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.
Highlights
The classical method for studying ribosomal interaction is the polysomal profile, based on the separation of cellular extracts on sucrose gradients[1] followed by the collection of fractions and the analysis by precipitation and Western blotting[2]
We provide evidence that the method that we have developed, named here iRIA is suitable for studying interactions on the ribosomal surfaces and their modulation
We used iRIA to answer two unresolved issues as proof-of-concept: to characterize eIF6 binding to 60S subunits and the number of ribosomes present in a cell after SBDS loss
Summary
The classical method for studying ribosomal interaction is the polysomal profile, based on the separation of cellular extracts on sucrose gradients[1] followed by the collection of fractions and the analysis by precipitation and Western blotting[2]. An high throughput proteomic study has identified a number of proteins that are associated with actively translating ribosomes (Integrin β 1, IGF2BP3, MARCKS), using a SILAC-Based approach[6]. In this case, it is not known which of these interactions occur at high affinity on the ribosomal platform. We provide evidence that the method that we have developed, named here iRIA is suitable for studying interactions on the ribosomal surfaces and their modulation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
