniela Brina, and Stefano Biffo. "Translational control by mTOR-independent routes: how eIF6 organizes metabolism." Biochemical Society Transactions 44, no. 6 (December 2, 2016): 1667–73. http://dx.doi.org/10.1042/bst20160179.
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Over the past few years, there has been a growing interest in the interconnection between translation and metabolism. Important oncogenic pathways, like those elicited by c-Myc transcription factor and mTOR kinase, couple the activation of the translational machinery with glycolysis and fatty acid synthesis. Eukaryotic initiation factor 6 (eIF6) is a factor necessary for 60S ribosome maturation. eIF6 acts also as a cytoplasmic translation initiation factor, downstream of growth factor stimulation. eIF6 is up-regulated in several tumor types. Data on mice models have demonstrated that eIF6 cytoplasmic activity is rate-limiting for Myc-induced lymphomagenesis. In spite of this, eIF6 is neither transcriptionally regulated by Myc, nor post-transcriptionally regulated by mTOR. eIF6 stimulates a glycolytic and fatty acid synthesis program necessary for tumor growth. eIF6 increases the translation of transcription factors necessary for lipogenesis, such as CEBP/β, ATF4 and CEBP/δ. Insulin stimulation leads to an increase in translation and fat synthesis blunted by eIF6 deficiency. Paradoxycally, long-term inhibition of eIF6 activity increases insulin sensitivity, suggesting that the translational activation observed upon insulin and growth factors stimulation acts as a feed-forward mechanism regulating lipid synthesis. The data on the role that eIF6 plays in cancer and in insulin sensitivity make it a tempting pharmacological target for cancers and metabolic diseases. We speculate that eIF6 inhibition will be particularly effective especially when mTOR sensitivity to rapamycin is abrogated by RAS mutations.
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Scagliola, Alessandra, Annarita Miluzio, Giada Mori, Sara Ricciardi, Stefania Oliveto, Nicola Manfrini, and Stefano Biffo. "Inhibition of eIF6 Activity Reduces Hepatocellular Carcinoma Growth: An In Vivo and In Vitro Study." International Journal of Molecular Sciences 23, no. 14 (July 13, 2022): 7720. http://dx.doi.org/10.3390/ijms23147720.
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Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity—eIFsixty-1, eIFsixty-4, and eIFsixty-6—and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.
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Scagliola, Alessandra, Annarita Miluzio, and Stefano Biffo. "Translational Control of Metabolism and Cell Cycle Progression in Hepatocellular Carcinoma." International Journal of Molecular Sciences 24, no. 5 (March 3, 2023): 4885. http://dx.doi.org/10.3390/ijms24054885.
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The liver is a metabolic hub characterized by high levels of protein synthesis. Eukaryotic initiation factors, eIFs, control the first phase of translation, initiation. Initiation factors are essential for tumor progression and, since they regulate the translation of specific mRNAs downstream of oncogenic signaling cascades, may be druggable. In this review, we address the issue of whether the massive translational machinery of liver cells contributes to liver pathology and to the progression of hepatocellular carcinoma (HCC); it represents a valuable biomarker and druggable target. First, we observe that the common markers of HCC cells, such as phosphorylated ribosomal protein S6, belong to the ribosomal and translational apparatus. This fact is in agreement with observations that demonstrate a huge amplification of the ribosomal machinery during the progression to HCC. Some translation factors, such as eIF4E and eIF6, are then harnessed by oncogenic signaling. In particular, the action of eIF4E and eIF6 is particularly important in HCC when driven by fatty liver pathologies. Indeed, both eIF4E and eIF6 amplify at the translational level the production and accumulation of fatty acids. As it is evident that abnormal levels of these factors drive cancer, we discuss their therapeutic value.
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Sanvito, Francesca, Simonetta Piatti, Antonello Villa, Mario Bossi, Giovanna Lucchini, Pier Carlo Marchisio, and Stefano Biffo. "The β4 Integrin Interactor p27BBP/eIF6 Is an Essential Nuclear Matrix Protein Involved in 60S Ribosomal Subunit Assembly." Journal of Cell Biology 144, no. 5 (March 8, 1999): 823–38. http://dx.doi.org/10.1083/jcb.144.5.823.
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p27BBP/eIF6 is an evolutionarily conserved protein that was originally identified as p27BBP, an interactor of the cytoplasmic domain of integrin β4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27BBP/eIF6, its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing β4 integrin, p27BBP/eIF6 is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of β4 integrin. Surprisingly, in the absence and in the presence of the β4 integrin subunit, p27BBP/eIF6 is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27BBP/eIF6 homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27BBP/eIF6 can rescue the lethal effect of the iihΔ yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27BBP/eIF6 in ribosome biogenesis or assembly rather than in translation. A further function related to the β4 integrin subunit may have evolved specifically in higher eukaryotic cells.
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Lin, Jinxin, Xihu Yu, Liping Xie, Puning Wang, Tuoyang Li, Yuanlv Xiao, Jiaming Zhou, et al. "eIF6 Promotes Colorectal Cancer Proliferation and Invasion by Regulating AKT-Related Signaling Pathways." Journal of Biomedical Nanotechnology 15, no. 7 (July 1, 2019): 1556–67. http://dx.doi.org/10.1166/jbn.2019.2792.
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Although abnormal expression of eukaryotic initiation factor 6 (eIF6) has been found in several human solid tumors, the functions and underlying mechanisms of eIF6 in the progression of colorectal cancer (CRC) still needs further elucidation. In the present study, large-scale gene analysis based on Oncomine and The Cancer Genome Atlas (TCGA) database revealed significantly higher baseline expression of eIF6 in colorectal cancer than in normal tissues. Furthermore, our Chinese cohort study revealed that high expression of eIF6 was correlated with aggressive characteristics and poor survival in CRC patients. Functional studies using magnetic nanoparticle extraction indicated that eIF6 was an oncogene in CRC cells. Regarding its mechanism, through Gene ontology (GO) and KEGG pathway analysis based on TCGA RNAseq database, we found that eIF6 can activate multiple AKT-related cancer signaling pathways, such as p-AKT\MMP1\cyclinD1\Bcl2-related signaling, to regulate cell proliferation, invasion, cell cycle and apoptosis in CRC. Collectively, these findings suggested that eIF6 can positively regulate AKT-related cancer signaling and enhance tumorigenicity in CRC, and may serve as a potential prognostic indicator and therapeutic target in CRC.
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Krassnig, Stefanie, Christina Wohlrab, Nicole Golob-Schwarzl, Andrea Raicht, Christoph Schatz, Anna Maria Birkl-Toeglhofer, Christina Skofler, et al. "A Profound Basic Characterization of eIFs in Gliomas: Identifying eIF3I and 4H as Potential Novel Target Candidates in Glioma Therapy." Cancers 13, no. 6 (March 23, 2021): 1482. http://dx.doi.org/10.3390/cancers13061482.
