Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits
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...
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doaj-1564496d7c2b46afb4b6e85ce4b854592020-11-25T01:33:22ZengMDPI AGCells2073-44092020-01-019117210.3390/cells9010172cells9010172Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal SubunitsElisa Pesce0Annarita Miluzio1Lorenzo Turcano2Claudia Minici3Delia Cirino4Piera Calamita5Nicola Manfrini6Stefania Oliveto7Sara Ricciardi8Renata Grifantini9Massimo Degano10Alberto Bresciani11Stefano Biffo12National Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyNational Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyDepartment of Translational and Discovery Research, IRBM S.p.A., Via Pontina km 30, 600, 00071 Pomezia (Roma), ItalyBiocrystallography Unit, Dept. of Immunology, Transplantation and Infectious Diseases, IRCCS Scientific Institute San Raffaele, Via Olgettina 58, 20132 Milan, ItalyNational Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyNational Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyNational Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyNational Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyNational Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyNational Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyBiocrystallography Unit, Dept. of Immunology, Transplantation and Infectious Diseases, IRCCS Scientific Institute San Raffaele, Via Olgettina 58, 20132 Milan, ItalyDepartment of Translational and Discovery Research, IRBM S.p.A., Via Pontina km 30, 600, 00071 Pomezia (Roma), ItalyNational Institute of Molecular Genetics, “Fondazione Romeo ed Enrica Invernizzi”, INGM, Via Francesco Sforza 35, 20122 Milan, ItalyEukaryotic 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.https://www.mdpi.com/2073-4409/9/1/172iriainitiationpolysomeseif4erack1shwachman–diamond syndromeeifsixty-i |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Elisa Pesce Annarita Miluzio Lorenzo Turcano Claudia Minici Delia Cirino Piera Calamita Nicola Manfrini Stefania Oliveto Sara Ricciardi Renata Grifantini Massimo Degano Alberto Bresciani Stefano Biffo |
spellingShingle |
Elisa Pesce Annarita Miluzio Lorenzo Turcano Claudia Minici Delia Cirino Piera Calamita Nicola Manfrini Stefania Oliveto Sara Ricciardi Renata Grifantini Massimo Degano Alberto Bresciani Stefano Biffo Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits Cells iria initiation polysomes eif4e rack1 shwachman–diamond syndrome eifsixty-i |
author_facet |
Elisa Pesce Annarita Miluzio Lorenzo Turcano Claudia Minici Delia Cirino Piera Calamita Nicola Manfrini Stefania Oliveto Sara Ricciardi Renata Grifantini Massimo Degano Alberto Bresciani Stefano Biffo |
author_sort |
Elisa Pesce |
title |
Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits |
title_short |
Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits |
title_full |
Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits |
title_fullStr |
Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits |
title_full_unstemmed |
Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits |
title_sort |
discovery and preliminary characterization of translational modulators that impair the binding of eif6 to 60s ribosomal subunits |
publisher |
MDPI AG |
series |
Cells |
issn |
2073-4409 |
publishDate |
2020-01-01 |
description |
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. |
topic |
iria initiation polysomes eif4e rack1 shwachman–diamond syndrome eifsixty-i |
url |
https://www.mdpi.com/2073-4409/9/1/172 |
work_keys_str_mv |
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