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

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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… Click to show full 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 fluorescence activated cell sorting.

Keywords: rna binding; consensus; reporter; rna; translational repression

Journal Title: RNA Biology
Year Published: 2022

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