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Glioblastoma (GBM) is an utterly devastating cerebral neoplasm and current therapies only marginally improve patients’ overall survival (OS). The PI3K/AKT/mTOR pathway participates in gliomagenesis through regulation of cell growth and proliferation. Since it is an upstream regulator of the rate-limiting translation initiation step of protein synthesis, controlled by eukaryotic initiation factors (eIFs), we aimed for a profound basic characterization of 17 eIFs to identify potential novel therapeutic targets for gliomas. Therefore, we retrospectively analyzed expressions of mTOR-related proteins and eIFs in human astrocytoma samples (WHO grades I–IV) and compared them to non-neoplastic cortical control brain tissue (CCBT) using immunoblot analyses and immunohistochemistry. We examined mRNA expression using qRT-PCR and additionally performed in silico analyses to observe the influence of eIFs on patients’ survival. Protein and mRNA expressions of eIF3B, eIF3I, eIF4A1, eIF4H, eIF5 and eIF6 were significantly increased in high grade gliomas compared to CCBT and partially in low grade gliomas. However, short OS was only associated with high eIF3I gene expression in low grade gliomas, but not in GBM. In GBM, high eIF4H gene expression significantly correlated with shorter patient survival. In conclusion, we identified eIF3I and eIF4H as the most promising targets for future therapy for glioma patients.
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Jaako, Pekka, Chi C. Wong, David Adams, and Alan J. Warren. "Attenuated Protein Synthesis Drives the Hematopoietic Defects in Shwachman-Diamond Syndrome." Blood 130, Suppl_1 (December 7, 2017): 876. http://dx.doi.org/10.1182/blood.v130.suppl_1.876.876.
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Abstract Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure and a striking propensity to develop poor prognosis myelodysplastic syndrome and acute myeloid leukemia. In 90 % of cases the disease is caused by biallelic mutations in the gene encoding SBDS. We have shown previously that SBDS is a cytoplasmic ribosome assembly factor that catalyzes the release of the eukaryotic initiation factor 6 (eIF6) from the subunit joining interface of 60S ribosomal subunit (Menne et al, 2007; Finch et al, 2011). Deficiency of SBDS therefore results in aberrant retention of eIF6 on the 60S subunits that in turn perturbs ribosomal subunit joining and the formation of translation-competent 80S ribosomes. However, the mechanism linking defective ribosome assembly to marrow failure and leukemia in SDS remain poorly understood. Lack of viable mouse models presents a barrier to progress in understanding SDS disease pathophysiology and to evaluate novel therapies. We hypothesized that induced overexpression of eIF6 would mimic the consequences of SBDS deficiency by reducing the cytoplasmic pool of free 60S subunits and impairing translation. To test this hypothesis we have generated a novel transgenic eIF6 mouse model for SDS using KH2 embryonic stem cells that constitutively express the M2-reverse tetracycline transactivator at the Rosa26 locus with the EIF6 gene targeted downstream of the Col1a1 locus. This strategy permits systemic doxycycline-inducible and graded overexpression of eIF6 through control of the transgene copy number. We have validated that eIF6 overexpression promotes an increase in eIF6-bound cytoplasmic 60S subunits with a concomitant reduction in 80S ribosomes and polysomes in c-kit+ hematopoietic progenitor cells isolated from the transgenic eIF6 mice, thereby recapitulating the ribosomal subunit joining defect observed in patients with SDS. In vitro, the hematopoietic progenitor cells exhibit a strict eIF6 dose-dependent expansion defect. In vivo, mice with graded eIF6 overexpression are viable but develop macrocytic anemia with reticulocytopenia, thrombocytosis and mild leukopenia. Bone marrow transfer experiments demonstrate that the phenotype is autonomous to the hematopoietic system. Longitudinal phenotypic analyses in primary and transplanted animals are ongoing. Flow cytometric analysis of the bone marrow from transgenic eIF6 mice reveals a significant increase in the frequencies of preCFU-E and CFU-E erythroid progenitor cells and erythroblasts, but a significant reduction in the frequency of reticulocytes. Furthermore, we observe a striking accumulation of abnormal orthochromatic erythroblast-like cells that appear to have failed to enucleate, comprising approximately 1.5 % of the total bone marrow cells. Amnis ImageStream analysis, which combines flow cytometry with fluorescent microscopy, reveals a significant decrease in the frequency of erythroblasts that are able to complete the enucleation process. To address the underlying mechanism, we hypothesized that by impairing the formation of translation-competent 80S ribosomes, eIF6 overexpression would reduce the global rate of protein synthesis. Indeed, O-propargyl-puromycin incorporation assays established that the erythroblasts from the transgenic eIF6 mice have an approximately 3-fold reduction in global protein synthesis rate. Furthermore, our preliminary data suggest that the erythroid phenotype is p53-independent. Finally, erythroblasts from the transgenic eIF6 mice show a significant increase in levels of reactive oxygen species, but the functional significance of this finding remains unclear. We conclude that reduced rates of global translation drive defective hematopoiesis in the transgenic eIF6 mice. Importantly, eIF6 overexpression in vivo phenocopies SBDS depletion in human CD34+ cells (Sen et al, 2011). Together with the recent discovery of DNAJC21 (the human homologue of the 60S ribosomal assembly factor JJJ1 in yeast) as an SDS disease gene, our data support the hypothesis that deregulated cytoplasmic 60S subunit maturation and reduced translation are the primary drivers of the hematopoietic defect in SDS. Our viable transgenic eIF6 mouse model provides a unique tool to further dissect the mechanisms that underlie bone marrow failure and malignant transformation in SDS and for the development of novel therapeutics. Disclosures No relevant conflicts of interest to declare.
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Basu, Uttiya, Kausik Si, Jonathan R. Warner, and Umadas Maitra. "The Saccharomyces cerevisiae TIF6 Gene Encoding Translation Initiation Factor 6 Is Required for 60S Ribosomal Subunit Biogenesis." Molecular and Cellular Biology 21, no. 5 (March 1, 2001): 1453–62. http://dx.doi.org/10.1128/mcb.21.5.1453-1462.2001.
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ABSTRACT Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. InSaccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.
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Kennedy, Alyssa L., Kasiani C. Myers, James Bowman, Christopher J. Gibson, Gwen M. Muscato, Robert Klein, Kaitlyn Ballotti, et al. "Distinct Genetic Pathways Define Leukemia Predisposition Versus Adaptive Clonal Hematopoiesis in Shwachman-Diamond Syndrome." Blood 136, Supplement 1 (November 5, 2020): 35–36. http://dx.doi.org/10.1182/blood-2020-137125.
