Direct and high throughput (HT) interactions on the ribosomal surface by iRIA

Pesce, Elisa; Minici, Claudia; Baβler, Jochen; Hurt, Ed; Degano, Massimo; Calamita, Piera; Biffo, Stefano

doi:10.1038/srep15401

Cited by 11 publications

(26 citation statements)

References 24 publications

(34 reference statements)

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“…This model is consistent with the accumulation of free 60S that we observed (Fig 2A). We recently developed a Ribosomes Interaction Assay (Fig 2G), able to quantify eIF6 binding sites on the 60S [40]. We immobilized equal ribosomes from Sbds R126T/R126T and wild type cells, and measured eIF6 binding sites.…”

Section: Resultsmentioning

confidence: 99%

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SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention

et al. 2017

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Ribosomopathies are a family of inherited disorders caused by mutations in genes necessary for ribosomal function. Shwachman-Diamond Bodian Syndrome (SDS) is an autosomal recessive disease caused, in most patients, by mutations of the SBDS gene. SBDS is a protein required for the maturation of 60S ribosomes. SDS patients present exocrine pancreatic insufficiency, neutropenia, chronic infections, and skeletal abnormalities. Later in life, patients are prone to myelodisplastic syndrome and acute myeloid leukemia (AML). It is unknown why patients develop AML and which cellular alterations are directly due to the loss of the SBDS protein. Here we derived mouse embryonic fibroblast lines from an SbdsR126T/R126T mouse model. After their immortalization, we reconstituted them by adding wild type Sbds. We then performed a comprehensive analysis of cellular functions including colony formation, translational and transcriptional RNA-seq, stress and drug sensitivity. We show that: 1. Mutant Sbds causes a reduction in cellular clonogenic capability and oncogene-induced transformation. 2. Mutant Sbds causes a marked increase in immature 60S subunits, limited impact on mRNA specific initiation of translation, but reduced global protein synthesis capability. 3. Chronic loss of SBDS activity leads to a rewiring of gene expression with reduced ribosomal capability, but increased lysosomal and catabolic activity. 4. Consistently with the gene signature, we found that SBDS loss causes a reduction in ATP and lactate levels, and increased susceptibility to DNA damage. Combining our data, we conclude that a cell-specific fragile phenotype occurs when SBDS protein drops below a threshold level, and propose a new interpretation of the disease.

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Section: Resultsmentioning

confidence: 99%

“…iRIA assay was performed as described in [40]. Briefly, 96-well plates were coated with a cellular extract diluted in 50 μL of PBS, 0,01% Tween-20, O/N at 4°C in humid chamber.…”

Section: Methodsmentioning

confidence: 99%

SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention

et al. 2017

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“…To assess whether increased transcription of genes related to ribosome biogenesis and rRNA processing observed in gene expression analysis experiments was accompanied by an effect in the translational machinery, we investigated changes in levels of free 60S subunits upon eIF6 overexpression. To this end, we performed the in vitro Ribosome Interaction Assay (iRIA) 33 . We found that the expression of eIF6 in larval eye discs (GMR>eIF6) led to a 25% reduction in free 60S sites when compared to control (GMRGAL4/+) ( Fig 6a).…”

Section: Eif6 Expression Reshapes the Transcriptome Increasing A Ribmentioning

confidence: 99%

The eukaryotic Initiation Factor 6 (eIF6) regulates ecdysone biosynthesis by modulating translation inDrosophila

Russo

Gatti

Alfieri

et al. 2017

Preprint

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22Increases in ribosomal proteins and initiation factors are often observed in tumours 23 and required during development. Haploinsufficient models have shown that such 24 elevation is essential for tumour growth. Models with increased gene dosage of 25 initiation factors, addressing the effects of their forced overexpression are lacking. 26The eukaryotic Initiation Factor 6 (eIF6) gene is amplified in some cancers and

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“…In this study, we developed a novel method for finding modulators of eIF6 binding to the 60S subunit [34] and performed an automated screening of small compounds. Here, we describe the screening and identification of three compounds that have a biological activity that can modulate the binding of eIF6 to the 60S.…”

Section: Introductionmentioning

confidence: 99%

Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits

Pesce

Miluzio

Turcano

et al. 2020

Cells

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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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Direct and high throughput (HT) interactions on the ribosomal surface by iRIA

Cited by 11 publications

References 24 publications

SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention

SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention

The eukaryotic Initiation Factor 6 (eIF6) regulates ecdysone biosynthesis by modulating translation inDrosophila

Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits

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