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

Abstract

Ribosomes function as platforms for binding of other molecules, but technologies for studying this process are lacking. Therefore we developed iRIA (in vitro Ribosomes Interaction Assay). In approach I, Artemia salina ribosomes spotted on solid phase are used for binding picomoles of analytes; in approach II, cellular extracts allow the measurement of ribosome activity in different conditions. We apply the method to analyze several features of eIF6 binding to 60S subunits. By approach I, we show that the off-rate of eIF6 from preribosomes is slower than from mature ribosomes and that its binding to mature 60S occurs in the nM affinity range. By approach II we show that eIF6 binding sites on 60S are increased with mild eIF6 depletion and decreased in cells that are devoid of SBDS, a ribosomal factor necessary for 60S maturation and involved in Swachman Diamond syndrome. We show binding conditions to immobilized ribosomes adaptable to HT and quantify free ribosomes in cell extracts. In conclusion, we suggest that iRIA will greatly facilitate the study of interactions on the ribosomal surface.

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

“…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.…”

“…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.…”

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

“…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).…”

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

“…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.…”