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Background: Shwachman-Diamond Syndrome (SDS) is a bone marrow failure disorder caused by impaired removal of EIF6 from the nascent 60S ribosome subunit, resulting in defective ribosome assembly. SDS patients have a high risk of myeloid neoplasms (MN) and the prognosis of those that develop MN is poor. Knowledge of the kinetics and functional consequences of somatic mutation acquisition in SDS may offer insight into mechanism of transformation and the potential for therapuetic intervention. Methods: We performed whole exome sequencing of 45 samples from 30 patients, and validated recurrent somatically mutated genes using targeted sequencing with error suppression in prospectively collected samples from 110 patients in the North American SDS Registry. We correlated mutation status with clinical outcome and performed functional studies to understand the consequence of somatic mutations in SDS. Results: We detected somatic mutations in 74 of 98 (76%) patients with germline biallelic SBDS mutations (median 2 mutations/patient, range 0-21). We found no mutations in patients with SDS-like disease; those who have clinical features of SDS without disease defining mutations. Of the 83 patients with SDS without a MN diagnosis, 60 (72%) had detectable clonal hematopoiesis (CH), 40 of whom had more than one mutation (median 3, range 1-21). The most frequently mutated genes were EIF6 (60/98, 61%),TP53 (44/98, 45%), PRPF8 (12/98, 12%), and CSNK1A1 (6/98, 6%). Among SDS patients with TP53 mutated CH, 90.9% (30 of 33) had concurrent EIF6 mutations. To determine whether EIF6 and TP53 mutations occur in the same or different clones, we performed single cell DNA sequencing. Among the 47 clones identified with either EIF6 or TP53 mutations, 24 had a sole EIF6 mutation, and 21 had a sole TP53 mutation, showing that these mutations arise in separate clones. To study the functional consequences of EIF6 missense mutations, we cloned 7 patient-derived mutations and generated cell lines expressing wild-type or mutant EIF6 cDNA. We found six mutants (I13N, R67W, G69S, P73R, A194T, G196R) reduced levels of EIF6 protein compared with wild type EIF6, despite comparable abundance of mRNA. The most common recurrent mutation, N106S, was found in 20% of patients and, by contrast to others listed above, did not change protein expression. This mutation is located at the EIF6/60S protein interface and disrupted the interaction of N106S-EIF6 with the 60S subunit as measured by polysome profiling followed by western blotting. To compare the effects of EIF6 versus TP53 somatic mutations in context of SDS deficient translation, we measured ribosome maturation and translation in SDS cells containing shRNAs targeting EIF6 or TP53. EIF6 knockdown ameliorated the SDS defect, reflected by improved ribosome joining (normalization of the 80:60s ratio) and enhanced protein translation (increased O-propargyl-puromycin incorporation), whereas TP53 knockdown had no effect. Knockdown of EIF6 in SDS deficient cells decreased p53 pathway activation as demonstrated by decreased CDKN1A expression. TP53 mutations were significantly associated with MN diagnosis (p=0.023), but were also common in SDS CH and typically stable over time. To identify the characteristics associated with transformation, we analyzed exomes from 7 patients with TP53 mutated myeloid malignancy for allelic imbalances at the TP53 locus and found that all 7 had biallelic alteration of TP53. Using single cell DNA sequencing from serial samples, we observed that TP53 LOH can precede transformation by several years and can distinguish pre-leukemic clones from indolent clones with monoallelic TP53 alterations. Somatic EIF6 mutations were not found in the leukemic clones. These results suggest early detection of TP53 LOH may distinguish clones with leukemic potential. Conclusions: In SDS, impairment of ribosome maturation drives selection of clones with somatic EIF6 or TP53 mutations. EIF6 mutations promote competitive fitness by rescuing the SDS ribosome defect and decreasing p53 pathway activation, and do not contribute to malignant transformation. TP53 mutations decrease checkpoint activation without affecting ribosome assembly. These results provide genetic evidence that germline SBDS deficiency causes a global, disease-specific HSC fitness constraint that drives parallel development of somatic CH and provides a mechanistic rationale for clinical surveillance. Disclosures Dale: Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Research Funding; X4 Pharmaceuticals: Honoraria. Gansner:Alnylam Pharmaceuticals: Current Employment, Current equity holder in private company. Edwards:Jazz Pharmaceuticals: Consultancy, Honoraria. Fleming:DISC Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy; Bluebird Bio: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding.
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Peng, Haoning, Mengyao Wang, Lu Li, Lunxu Liu, and Shensi Shen. "Abstract 3906: The mRNA translational plasticity of small cell lung carcinoma is associated with its phenotypic transdifferentiation." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3906. http://dx.doi.org/10.1158/1538-7445.am2023-3906.
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Abstract Small cell lung carcinoma(SCLC), classified as a recalcitrant cancer type, accounts for 13-15% of all lung cancers with a median overall survival less than 10 months in patients with extensive stage SCLC. SCLC was once considered as molecularly homogeneous due to nearly universal loss of TP53 and RB1, whereas subsets of MYC-driven, non-neuroendocrine (non-NE) SCLC variants, possess chemoresistance, suggesting its high plasticity and intertumoral heterogeneity related to rapid refractory to first-line chemotherapies. Current pathognomonic classification stratifies SCLC into two NE subtypes (ASCL1+, NEUROD1+) and two non-NE subtypes (POU2F3+ and triple negative), associated with different expression patterns of MYC family proteins. MYC is a master regulator of mRNA translation, whether translational regulation actively tunes SCLC plasticity and the associated chemosensitivity remains an enigma. Here, we delineate the translation landscape of NE and non-NE SCLC cells by using paired human NE SCLC cell line and constitutive or induced non-NE SCLC cells. As expected, non-NE SCLC cells are more resistant to the clinically relevant first-line chemotherapy than NE SCLC cells. With polysome profiling in combination with polysome RNA-seq, we showed differential expression at both transcription- and translation-level. However, transcription-level changes minimally overlapped with the translational changes between NE and non-NE SCLC cells, with a global reduction of translation activity observed in non-NE SCLC cells. Through analyzing the polysome profiles, we observed a striking accumulation of 60S monosomes and diminishment of 80S ribosomes derived from non-NE SCLC cells compared to those from NE SCLC cells. This unique translational pattern in non-NE SCLC cells was correlated with an increased expression of eIF6, a ‘sitting on the bench’ member of eukaryotic initiation factor (eIF) family. The eIF6 couples 60S monosome maturation and recycling, enrolling a quintessential role in functional 80S ribosome formation. Positive correlation between eIF6 expression and non-NE SCLC tumors was observed in 81 tumor samples derived from patients with limited stage SCLC and CCLE cell consortium, in which c-MYC expression was also positively correlated with eIF6 level. By using both ultracentrifugation and fraction analysis from polysome profiling, we found that less association of eIF6 with 60S may mitigate 80S ribosome assembly in non-NE SCLC cells. In addition, in vivo xenograft models showed that eIF6 expression was higher in residual tumors upon chemotherapy. Taken together, these data revealed an eIF6-dependent translational regulation in non-NE transdifferentiation of SCLC subtypes, suggesting an underappreciated function of translational regulation of SCLC plasticity and the potential therapeutic value of eIF6 for overcoming SCLC resistance to chemotherapy. Citation Format: Haoning Peng, Mengyao Wang, Lu Li, Lunxu Liu, Shensi Shen. The mRNA translational plasticity of small cell lung carcinoma is associated with its phenotypic transdifferentiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3906.
