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

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.

Automated summary

RNA-binding proteins play essential roles in regulating RNA processing and function. Translational repression assays provide a convenient and cost-effective method to study their interactions with specific RNA sequences. We optimized the assay by using linear RNA sequences instead of the previously reported hairpin type sequences, allowing for the analysis of a wider range of RNA-binding proteins. Multiplication of a consensus sequence greatly enhances the signal, enabling analysis through fluorescence intensity measurements and flow cytometry.