A guide to design and optimization of reporter assays for 3' untranslated region mediated regulation of mammalian messenger RNAs

Post-transcriptional regulatory mechanisms are pervasive in the control of gene expression. Regulatory sequences within transcripts can control RNA processing, localization, translation efficiency, and stability of the RNA. Regulation is mediated by a diverse set of RNA binding regulators, including proteins and RNAs, which interact with specific mRNA sequences that are often found in untranslated regions. The potential for vast post-transcriptional control exists: mammalian mRNAs contain extensive untranslated regions and their genomes encode many hundreds of RNA binding proteins and non-coding RNAs. Facile quantitative methods are necessary to study the activities and mechanisms of regulatory sequences and the RNA binding factors that recognize them. Here we discuss the design and implementation of luciferase-based reporter assays to measure the effect of regulatory RNA sequences on protein and RNA expression. Protocols are described for transfection of the reporter into cells, measurement of protein expression levels with luciferase activity assays, RNA purification, and measurement of mRNA levels by reverse-transcription and quantitative polymerase chain reaction. For each assay, troubleshooting of common problems and critical controls are discussed. We present our optimized techniques and data from studies that measure specific and direct repression (i.e. negative regulation) of mRNAs by members of the PUF family of RNA binding proteins in cultured human cells.