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Si, Kausik, and Umadas Maitra. "The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor." Molecular and Cellular Biology 19, no. 2 (February 1, 1999): 1416–26. http://dx.doi.org/10.1128/mcb.19.2.1416.
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ABSTRACT Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M r 25,550), designated TIF6, has been cloned and expressed inEscherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.
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Cai, Shao-xin, Wen-shu Chen, Wei Zeng, Xue-fei Cheng, Meng-bo Lin, and Jin-si Wang. "Roles of HDAC2, eIF5, and eIF6 in Lung Cancer Tumorigenesis." Current Medical Science 41, no. 4 (August 2021): 764–69. http://dx.doi.org/10.1007/s11596-021-2389-z.
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Zeman, Jakub, Yuzuru Itoh, Zdeněk Kukačka, Michal Rosůlek, Daniel Kavan, Tomáš Kouba, Myrte E. Jansen, Mahabub P. Mohammad, Petr Novák, and Leoš S. Valášek. "Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes." Nucleic Acids Research 47, no. 15 (July 10, 2019): 8282–300. http://dx.doi.org/10.1093/nar/gkz570.
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Abstract eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.
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Singh, Sharon, Lionel Blanc, Adrianna Henson, Gulay Sezgin, Steven R. Ellis, and Johnson M. Liu. "Suppression of the Hematopoietic Defect in TF-1 Cells Depleted of Shwachman-Diamond Syndrome Protein: Correlation with Decreased eIF6 Levels." Blood 120, no. 21 (November 16, 2012): 1270. http://dx.doi.org/10.1182/blood.v120.21.1270.1270.
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Abstract Abstract 1270 Shwachman Diamond syndrome (SDS) is a rare autosomal recessive bone marrow failure syndrome mainly characterized by neutropenia, exocrine pancreatic insufficiency and an increased risk of myelodysplastic syndrome and leukemia. The phenotype in patients is variable for unclear reasons, but approximately 90% of patients have biallelic mutations in the SBDS gene. At least one action of the SBDS protein is to couple with the GTPase ELF1 to facilitate release of the eIF6 protein from the 60S ribosome subunit, thus enabling joining of the 60S and 40S ribosome subunits, a function that has prompted many to consider SDS a “ribosomopathy”. We created a cellular model of SDS using TF-1 erythroleukemia cells transduced with lentiviral vectors containing two different shRNAs against SBDS or a scrambled sequence. Clones were grown under puromycin selection and a clone from each shRNA was selected. In each clone, knockdown of SBDS was verified at the protein level by western blot, and expression levels of SBDS were less than 1%. Both clones underwent differentiation to either myeloid or erythroid colonies by culturing in GM-CSF or erythropoietin, respectively. The 2–12 clone had a significant decrease in the number and size of both myeloid and erythroid colonies (see Table) when compared with the scrambled shRNA control. In contrast, the 1–7 clone had the same number of myeloid and erythroid colonies as the control. Previous work by other investigators in SDS yeast models revealed that missense mutations in the anti-association factor, Tif6 suppress the slow growth phenotype of SDS-mutant yeast cells. In exploring the molecular basis for the difference in phenotype observed in our TF-1 cells, we therefore focused on eIF6, the human ortholog of Tif6. The 2–12 clone had similar expression of the eIF6 protein when compared to the scrambled control. However, the 1–7 clone had a significantly decreased amount of eIF6 protein compared to the control. DNA sequencing did not reveal any mutations in the eIF6 gene, and quantitative RT-PCR showed similar levels of eIF6 mRNA transcripts, suggesting that the differences in eIF6 protein levels may be due to post-translational modifications. Pressato and colleagues (Br J Haematol 157:503, 2012) have recently speculated that the relatively benign course of SDS patients with a deletion of chromosome 20q may be due to loss of the eIF6 protein (whose gene is located on 20q). Our findings add to the hypothesis that antagonizing eIF6 may modify or rescue the SDS phenotype, possibly by reducing the requirement of SBDS in giving rise to 60S subunits lacking eIF6. Scramble colonies +/− SE 2–12 colonies +/−SE 1–7 colonies +/− SE Myeloid 131+/−4.4 112+/−3.5 p<0.01 135+/−6.8 p=0.64 Erythroid 89+/−8.4 48+/−4 p<0.001 89+/− 4.7 p=0.94 Disclosures: No relevant conflicts of interest to declare.
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Chierchia, Loredana, Margherita Tussellino, Domenico Guarino, Rosa Carotenuto, Nadia DeMarco, Chiara Campanella, Stefano Biffo, and Maria Carmela Vaccaro. "Cytoskeletal proteins associate with components of the ribosomal maturation and translation apparatus in Xenopus stage I oocytes." Zygote 23, no. 5 (September 17, 2014): 669–82. http://dx.doi.org/10.1017/s0967199414000409.
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SummaryActin-based cytoskeleton (CSK) and microtubules may bind to RNAs and related molecules implicated in translation. However, many questions remain to be answered regarding the role of cytoskeletal components in supporting the proteins involved in steps in the maturation and translation processes. Here, we performed co-immunoprecipitation and immunofluorescence to examine the association between spectrins, keratins and tubulin and proteins involved in 60S ribosomal maturation and translation in Xenopus stage I oocytes, including ribosomal rpl10, eukaryotic initiation factor 6 (Eif6), thesaurins A/B, homologs of the eEF1α elongation factor, and P0, the ribosomal stalk protein. We found that rpl10 and eif6 cross-reacted with the actin-based CSK and with tubulin. rpl10 co-localizes with spectrin, particularly in the perinuclear region. eif6 is similarly localized. Given that upon ribosomal maturation, the insertion of rpl10 into the 60S subunit occurs simultaneously with the release of eif6, one can hypothesise that actin-based CSK and microtubules provide the necessary scaffold for the insertion/release of these two molecules and, subsequently, for eif6 transport and binding to the mature 60S subunit. P0 and thesaurins cross-reacted with only spectrin and cytokeratins. Thesaurins aggregated at the oocyte periphery, rendering this a territory favourable site for protein synthesis; the CSK may support the interaction between thesaurins and sites of the translating ribosome. Moreover, given that the assembly of the ribosome stalk, where P0 is located, to the 60S subunit is essential for the release of eif6, it can be hypothesised that the CSK can facilitate the binding of the stalk to the 60S.
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Burwick, Nicholas, Scott Coats, and Akiko Shimamura. "SBDS and eIF6 Modulate Ribosome Subunit Joining in Shwachman Diamond Syndrome,." Blood 118, no. 21 (November 18, 2011): 3438. http://dx.doi.org/10.1182/blood.v118.21.3438.3438.
