A translational repression reporter assay for the analysis of RNA-binding protein consensus sites

Jessica Nowacki; Mateo Malenica; Stefan Schmeing; Damian Schiller; Benjamin Buchmuller; Gulshan Amrahova; Peter 't Hart

doi:10.1080/15476286.2023.2192553

A translational repression reporter assay for the analysis of RNA-binding protein consensus sites

RNA Biol. 2023 Jan;20(1):85-94. doi: 10.1080/15476286.2023.2192553.

Authors

Jessica Nowacki¹, Mateo Malenica¹, Stefan Schmeing¹, Damian Schiller², Benjamin Buchmuller², Gulshan Amrahova¹, Peter 't Hart¹

Affiliations

¹ Chemical Genomics Centre of the Max Planck Society, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
² Faculty of Chemistry and Chemical Biology, Technical University of Dortmund, Dortmund, Germany.

Abstract

RNA-binding proteins are essential regulators of RNA processing and function. Translational repression assays can be used to study how they interact with specific RNA sequences by insertion of such a consensus sequence into the 5' untranslated region of a reporter mRNA and measuring reporter protein translation. The straightforward set-up of these translational repression assays avoids the need for the isolation of the protein or the RNA providing speed, robustness and a low-cost method. Here, we report the optimization of the assay to function with linear RNA sequences instead of the previously reported hairpin type sequences to allow the study of a wider variety of RNA-binding proteins. Multiplication of a consensus sequence strongly improves the signal allowing analysis by both fluorescence intensity measurements and flow cytometry.

Keywords: Binding studies; Protein-RNA interactions; RNA-binding proteins; Translation; Translational repression assay.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Protein Biosynthesis*
RNA, Messenger / genetics
RNA, Messenger / metabolism
RNA-Binding Proteins* / genetics
RNA-Binding Proteins* / metabolism

Substances

RNA-Binding Proteins
RNA, Messenger

Grants and funding

This work was funded via the Chemical Genomics Centre of the Max Planck Society which is supported by AstraZeneca, Merck KGaA, Pfizer Inc, and the Max Planck Society. Gulshan Amrahova was funded by a scholarship from the German Academic Exchange Service (DAAD).