Protocol for in vitro transcribing mRNAs with defined poly(A)-tail lengths and visualizing sequential PABP binding

Abstract

Summary: Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation. Biochemical assays offer significant advantages for this purpose. Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach. We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of Cytoplasmic Poly(A)-Binding Proteins (PABPCs) to these poly(A) tails. We detail quality control steps throughout the procedure.For complete details on the use and execution of this protocol, please refer to Grandi et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Bioinformatics
• Cell-based Assays
• Molecular Biology
• Gene Expression