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Abstract Abstract 3438 Shwachman Diamond syndrome (SDS) is an autosomal recessive marrow failure syndrome with a predisposition to leukemia. Over 90% of SDS patients harbor biallelic mutations in the SBDS gene. SBDS has been implicated in several cellular functions including ribosome biogenesis and mitotic spindle stabilization. Deletion of SBDS orthologues in yeast results in a severe slow growth phenotype and depressed polysomes. Homozygous deletion of Sbds in murine models results in early embryonic lethality, while conditional deletion of Sbds in mouse liver demonstrates accumulation of 40S and 60S subunits and halfmer formation consistent with impaired ribosome joining. SBDS facilitates the release of eIF6, a factor that prevents ribosome joining. The dramatic phenotypic and polysome changes noted in these experimental models were not observed in cells derived from SDS patients. SDS patient cells have only a mildly reduced growth rate compared to heatlhy controls, and polysome profiles do not demonstrate depressed polysomes or halfmer formation. Since complete abrogation of SBDS expression is lethal and biallelic null mutations in SBDS have not been reported, we examined the role of SBDS and eIF6 in SDS patients and human cell models. We first investigated whether ribosome subunit homeostasis is impaired in SDS patient cells. We find that the 60S:40S ribosomal subunit ratio is consistently reduced in bone marrow stromal cells from SDS patients of different genotypes (n=4). This impairment in 60S:40S ratio is demonstrated in both SDS patient stromal cells and patient lymphoblasts. Stable lentiviral knockdown of SDS in normal marrow stromal cells recapitulates the reduction in 60S:40S ratio. SBDS and eIF6 co-sediment in polysome gradients of human SDS cells. This co-sedimentation is specific for the 60S ribosomal subunit. Since eIF6 has a role as an anti-joining factor, we next developed an in vitro assay to test for ribosome subunit joining in human cells. In this assay, we validate that over-expression of eIF6 results in reduced ribosome joining, and eIF6 knockdown promotes ribosome joining. Moreover, we find that SDS patient stromal cells and patient lymphoblasts both demonstrate impaired ribosome subunit joining, compared with healthy controls. Importantly, the addition of wild type SBDS or depletion of eIF6 improve ribosome joining in SDS patient cells. We demonstrate that the amino terminal sequences of SBDS are necessary but not sufficient for the association of SBDS with the 60S ribosomal subunit. Insertion of a patient-derived N-terminal SBDS point mutation also results in decreased association of SBDS with the 60S ribosomal subunit. These structure-function studies may help to inform genotype:phenotype correlations in SDS. The role of defective ribosome joining in promoting the SDS hematopoietic phenotype is of particular interest. Ongoing studies are interrogating the role of eIF6 modulation on the hematopoietic phenotype in SBDS- depleted cells. Insights garnered from these experiments will help inform the development of novel agents to improve the hematopoetic defect in human SDS. Disclosures: No relevant conflicts of interest to declare.
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Kawashima, Nozomu, Usua Oyarbide, and Seth J. Corey. "Loss of EFL1 Which Causes a Shwachman-Diamond Syndrome Reduces Cell Proliferation and Alters Transcriptional Profiling during Neutrophil Differentiation." Blood 142, Supplement 1 (November 28, 2023): 2729. http://dx.doi.org/10.1182/blood-2023-187549.
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Introduction. Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. Patients with SDS are predisposed to develop myeloid malignancy. Almost all individuals with SDS harbor biallelic mutations in SBDS. The partner of SBDS is EFL1, a ribosomal protein with GTPase activity. More than a dozen patients have been found with SDS due to biallelic mutations in EFL1. EFL1 serves to release EIF6, facilitating the assembly of the 80S ribosome from 60S and 40S ribosome subunits. Our lab has generated both sbds and efl1 null zebrafish, which phenocopy much of the human syndrome. We recently reported that Sbds and Efl1 depletion leads to upregulation of tp53 and cdkn1a ( p21) and accumulation of Eif6 in SDS zebrafish models, which is hypothesized to cause bone marrow failure. However, the mechanism(s) of EFL1's loss-of-function leading to neutropenia is not known. Methods. We used CRISPR/Cas9 editing to generate mutations in exon 17 of Efl1 of 32Dcl3, an IL-3-dependent murine myeloid cell line that can be differentiated to mature neutrophils. We analyzed the effects of Efl1 mutations on cell proliferation, survival, and differentiation with G-CSF. Immunoblotting and qPCR-based gene expression analysis were performed to identify affected mechanistic pathways. Results. Two clones were isolated and were sequenced to verify biallelic deleterious mutations. A clone with compound heterozygous inframe deletion and frameshift mutations showed trace expression of EFL1 (EFL1-KD) and the other with compound heterozygous frameshift mutations showed no expression (EFL1-KO). They presented a myeloblast-like morphology indistinguishable from parental cells. They proliferated significantly less than parental cells, although there was no increased apoptosis. Both EFL1-KD and KO cells showed significantly increased protein expression of EIF6 and transcriptional expression of Trp53 and Cdkn1a. EFL1 was downregulated during differentiation of parental 32Dcl3 cells to neutrophils, whereas EIF6 levels did not change. Withdrawal and replenishment of IL-3 from culturing media did not alter EFL1 protein expression. When EFL1-KD and KO were cultured in differentiating media, they did not proliferate and underwent greater apoptosis, particularly after 3 days of differentiation compared to parental 32Dcl3. The surviving EFL1-KD and KO matured into neutrophils after 7 days. Gene profiling for neutrophil differentiation identified equivalent expression of key transcriptional factors ( Cebpa, Cebpe, and Spi1) of EFL1-KD and KO in the maintenance media, except for Gfi1 which was two-fold and three-fold high in EFL1-KD and KO compared to parental cells, respectively. G-CSF induced significantly higher expression of Cebpa and lower expression of Cebpe in EFL1-KO from those in parental cells, respectively. Based on the observation that somatic genetic rescue by EIF6 mutations occurs in human SDS patients and animal models, we transfected 32Dcl3 cells with siRNA against Eif6. In parental 32Dcl3 cells, reduction of EIF6 caused slow cell growth; however, transfection of siRNA to Eif6 partially rescued proliferation of EFL1-KD and KO in both maintenance and differentiating media. Conclusions. Our results suggested that imbalance of late maturing factors of ribosome associated with SDS may cause decreased proliferation in myeloid-committed cells, thereby leading to neutropenia in SDS. Newly established Efl1-deficient cells recapitulated key observation from SDS patients, with an increased proapoptotic profile in hematopoietic cells. Because EFL1 has only been recognized recently as a rare cause of SDS, we do not know if EFL1 deficiency serves as a leukemia predisposition syndrome. We speculate that the aberrant normal neutrophil differentiation may form the foundation for myeloid neoplasia in SDS.
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Das, Supratik, and Umadas Maitra. "Mutational Analysis of Mammalian Translation Initiation Factor 5 (eIF5): Role of Interaction between the β Subunit of eIF2 and eIF5 in eIF5 Function In Vitro and In Vivo." Molecular and Cellular Biology 20, no. 11 (June 1, 2000): 3942–50. http://dx.doi.org/10.1128/mcb.20.11.3942-3950.2000.
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ABSTRACT Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S–eIF3–AUG–Met-tRNAf–eIF2–GTP) to promote the hydrolysis of ribosome-bound GTP. eIF5 also forms a complex with eIF2 by interacting with the β subunit of eIF2. In this work, we have used a mutational approach to investigate the importance of eIF5-eIF2β interaction in eIF5 function. Binding analyses with recombinant rat eIF5 deletion mutants identified the C terminus of eIF5 as the eIF2β-binding region. Alanine substitution mutagenesis at sites within this region defined several conserved glutamic acid residues in a bipartite motif as critical for eIF5 function. The E346A,E347A and E384A,E385A double-point mutations each caused a severe defect in the binding of eIF5 to eIF2β but not to eIF3-Nip1p, while a eIF5 hexamutant (E345A,E346A,E347A,E384A,E385A,E386A) showed negligible binding to eIF2β. These mutants were also severely defective in eIF5-dependent GTP hydrolysis, in 80S initiation complex formation, and in the ability to stimulate translation of mRNAs in an eIF5-dependent yeast cell-free translation system. Furthermore, unlike wild-type rat eIF5, which can functionally substitute for yeast eIF5 in complementing in vivo a genetic disruption of the chromosomal copy of the TIF5 gene, the eIF5 double-point mutants allowed only slow growth of this ΔTIF5 yeast strain, while the eIF5 hexamutant was unable to support cell growth and viability of this strain. These findings suggest that eIF5-eIF2β interaction plays an essential role in eIF5 function in eukaryotic cells.
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Chendrimada, Thimmaiah P., Kenneth J. Finn, Xinjun Ji, David Baillat, Richard I. Gregory, Stephen A. Liebhaber, Amy E. Pasquinelli, and Ramin Shiekhattar. "MicroRNA silencing through RISC recruitment of eIF6." Nature 447, no. 7146 (May 16, 2007): 823–28. http://dx.doi.org/10.1038/nature05841.
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Valášek, Leoš, Klaus H. Nielsen, Fan Zhang, Christie A. Fekete, and Alan G. Hinnebusch. "Interactions of Eukaryotic Translation Initiation Factor 3 (eIF3) Subunit NIP1/c with eIF1 and eIF5 Promote Preinitiation Complex Assembly and Regulate Start Codon Selection." Molecular and Cellular Biology 24, no. 21 (November 1, 2004): 9437–55. http://dx.doi.org/10.1128/mcb.24.21.9437-9455.2004.
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ABSTRACT The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the eIF2-GTP-Met- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{i}}^{\mathit{Met}}\) \end{document} ternary complex (TC) to form the multifactor complex (MFC). We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui− phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui− phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui− phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo.
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Oyarbide Cuervas-Mons, Usua, Matthew Snyderman, Jacek Topczewski, and Seth J. Corey. "Expression of Human SBDSR126T in Sbds Null Background Shows Eif6 Dysregulation: An Adult Zebrafish Model for Shwachman-Diamond Syndrome." Blood 134, Supplement_1 (November 13, 2019): 3737. http://dx.doi.org/10.1182/blood-2019-129014.
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Background. Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency, skeletal defects, neutropenia, and an increased risk of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). SDS occurs in 1/75,000 births, and biallelic mutations in the SBDS gene account for ~90% of patients. The SBDS protein is highly conserved. SBDS interacts physically with EFL1 to release EIF6 from the cytoplasmic pre-60S ribosomal subunit and promote the assembly of the mature 80S ribosome. The SBDS R126T allele is found in combination with the common K62X mutation in some SDS patient. A recent study showed that the SBDSR126T is not able to activate the GTPase activity of the EFL1, affecting the release of EIF6 from the 60S surface. Methods. We created a zebrafish knockout line that phenocopies the SDS with neutropenia, pancreas atrophy, small size (Figure 1A), and decreased 80S ribosomes. To rescue those fish from early mortality, we generated a new transgenic line Tg(ubi:SBDSR126T:pA) expressing the missense variant R126T, a disease-associated allele. Results. The sbds knockout fish die after 21 days post fertilization (dpf), corresponding to an early juvenile stage. However, the SBDSR126T transgenic line in the background of the sbds knockout can live for at least 12 months. This is in strong contrast to the mouse SbdsR126T/R126T line that do not survive to birth. Transgenically-rescued fish displayed a small size phenotype resembling SDS (Figure 1B). Levels of ribosomal proteins Rpl5 and Rpl11 were lower in the sbds knockout at 21 dpf but they were normal in the transgenic line at 6 months. We also observed a concordant regulation of Sbds and Eif6 expression (Figure 1C,D). sbds null fish showed a significant upregulation of cdkn1a, while in their transgenic siblings levels were normal (Figure 1E). Moreover, mpx was upregulated in the transgenic line with the null background (Figure 1F). Analysis of neutrophil and monocyte counts are being performed and will be reported. Conclusions. Our novel SBDSR126T zebrafish model survives until adulthood, which will allow us to carry out a number of informative assays such as stress response, gene expression, and polysome profiles in different organs. Rpl5 and Rpl11 levels are affected in sbds mutants but not in the transgenic line. Activation of cdkn1a (p21) in sbds mutants might lead to apoptosis and death. The normal levels of cdkn1a in the transgenic line might be non-deleterious, as loss of Tp53 activation can rescue some models of bone marrow failure. In addition, loss of sbds or expression of SBDSR126T affect Eif6 levels in zebrafish. Importantly, some patients with SBDS deficiency acquire interstitial deletions of chromosome 20, resulting in the loss of the EIF6 gene. This might be a potential mechanism to suppress the defect in ribosome biogenesis by reducing the copy number of the EIF6 gene and has been related to a lower risk of MDS/AML comparing to other SDS patients. Our adult model of Shwachman-Diamond Syndrome can provide new insights into the pathogenesis of SDS and its progression to malignancy, which can be used to identify novel targets for AML/MDS therapy. Figure 1 Disclosures No relevant conflicts of interest to declare.
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Tan, Shengjiang, Laëtitia Kermasson, Angela Hoslin, Pekka Jaako, Alexandre Faille, Abraham Acevedo-Arozena, Etienne Lengline, et al. "EFL1 mutations impair eIF6 release to cause Shwachman-Diamond syndrome." Blood 134, no. 3 (July 18, 2019): 277–90. http://dx.doi.org/10.1182/blood.2018893404.
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Abstract Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.
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Niepmann, Michael, and Gesche K. Gerresheim. "Hepatitis C Virus Translation Regulation." International Journal of Molecular Sciences 21, no. 7 (March 27, 2020): 2328. http://dx.doi.org/10.3390/ijms21072328.
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Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5’-untranslated region (5′UTR) and part of the core protein coding sequence, and by the 3′UTR. The 5′UTR has some highly conserved structural regions, while others can assume different conformations. The IRES can bind to the ribosomal 40S subunit with high affinity without any other factors. Nevertheless, IRES activity is modulated by additional cis sequences in the viral genome, including the 3′UTR and the cis-acting replication element (CRE). Canonical translation initiation factors (eIFs) are involved in HCV translation initiation, including eIF3, eIF2, eIF1A, eIF5, and eIF5B. Alternatively, under stress conditions and limited eIF2-Met-tRNAiMet availability, alternative initiation factors such as eIF2D, eIF2A, and eIF5B can substitute for eIF2 to allow HCV translation even when cellular mRNA translation is downregulated. In addition, several IRES trans-acting factors (ITAFs) modulate IRES activity by building large networks of RNA-protein and protein–protein interactions, also connecting 5′- and 3′-ends of the viral RNA. Moreover, some ITAFs can act as RNA chaperones that help to position the viral AUG start codon in the ribosomal 40S subunit entry channel. Finally, the liver-specific microRNA-122 (miR-122) stimulates HCV IRES-dependent translation, most likely by stabilizing a certain structure of the IRES that is required for initiation.
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Ceci, Marcello, Nina Offenhäuser, Pier Carlo Marchisio, and Stefano Biffo. "Formation of nuclear matrix filaments by p27BBP/eIF6." Biochemical and Biophysical Research Communications 295, no. 2 (July 2002): 295–99. http://dx.doi.org/10.1016/s0006-291x(02)00671-x.
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Mohammad-Qureshi, Sarah S., Martin D. Jennings, and Graham D. Pavitt. "Clues to the mechanism of action of eIF2B, the guanine-nucleotide-exchange factor for translation initiation." Biochemical Society Transactions 36, no. 4 (July 22, 2008): 658–64. http://dx.doi.org/10.1042/bst0360658.
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A variety of cellular processes rely on G-proteins, which cycle through active GTP-bound and inactive GDP-bound forms. The switch between these states is commonly regulated by GEFs (guanine-nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Although G-proteins have structural similarity, GEFs are very diverse proteins. A complex example of this system is seen in eukaryotic translation initiation between eIF (eukaryotic initiation factor) 2, a G-protein, its five-subunit GEF, eIF2B, and its GAP, eIF5. eIF2 delivers Met-tRNAi (initiator methionyl-tRNA) to the 40S ribosomal subunit before mRNA binding. Upon AUG recognition, eIF2 hydrolyses GTP, aided by eIF5. eIF2B then re-activates eIF2 by removing GDP, thereby promoting association of GTP. In the present article, we review data from studies of representative G-protein–GEF pairs and compare these with observations from our research on eIF2 and eIF2B to propose a model for how interactions between eIF2B and eIF2 promote guanine nucleotide exchange.
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Gartmann, Marco, Michael Blau, Jean-Paul Armache, Thorsten Mielke, Maya Topf, and Roland Beckmann. "Mechanism of eIF6-mediated Inhibition of Ribosomal Subunit Joining." Journal of Biological Chemistry 285, no. 20 (March 31, 2010): 14848–51. http://dx.doi.org/10.1074/jbc.c109.096057.
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Romano, Nicla, Sara Ricciardi, Paolo Gallo, and Marcello Ceci. "Upregulation of eIF6 inhibits cardiac hypertrophy induced by phenylephrine." Biochemical and Biophysical Research Communications 495, no. 1 (January 2018): 601–6. http://dx.doi.org/10.1016/j.bbrc.2017.11.046.
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Singh, Chingakham Ranjit, Cynthia Curtis, Yasufumi Yamamoto, Nathan S. Hall, Dustin S. Kruse, Hui He, Ernest M. Hannig, and Katsura Asano. "Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation." Molecular and Cellular Biology 25, no. 13 (July 1, 2005): 5480–91. http://dx.doi.org/10.1128/mcb.25.13.5480-5491.2005.
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ABSTRACT The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts− phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd− phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNAi Met ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn− phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.
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Ye, Caiyong, Bochao Liu, Huimei Lu, Jingmei Liu, Arnold B. Rabson, Estela Jacinto, Dimitri G. Pestov, and Zhiyuan Shen. "BCCIP is required for nucleolar recruitment of eIF6 and 12S pre-rRNA production during 60S ribosome biogenesis." Nucleic Acids Research 48, no. 22 (November 27, 2020): 12817–32. http://dx.doi.org/10.1093/nar/gkaa1114.
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Abstract Ribosome biogenesis is a fundamental process required for cell proliferation. Although evolutionally conserved, the mammalian ribosome assembly system is more complex than in yeasts. BCCIP was originally identified as a BRCA2 and p21 interacting protein. A partial loss of BCCIP function was sufficient to trigger genomic instability and tumorigenesis. However, a complete deletion of BCCIP arrested cell growth and was lethal in mice. Here, we report that a fraction of mammalian BCCIP localizes in the nucleolus and regulates 60S ribosome biogenesis. Both abrogation of BCCIP nucleolar localization and impaired BCCIP–eIF6 interaction can compromise eIF6 recruitment to the nucleolus and 60S ribosome biogenesis. BCCIP is vital for a pre-rRNA processing step that produces 12S pre-rRNA, a precursor to the 5.8S rRNA. However, a heterozygous Bccip loss was insufficient to impair 60S biogenesis in mouse embryo fibroblasts, but a profound reduction of BCCIP was required to abrogate its function in 60S biogenesis. These results suggest that BCCIP is a critical factor for mammalian pre-rRNA processing and 60S generation and offer an explanation as to why a subtle dysfunction of BCCIP can be tumorigenic but a complete depletion of BCCIP is lethal.
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Basu, Uttiya, Kausik Si, Haiteng Deng, and Umadas Maitra. "Phosphorylation of Mammalian Eukaryotic Translation Initiation Factor 6 and Its Saccharomyces cerevisiae Homologue Tif6p: Evidence that Phosphorylation of Tif6p Regulates Its Nucleocytoplasmic Distribution and Is Required for Yeast Cell Growth." Molecular and Cellular Biology 23, no. 17 (September 1, 2003): 6187–99. http://dx.doi.org/10.1128/mcb.23.17.6187-6199.2003.
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ABSTRACT The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.
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Burwick, Nicholas, Scott A. Coats, Tomoka Nakamura, and Akiko Shimamura. "Impaired ribosomal subunit association in Shwachman-Diamond syndrome." Blood 120, no. 26 (December 20, 2012): 5143–52. http://dx.doi.org/10.1182/blood-2012-04-420166.
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Abstract Shwachman-Diamond syndrome (SDS) is an autosomal-recessive marrow failure syndrome with a predisposition to leukemia. SDS patients harbor biallelic mutations in the SBDS gene, resulting in low levels of SBDS protein. Data from nonhuman models demonstrate that the SBDS protein facilitates the release of eIF6, a factor that prevents ribosome joining. The complete abrogation of Sbds expression in these models results in severe cellular and lethal physiologic abnormalities that differ from the human disease phenotype. Because human SDS cells are characterized by partial rather than complete loss of SBDS expression, we interrogated SDS patient cells for defects in ribosomal assembly. SDS patient cells exhibit altered ribosomal profiles and impaired association of the 40S and 60S subunits. Introduction of a wild-type SBDS cDNA into SDS patient cells corrected the ribosomal association defect, while patient-derived SBDS point mutants only partially improved subunit association. Knockdown of eIF6 expression improved ribosomal subunit association but did not correct the hematopoietic defect of SBDS-deficient cells. In summary, we demonstrate an SBDS-dependent ribosome maturation defect in SDS patient cells. The role of ribosomal subunit joining in marrow failure warrants further investigation.
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Naranda, T., S. E. MacMillan, T. F. Donahue, and J. W. Hershey. "SUI1/p16 is required for the activity of eukaryotic translation initiation factor 3 in Saccharomyces cerevisiae." Molecular and Cellular Biology 16, no. 5 (May 1996): 2307–13. http://dx.doi.org/10.1128/mcb.16.5.2307.
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A genetic reversion analysis at the HIS4 locus in Saccharomyces cerevisiae has identified SUI1 as a component of the translation initiation complex which plays an important role in ribosomal recognition of the initiator codon. SUI1 is an essential protein of 12.3 kDa that is required in vivo for the initiation of protein synthesis. Here we present evidence that SUI1 is identical to the smallest subunit, p16, of eukaryotic translation initiation factor 3 (eIF-3) in S. cerevisiae. SUI1 and eIF3-p16 comigrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cross-react with anti-SUI1 and anti-eIF3 antisera. Anti-SUI1 antisera immunoprecipitate all of the subunits of eIF3, whereas antisera against the eIF3 complex and the individual PRT1 and GCD10 subunits of eIF3 immunoprecipitate SUI1. Finally, the N-terminal amino acid sequence of a truncated form of eIF3-p16 matches the sequence of SUI1. eIF3 isolated from a sui1(ts) strain at 37 degrees C lacks SUI1 and fails to exhibit eIF3 activity in the in vitro assay for methionyl-puromycin synthesis. A free form of SUI1 separate from the eIF3 complex is found in S. cerevisiae but lacks activity in the in vitro assay. The results, together with prior genetic experiments, indicate that SUI1 is essential for eIF3 activity and functions as part of eIF3 and in concert with eIF2 to promote eIF2-GTP-Met-tRNAi ternary complex recognition of the initiator codon.
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Browning, K. S. "Plant translation initiation factors: it is not easy to be green." Biochemical Society Transactions 32, no. 4 (August 1, 2004): 589–91. http://dx.doi.org/10.1042/bst0320589.
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Plants have significant differences in some of the ‘parts’ of the translational machinery. There are two forms of eukaryotic initiation factor (eIF) 4F, eIF3 has two novel subunits, eIF4B is poorly conserved, and eIF2 kinases and eIF4E binding proteins (4E-BP) are yet to be discovered. These differences suggest that plants may regulate their translation in unique ways.
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GOLOB-SCHWARZL, NICOLE, PHILIP PUCHAS, MARGIT GOGG-KAMERER, WILKO WEICHERT, BENJAMIN GÖPPERT, and JOHANNES HAYBAECK. "New Pancreatic Cancer Biomarkers eIF1, eIF2D, eIF3C and eIF6 Play a Major Role in Translational Control in Ductal Adenocarcinoma." Anticancer Research 40, no. 6 (June 2020): 3109–18. http://dx.doi.org/10.21873/anticanres.14292.
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Weis, Félix, Emmanuel Giudice, Mark Churcher, Li Jin, Christine Hilcenko, Chi C. Wong, David Traynor, Robert R. Kay, and Alan J. Warren. "Mechanism of eIF6 release from the nascent 60S ribosomal subunit." Nature Structural & Molecular Biology 22, no. 11 (October 19, 2015): 914–19. http://dx.doi.org/10.1038/nsmb.3112.
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Vaccaro, Maria Carmela, Marta Cuccaro, Nadia De Marco, and Chiara Campanella. "Expression of p27BBP/eIF6 is highly modulated duringXenopus laevis embryogenesis." Molecular Reproduction and Development 73, no. 4 (2006): 482–90. http://dx.doi.org/10.1002/mrd.20449.
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ASANO, K., L. PHAN, L. VALASEK, L. W. SCHOENFELD, A. SHALEV, J. CLAYTON, K. NIELSEN, T. F. DONAHUE, and A. G. HINNEBUSCH. "A Multifactor Complex of eIF1, eIF2, eIF3, eIF5, and tRNAiMet Promotes Initiation Complex Assembly and Couples GTP Hydrolysis to AUG Recognition." Cold Spring Harbor Symposia on Quantitative Biology 66 (January 1, 2001): 403–16. http://dx.doi.org/10.1101/sqb.2001.66.403.
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Pérez-Juárez, Jesús, Juana Virginia Tapia-Vieyra, Gabriel Gutiérrez-Magdaleno, and Nuria Sánchez-Puig. "Altered Conformational Landscape upon Sensing Guanine Nucleotides in a Disease Mutant of Elongation Factor-like 1 (EFL1) GTPase." Biomolecules 12, no. 8 (August 19, 2022): 1141. http://dx.doi.org/10.3390/biom12081141.
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The final maturation step of the 60S ribosomal subunit requires the release of eukaryotic translation initiation factor 6 (human eIF6, yeast Tif6) to enter the pool of mature ribosomes capable of engaging in translation. This process is mediated by the concerted action of the Elongation Factor-like 1 (human EFL1, yeast Efl1) GTPase and its effector, the Shwachman-Bodian-Diamond syndrome protein (human SBDS, yeast Sdo1). Mutations in these proteins prevent the release of eIF6 and cause a disease known as Shwachman–Diamond Syndrome (SDS). While some mutations in EFL1 or SBDS result in insufficient proteins to meet the cell production of mature large ribosomal subunits, others do not affect the expression levels with unclear molecular defects. We studied the functional consequences of one such mutation using Saccharomyces cerevisiae Efl1 R1086Q, equivalent to human EFL1 R1095Q described in SDS patients. We characterised the enzyme kinetics and energetic basis outlining the recognition of this mutant to guanine nucleotides and Sdo1, and their interplay in solution. From our data, we propose a model where the conformational change in Efl1 depends on a long-distance network of interactions that are disrupted in mutant R1086Q, whereby Sdo1 and the guanine nucleotides no longer elicit the conformational changes previously described in the wild-type protein. These findings point to the molecular malfunction of an EFL1 mutant and its possible impact on SDS pathology.
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Nielsen, Klaus H., Leos Valášek, Caroah Sykes, Antonina Jivotovskaya, and Alan G. Hinnebusch. "Interaction of the RNP1 Motif in PRT1 with HCR1 Promotes 40S Binding of Eukaryotic Initiation Factor 3 in Yeast." Molecular and Cellular Biology 26, no. 8 (April 15, 2006): 2984–98. http://dx.doi.org/10.1128/mcb.26.8.2984-2998.2006.
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ABSTRACT We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts− phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts− phenotype is exacerbated by tif32-Δ6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Δ impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG recognition in vivo.